CN114286858B - 叶酸生产菌株及其制备和应用 - Google Patents
叶酸生产菌株及其制备和应用 Download PDFInfo
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- CN114286858B CN114286858B CN202080059788.7A CN202080059788A CN114286858B CN 114286858 B CN114286858 B CN 114286858B CN 202080059788 A CN202080059788 A CN 202080059788A CN 114286858 B CN114286858 B CN 114286858B
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Abstract
本发明提供了一种叶酸生产菌株及其制备和应用,特别是降低了本发明工程菌株中内源性folC基因的表达水平,引入外源性folC基因,与出发菌株相比,工程菌株中叶酸、其前体或其中间体的生产能力显著提高。
Description
技术领域
本发明涉及生物技术工程领域,具体涉及一种产生叶酸的菌株及其制备和应用。
背景
叶酸是叶酸及其多种衍生物的总称;它们在氧化状态、蝶啶环的单碳取代和γ-连接的谷氨酸残基的数量上有所不同(如图1所示)。叶酸的蝶啶部分可以以三种氧化状态存在:完全氧化(叶酸),或还原的7,8-二氢叶酸(DHF),或5,6,7,8-四氢叶酸(THF)(参见结构式I)。THF是维生素的共酶活性形式,它接受、转移和提供C1基团,这些C1基团连接在N5或N10位置或通过桥接这些位置。C1基团的氧化态也不同,叶酸以甲酸衍生物(5-甲酰基-THF(5-FTHF或亚叶酸)、10-甲酰基-THF、5,10-亚甲基-THF和5-forminino-THF)、甲醇(5-甲基-THF)或甲醛(5,10-亚甲基-THF)。此外,大多数天然存在的叶酸以γ-连接的聚谷氨酸结合物的形式存在。
叶酸(蝶酰-L-谷氨酸)是一种合成化合物,自然界中不存在。叶酸作为辅酶没有活性,必须在细胞内经过几个代谢步骤才能转化为具有代谢活性的THF形式。然而,叶酸是商业上最重要的叶酸化合物,工业上通过化学合成生产。哺乳动物不能合成叶酸,并依赖膳食补充剂来维持叶酸的正常水平。低叶酸状态可能是由于饮食摄入量低、摄入叶酸吸收不良以及由于遗传缺陷或药物相互作用引起的叶酸代谢改变。大多数国家已经通过叶酸补充剂或强化食品确定了叶酸的推荐摄入量。膳食补充剂中使用的叶酸包括叶酸、亚叶酸(5-FTHF、甲酰四氢叶酸)或5-MTHF(Scaglione和Panzavolta 2014)。目前生产两种盐形式的5-MTHF作为补充剂。默克密理博生产一种5-MTHF的钙盐,它是一种稳定的结晶形式,是天然存在的主要叶酸形式。Gnosis S.p.A.开发了一种(6S)-5-MTHF的氨基葡萄糖盐并获得了专利,品牌名为/>
目前,叶酸在工业上主要是通过化学合成生产的,而与其他维生素不同,由于当前细菌菌株产生的叶酸产量低,工业规模的微生物叶酸生产并未得到利用(Rossi等人,2016年)。虽然化学产生的叶酸不是天然存在的分子,但人类能够通过二氢叶酸还原酶(DHFR)的作用将其代谢成生物活性形式的叶酸。有几个原因支持用微生物发酵替代化学合成方法来商业化生产叶酸:首先,还原形式的叶酸可以由微生物产生,人类可以更有效地利用。最重要的是,原则上,单步发酵过程比多阶段化学过程更高效、更环保。
以前的研究已经完成以阐明微生物中叶酸的产生。大多数用于生产叶酸的微生物应用仅限于发酵乳制品的强化和产生叶酸的益生菌。还进行了培养条件的优化以提高叶酸的合成,达到约150μg/g的叶酸产量(Hjortmo等人,2008;Sybesma等人,2003b)。一些研究已经描述了乳酸菌(Sybesma等人,2003a)、酵母菌(Walkey等人,2015年)或丝状真菌(Serrano-Amatriain等人,2016年)的转基因菌株,它们能够产生叶酸,滴度高达6.6mg/L。另一种成功用于微生物生产叶酸的方法是在对氨基苯甲酸(pABA)存在下培养酵母或细菌菌株。在这些培养物的上清液中测得总叶酸含量高达22mg/L。
因此,迫切需要开发一种新的叶酸生产菌株,以提高叶酸、其盐、其前体或其中间体的生产能力。
发明内容
本发明的目的是提供一种产生叶酸的菌株及其制备和应用。
在本发明的第一方面,提供了一种用于合成叶酸、其盐、其前体或其中间体的基因工程菌株,其中所述工程菌株中内源性folC基因的表达水平降低,并且引入外源folC基因并且所述工程菌株与其出发菌株相比具有显著提高的叶酸、其前体或其中间体的生产能力。
在另一优选实施方式中,叶酸、其盐、其前体或其中间体的结构式如式I所示:
其中,当a为单键时,a’为无或当a’为单键时,a为无;
当b是单键时,b'是无或当b'是单键时,b是无;
R1选自下组:-H、-CH3(5-甲基)、-CHO(5-甲酰基)、-CH=或=CH-(5,10-亚甲基)、-CH2-(5,10-亚甲基)、-CH=NH(5-亚胺甲基)、或其组合;
R2选自下组:-H、-CHO(10-甲酰基)、-CH=、=CH-(5,10-亚甲基)、-CH2-(5,10-亚甲基)、或其组合。
在另一个优选的实施方式中,所述工程菌株的出发菌株选自下组:大肠杆菌、乳酸乳球菌、枯草芽孢杆菌、无名假丝酵母和棉囊阿舒霉。
在另一个优选的实施方案中,所述工程菌株的出发菌株包括枯草芽孢杆菌。
在另一个优选的实施方案中,所述基因工程菌株是细菌。
在另一个优选的实施方案中,所述基因工程菌株是芽孢杆菌属的细菌。
在另一个优选的实施方案中,所述基因工程菌株是枯草芽孢杆菌的细菌。
在另一个优选的实施方案中,内源folC基因的表达水平降低是指与出发菌株(野生型)相比,所述工程菌株中内源性folC基因的表达水平降低至少50%,优选降低至少60%、70%、80%、90%或100%。
在另一个优选的实施方案中,外源性folC基因来源于棉囊阿舒霉或罗伊氏乳杆菌。
在另一个优选的实施方案中,外源性folC基因的表达产物包含选自下组的多肽或其衍生多肽:二氢叶酸合酶(DHFS-EC 6.3.2.12)。
在另一个优选的实施方案中,二氢叶酸合酶的氨基酸序列如SEQ ID NO.:22或23所示。
在另一个优选的实施方案中,编码二氢叶酸合酶的多核苷酸序列如SEQ ID NO.:24或25所示。
在另一个优选的实施方案中,外源性folC基因包括与外源性folC基因≥80%同一性的基因,优选≥90%,更优选≥95%,更优选≥98%,更优选≥99%(注:在核苷酸水平上)。
在另一个优选的实施方案中,外源性folC基因如SEQ ID NO.:24或25所示。
在另一个优选的实施方案中,所述二氢叶酸合酶包含与SEQ ID NO:22或23的具有至少70%,例如至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的序列同一性的氨基酸序列。
在另一个优选实施方式中,“显著提高”是指与出发菌株相比,工程菌株的叶酸发酵产量至少大于0.01g/L,优选至少0.01-0.1g/L;更优选地,至少0.1-1g/L,根据发酵液的体积,每升;和/或
“显著提高”是指与出发菌株相比,工程菌株的叶酸生产能力增加或提高100%;优选200-50000%。
在另一个优选的实施方案中,“显著提高”是指与出发菌株相比,工程菌株中的叶酸生产能力增加或提高至少50%,例如至少100%、至少200%、至少500%、至少1000%、至少2000%、至少5000%、至少10000%、至少20000%或至少50000%,与起始菌株相比。
在另一个优选实施方案中,在工程菌株中引入或上调编码叶酸生物合成酶的基因。
在另一个优选的实施方案中,上调是指与出发菌株(野生型)相比,在引入或上调叶酸生物合成基因的工程菌株中,叶酸生物合成基因的表达水平增加至少80%,更优选增加至少100%、200%、300%、400%、500%、600%或800%。
在另一个优选的实施方案中,上调是指与出发菌株(野生型)相比,在引入或上调叶酸生物合成基因的工程菌株中,与出发菌株(野生型)相比,叶酸生物合成基因的表达水平具有至少50%,例如至少100%、至少200%、至少500%、至少1000%、至少2000%、至少5000%、至少10000%、至少20000%或至少50000%。
在另一个优选的实施方案中,叶酸生物合成基因选自下组:folE/mtrA、folB、folK、folP/sul、folA/dfrA、或其组合。
在另一个优选的实施方案中,叶酸生物合成基因是选自folE/mtrA、folB、folK、folP/sul、folA/dfrA的至少一种基因(例如至少两种、至少三种、至少四种或至少五种基因)。
在另一个优选的实施方案中,叶酸生物合成基因来源于细菌或真菌,优选选自芽孢杆菌属、乳球菌属和阿舒囊霉属。
在另一个优选的实施方案中,叶酸生物合成基因来自细菌,优选来自芽孢杆菌属物种的细菌,最优选来自枯草芽孢杆菌或乳酸乳球菌或棉阿舒囊霉。
在另一个优选的实施方案中,叶酸生物合成基因的表达产物包含选自下组的多肽或其衍生物:GTP环化水解酶、7,8-二氢新蝶呤醛缩酶、2-氨基-4-羟基-6-羟甲基二氢蝶啶焦磷酸激酶、二氢蝶酸合酶、二氢叶酸还原酶、或其组合。
在另一个优选的实施方案中,叶酸生物合成基因的表达产物是至少一种参与叶酸生物合成的酶。
在另一个优选的实施方案中,参与叶酸生物合成的至少一种酶对于基因工程微生物是异源的。
在另一个优选的实施方案中,参与叶酸生物合成的至少一种酶来源于细菌或真菌,优选选自芽孢杆菌属、乳球菌属、希瓦氏菌属、弧菌属和阿舒囊霉属。
在另一个优选的实施方案中,参与叶酸生物合成的至少一种酶来源于枯草芽孢杆菌、乳酸乳杆菌、紫色希瓦氏菌(Shewanella violacea)、需钠弧菌(Vibrio natriegens)或棉阿舒囊霉(Ashbya gossypii)。
在另一个优选的实施方案中,具有GTP环化水解酶活性的多肽包含与SEQ ID NO:7具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,具有7,8-二氢新蝶呤醛缩酶活性的多肽包含与SEQID NO:8具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少具有98%或至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,具有2-氨基-4-羟基-6-羟甲基-二氢蝶啶焦磷酸激酶活性的多肽包含与SEQ ID NO:9具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,具有二氢蝶酸合酶活性的多肽包含与SEQ ID NO:10具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,具有二氢叶酸还原酶活性的多肽包含与SEQ ID NO:12具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,GTP环化水解酶的氨基酸序列如SEQ ID NO.:7所示。
在另一个优选的实施方案中,GTP环化水解酶的编码序列如SEQ ID NO.:1所示。
在另一个优选的实施方案中,7,8-二氢新蝶呤醛缩酶氨基酸序列如SEQ ID NO.:2所示。
在另一个优选的实施方案中,7,8-二氢新蝶呤醛缩酶的编码序列如SEQ ID NO.:8所示。
在另一个优选的实施方案中,2-氨基-4-羟基-6-羟甲基二氢蝶啶焦磷酸激酶的氨基酸序列如SEQ ID NO.:3所示。
在另一个优选的实施方案中,2-氨基-4-羟基-6-羟甲基二氢蝶啶焦磷酸激酶的编码序列如SEQ ID NO.:9所示。
在另一个优选的实施方案中,二氢蝶酸合酶的氨基酸序列如SEQ ID NO.:4所示。
在另一个优选的实施方案中,二氢蝶酸合酶的编码序列如SEQ ID NO.:10所示。
在另一个优选的实施方案中,二氢叶酸还原酶的氨基酸序列如SEQ ID NO.:6所示。
在另一个优选的实施方案中,二氢叶酸还原酶的编码序列如SEQ ID NO.:12所示。
在另一个优选的实施方案中,工程菌株通过以下方法获得:
(a)降低出发菌株中内源性folC基因的表达水平和/或活性,并引入外源性folC基因。
在另一个优选的实施方案中,所述方法还包括在出发菌株中引入或上调叶酸生物合成基因的步骤(b)。
在另一个优选的实施方案中,生产能力包括:发酵产量(生产率)。
在第二方面,提供了一种叶酸、其盐、其前体或其中间体的制备方法,包括以下步骤:
(i)提供权利要求1所述的工程菌株;
(ii)培养步骤(i)所述的工程菌株,从而获得含有叶酸、其盐、其前体或其中间体的一种或多种化合物的发酵产物;
(iii)任选地,对步骤(ii)中得到的发酵产物进行分离纯化,进一步获得叶酸、其盐、其前体或其中间体的一种或多种化合物;
(iv)任选地,将步骤(ii)或(iii)中得到的产物经酸性或碱性条件进一步得到叶酸、其盐、其前体或其中间体的不同化合物;
其中叶酸、其盐、其前体或其中间体的结构式如式I所示:
并且R1、R2、a、a'、b、b'如上定义。
在另一个优选的实施方案中,叶酸、其盐、其前体或其中间体是叶酸。
另一方面,提供了一种制备叶酸、其前体或其中间体的方法,包括以下步骤:
(i)提供权利要求1所述的工程菌株;
(ii)培养步骤(i)所述的工程菌株,从而得到含叶酸的发酵产物;
(iii)任选地,对步骤(ii)中得到的发酵产物进行分离纯化,进一步得到叶酸、其前体或其中间体。
在另一个优选的实施方案中,叶酸、其盐、其前体或其中间体的结构式如式I所示:
其中,当a为单键时,a’为无或当a’为单键时,a为无;
当b是单键时,b'是无或当b'是单键时,b是无;
R1选自下组:-H、-CH3(5-甲基)、-CHO(5-甲酰基)、-CH=或=CH-(5,10-亚甲基)、-CH2-(5,10-亚甲基)、-CH=NH(5-亚胺甲基)、或其组合;
R2选自下组:-H、-CHO(10-甲酰基)、-CH=、=CH-(5,10-亚甲基)、-CH2-(5,10-亚甲基)、或其组合。
在另一个优选的实施方案中,工程菌株的培养温度为32-42℃,优选34-39℃,更优选36-39℃,例如约37℃。
在另一个优选的实施方案中,工程菌株的培养时间为10-70h,优选24-60h,更优选36-50h。
在另一个优选的实施方案中,工程菌株培养的pH为6-8,优选6.5-7.5,更优选6.8-7.2。
在另一个优选的实施方案中,所述方法还包括在步骤(ii)的培养过程中添加对氨基苯甲酸(PABA)的步骤。
在另一个优选的实施方案中,对氨基苯甲酸(PABA)选自下组:对氨基苯甲酸钾、对氨基苯甲酸钠、对氨基苯甲酸甲酯、对氨基苯甲酸乙酯、对氨基苯甲酸丁酯、或其组合。
在另一个优选的实施方案中,进一步包括将步骤(i)或(ii)或(iii)中得到的产物置于酸性或碱性条件下,进一步得到衍生化合物。
在第三方面,提供了本发明第一方面所述的工程菌株的制备方法,包括以下步骤:
(a)降低出发菌株中内源性folC基因的表达水平,并引入外源性folC基因,从而获得权利要求1所述的工程菌株。
在另一个优选的实施方案中,所述方法还包括在出发菌株中引入或上调叶酸合成调控基因的步骤(b)。
在另一个优选实施方式中,所述方法包括以下步骤:
(a1)敲除宿主细胞中的内源性folC基因;
(b1)培养所述宿主细胞;和
所述方法包括以下步骤:
(a2)提供携带外源性folC基因的表达载体;
(b2)将所述表达载体转入到宿主细胞中;
(c2)培养所述宿主细胞。
在另一个优选的实施方案中,所述载体是质粒、粘粒或核酸片段。
在第四方面,提供了本发明第一方面所述的工程菌株的用途,其用作用于发酵生产叶酸、其盐、其前体或其中间体的工程菌株。
在第五方面,提供了一种基因工程微生物,优选细菌或酵母,其已被修饰为i)与其他方面相同的微生物(参考微生物)相比,编码具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽的内源基因的表达水平降低,和ii)表达仅具有二氢叶酸合酶活性的异源多肽。
在另一个优选的实施方案中,与其他相同的微生物相比,内源性基因的表达水平降低至少50%,例如降低至少60%、至少70%、至少80%、至少90%或至少100%。
在另一个优选的实施方案中,编码具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽的内源基因已经失活。
在另一个优选的实施方案中,编码具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽的内源基因已通过部分或整个基因序列的缺失而失活。
在另一个优选的实施方案中,编码具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽的内源基因是基因folC。
在另一个优选的实施方案中,编码具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽的内源基因是内源基因folC。
在另一个优选的实施方案中,编码具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽的内源基因包含与SEQ ID NO:5所示的核酸序列具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的序列同一性的核酸序列。
在另一个优选的实施方案中,由内源基因编码的同时具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽包含与SEQ ID NO:11所示的氨基酸序列具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,仅具有二氢叶酸合酶活性的异源多肽来源于细菌或真菌,优选地选自罗伊氏乳杆菌和棉阿舒囊霉。
在另一个优选的实施方案中,仅具有二氢叶酸合酶活性的异源多肽包含与SEQ IDNO:22或23的具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,与其他方面相同的微生物(参考微生物)相比,基因工程微生物已被进一步修饰以具有显著提高的叶酸、其前体或其中间体的生产能力。
在另一个优选的实施方案中,与其他方面相同的微生物相比,叶酸、其前体或其中间体的生产能力增加至少50%,例如至少100%、至少200%、至少500%、至少1000%、至少2000%、至少5000%、至少10000%、至少20000%或至少50000%。
在另一个优选的实施方案中,与其他方面相同的微生物相比,基因工程微生物已被进一步修饰以具有增加的至少一种基因(例如至少两种、至少三种、至少四种或至少五种基因)的表达水平,该基因编码参与叶酸生物合成的酶。
在另一个优选的实施方案中,与其他方面相同的微生物相比,编码参与叶酸生物合成的酶的至少一种基因(例如至少两种、三种、四种或五种基因)的表达水平增加至少50%,例如至少100%、至少200%、至少500%、至少1000%、至少2000%、至少5000%、至少10000%、至少20000%或至少50000%。
在另一个优选的实施方案中,编码参与叶酸生物合成的酶的至少一种基因选自下组:folE/mtrA、folB、folK、folP/sul和folA/dfrA。
在另一个优选的实施方案中,参与叶酸生物合成的酶选自下组:具有GTP环化水解酶活性的多肽、具有7,8-二氢新蝶呤醛缩酶活性的多肽、具有2-氨基-4-羟基-6-羟甲基二氢蝶啶焦磷酸激酶活性的多肽、具有二氢蝶酸合酶活性的多肽和具有二氢叶酸还原酶活性的多肽。
在另一个优选的实施方案中,编码参与叶酸生物合成的酶的至少一种基因对于基因工程微生物是异源的。
在另一个优选的实施方案中,编码参与叶酸生物合成的酶的至少一种基因来源于细菌或真菌,优选选自芽孢杆菌属、乳球菌属和阿舒囊霉属。
在另一个优选的实施方案中,所述至少一种编码参与叶酸生物合成的酶的基因来源于选自枯草芽孢杆菌、乳酸乳杆菌和棉阿舒囊霉的细菌或真菌。
在另一个优选的实施方案中,与其他方面相同的微生物相比,基因工程微生物已被进一步修饰以具有增加的参与叶酸生物合成的至少一种酶(例如至少两种、至少三种、至少四种或至少五种酶)的表达水平。
在另一个优选的实施方案中,所述至少一种参与叶酸生物合成的酶选自下组:具有GTP环化水解酶活性的多肽、具有7,8-二氢新蝶呤醛缩酶活性的多肽、具有2-氨基-4-羟基-6-羟甲基二氢蝶啶焦磷酸激酶活性的多肽、具有二氢蝶酸合酶活性的多肽和具有二氢叶酸还原酶活性的多肽。
在另一个优选的实施方案中,参与叶酸生物合成的至少一种酶对于基因工程微生物是异源的。
在另一个优选的实施方案中,参与叶酸生物合成的至少一种酶来源于细菌或真菌,优选选自芽孢杆菌属、乳球菌属、希瓦氏菌属、弧菌属和阿舒囊霉属。
在另一个优选的实施方案中,参与叶酸生物合成的至少一种酶来源于枯草芽孢杆菌、乳酸乳杆菌、紫色希瓦氏菌(Shewanella violacea)、需钠弧菌(Vibrio natriegens)或棉阿舒囊霉(Ashbya gossypii)。
在另一个优选的实施方案中,具有GTP环化水解酶活性的多肽包含具有与SEQ IDNO:7的至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,具有7,8-二氢新蝶呤醛缩酶活性的多肽包含与SEQID NO:8具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少具有98%或至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,具有2-氨基-4-羟基-6-羟甲基-二氢蝶啶焦磷酸激酶活性的多肽包含与SEQ ID NO:9具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,具有二氢蝶酸合酶活性的多肽包含与SEQ ID NO:10具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,具有二氢叶酸还原酶活性的多肽包含与SEQ ID NO:12具有至少70%、例如至少80%、至少85%、至少90%、至少95%、至少98%或至少99%的序列同一性的氨基酸序列。
在另一个优选的实施方案中,基因工程微生物是细菌。
在另一个优选的实施方案中,基因工程微生物是芽孢杆菌属的细菌。
在另一个优选的实施方案中,基因工程微生物是枯草芽孢杆菌的细菌。
在第六方面,提供了一种制备叶酸或其盐、其前体或其中间体的方法,包括i)在培养基中,在合适的培养条件下培养本发明第五方面所述的基因工程微生物以获得含有所述叶酸、其前体或其中间体的发酵产物;ii)任选地,分离和/或纯化所述叶酸、其前体或其中间体。
在另一个优选的实施方案中,步骤i)在32至42℃,优选34至39℃,更优选36至39℃,例如约37℃的培养温度下进行。
在另一个优选的实施方案中,步骤i)进行10至70小时,优选24至60小时,更优选36至50小时的时间。
在另一个优选的实施方案中,其中步骤i)在pH为6至8,优选6.5至7.5,更优选6.8至7.2的范围内进行。
在另一个优选的实施方案中,叶酸或其盐、其前体或其中间体是式I化合物:
其中,当a为单键时,a’为无或当a’为单键时,a为无;
当b是单键时,b'是无或当b'是单键时,b是无;
R1选自下组:-H、-CH3(5-甲基)、-CHO(5-甲酰基)、-CH=或=CH-(5,10-亚甲基)、-CH2-(5,10-亚甲基)和-CH=NH(5-亚胺甲基);
R2选自下组:-H、-CHO(10-甲酰基)、-CH=、=CH-(5,10-亚甲基)和-CH2-(5,10-亚甲基)。
在另一个优选的实施方案中,还包括在培养步骤(i)中添加对氨基苯甲酸(PABA)的步骤。
在另一个优选的实施方案中,对氨基苯甲酸(PABA)选自:对氨基苯甲酸钾、对氨基苯甲酸钠、对氨基苯甲酸甲酯、对氨基苯甲酸乙酯、对氨基苯甲酸丁酯、或其组合。
在另一个优选的实施方案中,还包括将步骤(i)或(ii)中得到的产物置于酸性或碱性条件下,进一步得到衍生化合物。
在另一个优选的实施方案中,包括以下步骤:(a)与其他方面相同的微生物(参考微生物)相比,降低编码具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽的内源基因的表达水平,和b)表达仅具有二氢叶酸合酶活性的异源多肽。
在另一个优选的实施方案中,包括以下步骤:aa)失活所述微生物中编码具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽的内源基因,例如通过删除部分或整个基因序列;和/或bb)将包含编码仅具有二氢叶酸合酶活性的异源多肽的核酸序列的外源核酸分子引入所述微生物中。
应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。
附图说明
图1显示了叶酸的核心结构。在天然叶酸中,蝶呤环以四氢形式(如图所示)或7,8-二氢形式存在。该环在化学产生的叶酸中被完全氧化。叶酸通常具有与第一个谷氨酸相连的最多约8个残基的γ连接的聚谷氨酰尾。一个碳单元(甲酰基、甲基等)可以与N5和/或N10位置偶联,从而合成5-甲酰基叶酸、10-甲酰基叶酸或5-甲基叶酸。
图2显示了由乳酸乳球菌基因组成的叶酸操纵子示例的示意图。
图3显示了由棉阿舒囊霉(A.gossypii)基因组成的叶酸操纵子示例的示意图。
图4显示了由枯草芽孢杆菌基因组成的叶酸操纵子示例的示意图。
图5显示了在Pveg启动子下具有四环素抗性基因(TetR)、异源folC2-LR或folC2-AG基因的FolC干扰表达盒的示意图,侧翼同源末端用于天然folC靶基因的破坏。用于DNA干扰表达盒的PCR扩增的引物的位置用线表示。
图6显示了10-甲酰基叶酸标准品的色谱图。黑色:UV信号,红色:MS/MS信号。
图7显示了在CE 20V下源自m/z 470的SRM片段。
图8显示了5-甲酰基-THF标准品的色谱图。黑色:UV信号,红色:MS/MS信号。
图9显示了在CE 20V下源自m/z 474的SRM片段。
图10显示了5-甲基-THF标准品的色谱图。黑色:UV信号,红色:MS/MS信号。
图11显示了在CE 20V下源自m/z 460的SRM片段和发酵液样品的色谱图。黑色:UV信号,红色:MS扫描信号。
图12显示了在CE 20V下源自m/z 472的SRM片段。在RT=10分钟的新峰的特性被确定为10-二氢-甲酰基叶酸。
图13显示了发酵液样品的色谱图。黑色:UV信号,红色:MS扫描信号。
图14显示了10-甲酰基二氢叶酸在氧气存在下氧化成10-甲酰基叶酸的示意图,10-甲酰基二氢叶酸在过氧化氢存在下氧化成10-甲酰基叶酸的示意图和在高碘酸钠存在下10-甲酰基二氢叶酸氧化成10-甲酰基叶酸的示意图。
图15显示了10-甲酰基叶酸在酸性介质中脱甲酰基为叶酸的示意图。
图16显示了10-甲酰基叶酸在碱性介质中脱甲酰基为叶酸的示意图。
图17显示了叶酸生产的生物过程概况。叶酸(mg/L):满星;葡萄糖浓度(g/L):空方块;乙偶姻浓度(g/L):全方块;PABA浓度(mg/L):空心圆圈;PABA进料(mg/L):竖线;光密度:整圆。
图18显示了枯草芽孢杆菌菌株w.t.168、菌株VBB38、菌株FL21和FL23在摇床5ml放大实验的总叶酸生产滴度。
详述
经过广泛深入的研究和大量筛选,发明人意外地发现,如果在出发菌株中降低内源性folC基因的表达水平,同时引入外源性folC基因,并且在生物合成的叶酸上只添加了一种谷氨酸,叶酸、其盐、其前体或其中间体的生产能力显著提高。此外,本发明人还发现,在出发菌株中引入或上调叶酸生物合成基因(例如,folE/mtrA、folB、folK、folP/sul、folA/dfrA)也可以显著提高叶酸、其盐、其前体或其中间体的生产能力。发明人还意外地发现,在如上所述获得的菌株的培养过程中添加对氨基苯甲酸(PABA)可以显著进一步提高叶酸、其盐、其前体或其中间体的生产能力。在此基础上,发明人完成了本发明。
如本文所用,“异源”是指多肽通常不在宿主生物中发现或由宿主生物制造(即表达),而是源自不同物种。
如本文所用,“失活”是指所讨论的基因不再表达功能性蛋白质。由于部分或整个基因序列的缺失、基因阅读框的移位、错义/无义突变的引入,或基因邻近区域的修饰,包括控制基因表达的序列,例如启动子、增强子、弱化子、核糖体结合位点等,修饰的DNA区域可能无法自然表达基因。优选地,目的基因因部分或整个基因序列的缺失而失活,例如通过基因替换。
可以通过PCR、Southern印迹等众所周知的方法检测细菌染色体上基因的存在与否。此外,可以通过使用各种众所周知的方法测量从基因转录的mRNA量来估计基因表达水平,包括Northern印迹、定量RT-PCR等。该基因编码的蛋白质的量可以通过公知的方法进行测定,包括SDS-PAGE,然后进行免疫印迹分析(Western印迹分析)等。
在本发明中,术语“基因工程菌株”和“基因工程微生物”可以互换使用。
出发菌株
如本文所用,术语“本发明的出发菌株”或“本发明的出发微生物”可以互换使用,是指在其基因组中编码具有二氢叶酸合酶活性和叶酰聚谷氨酸合成酶活性的多肽的任何细菌或真菌,例如任何芽孢杆菌属物种,例如枯草芽孢杆菌。
在一个优选的实施方案中,出发菌株从工业微生物遗传和选择研究所的俄罗斯国家工业微生物保藏中心获得或购买,编号为VKPM B-2116,通用交叉名称为VNIIGenetika-304或VBB38。
本发明的出发菌株的生理和生化特性是:核黄素生物合成的失调、嘌呤碱基生物合成的失调、在8-氮鸟嘌呤存在下生长的能力,在玫瑰黄色素存在下生长的能力。
应当理解,出发菌株不仅包括编号为VKPM B-2116的菌株。该菌株还包括其衍生菌株。
叶酸、其盐、其前体或其中间体
在本发明中,叶酸、其盐、其前体或其中间体如式I所示:
其中,当a为单键时,a’为无或当a’为单键时,a为无;
当b是单键时,b'是无或当b'是单键时,b是无;
R1选自下组:-H、-CH3(5-甲基)、-CHO(5-甲酰基)、-CH=或=CH-(5,10-亚甲基)、-CH2-(5,10-亚甲基)、-CH=NH(5-亚胺甲基)、或其组合;
R2选自下组:-H、-CHO(10-甲酰基)、-CH=、=CH-(5,10-亚甲基)、-CH2-(5,10-亚甲基)、或其组合。
叶酸是B族维生素中的一种重要维生素,广泛用于食品和动物饲料的强化以及膳食补充剂的生产。叶酸通常在怀孕期间被女性用作补充剂,以降低婴儿神经管缺陷的风险。长期补充也与中风和心血管疾病风险的小幅降低有关。
“叶酸”是用于命名多种维生素形式的术语,即叶酸及其同系物,包括四氢叶酸(维生素的活化形式)、甲基四氢叶酸(血清中的主要形式)、亚甲基四氢叶酸、亚叶酸,和叶酸。
传统的叶酸生产基于化学合成。主要三种成分,2,4,5-三氨基-6-羟基嘧啶、1,1,3-三氯丙酮和N-(4-氨基苯甲酰基)-L-谷氨酸经酸沉碱精制缩合生成蝶酸单谷氨酸盐。这种叶酸化学生产工艺存在收率低、产生大量废水、环境污染严重等缺点。
发明人发现,通过对出发菌株进行基因工程改造,可以显著提高该菌株中叶酸、其盐、其前体或其中间体的生产能力。
本发明的“叶酸、其盐、其前体或其中间体的生产能力”是指叶酸类化合物、其盐、其前体或其中间体的生产能力,即其相当于其前体或其中间体的“工业生产等级”、“工业潜力”、“工业生产能力”、“生产能力”,可以互换使用,指发酵产量至少为0.01g/L,优选至少0.05–0.1g/L;更优选至少0.5-1g/L,以发酵液的总体积计的在此范围内的任意整数和非整数值,在此不再赘述。
本发明的实验表明,本发明的基因工程菌株(如枯草芽孢杆菌)显著提高了叶酸及其盐、其前体或其中间体的合成能力,摇瓶实验产量可达333mg/L。在野生型菌株(如枯草芽孢杆菌)中,叶酸及其前体或其中间体的合成能力很低,产量只能达到0.31mg/L。这是非常出乎意料的。
folC基因
在一些细菌,例如枯草芽孢杆菌中,向二氢蝶酸(二氢叶酸合成酶(DHFS)活性,EC6.3.2.12)添加L-谷氨酸,随后通过γ羧基向四氢叶酸添加L-谷氨酸(叶酰聚谷氨酸合成酶(FPGS)活性,EC 6.3.2.17),由相同的酶FolC催化。相反,在真核生物和其他一些细菌中,DHFS和FPGS酶活性编码在不同的基因中。与许多其他细菌一样,枯草芽孢杆菌将γ连接的聚谷氨酸尾巴添加到叶酸中,以增加溶解度并防止这种必需的辅因子流失到环境中。因此,枯草芽孢杆菌FolC具有叶酰聚谷氨酸合成酶(FPGS)活性,除了在叶酸从头生物合成途径中作为二氢叶酸合酶的作用外,它还通过其γ-羧基催化叶酸的聚谷氨酰化。叶酸聚阴离子不能输出细胞,导致细胞内滞留增强(Sybesma等,2003c)。此外,FPGS酶的产物叶酰聚谷氨酸是叶酸生物合成酶的强抑制剂(McGuire和Bertino,1981)。因此,为了增加叶酸的产量,我们通过敲除天然folC基因,并将其替换为仅编码必需二氢叶酸合成酶(DHFS)活性的异源性folC基因,取消了叶酸的聚谷氨酰化,导致添加只有一个必需的谷氨酸部分。仅具有二氢叶酸合成酶(DHFS)而没有叶酰聚谷氨酸(FGPS)合成酶活性的FolC同源物可以在许多细菌物种如罗伊氏乳杆菌和许多真核生物如棉阿舒囊霉中找到。
叶酸生物合成基因
在本发明中,叶酸生物合成基因包括folE/mtrA、folB、folK、folP/sul和folA/dfrA。
叶酸分子包含一个蝶呤部分,源自鸟苷三磷酸(GTP),与对氨基苯甲酸(pABA)和至少一个谷氨酸分子结合。因此,叶酸的从头生物合成需要三种前体:GTP、pABA和谷氨酸。
叶酸生物合成通过GTP在四个连续步骤中转化为6-羟甲基-7,8-二氢蝶呤焦磷酸盐(DHPPP)进行。第一步由GTP环化水解酶I(EC 3.5.4.16)(基因folE/mtrA)催化,涉及GTP的广泛转化,形成蝶呤环结构。去磷酸化后,蝶呤分子经历醛缩酶(EC 4.1.2.25)(基因folB)和焦磷酸激酶反应(EC 2.7.6.3)(基因folK),产生活化的焦磷酸化的DHPPP。在二氢蝶酸合酶(EC 2.5.1.15)(基因folP/sul)催化下,对氨基苯甲酸(pABA)与DHPPP第一次缩合以产生二氢蝶酸。第二次缩合是谷氨酸与二氢蝶酸通过二氢叶酸合酶(DHFS)(EC6.3.2.12)(基因folC)反应形成二氢叶酸。然后,DHF被DHF还原酶-DHFR(EC 1.5.1.3)(基因folA/dfrA)还原为具有生物活性的辅因子四氢叶酸(THF)。
本发明中叶酸生物合成基因的信息见表1。
表1.叶酸生物合成基因
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工程菌株及其制备方法
本发明的“工程菌”、“工程菌株”和“基因工程菌株”可以互换使用,均指降低内源性folC基因的表达水平,以及引入外源性folC基因。在一个优选的实施方案中,叶酸合成调控基因(例如,folE/mtrA、folB、folK、folP/sul、folA/dfrA)也可以被引入或上调。
其中,本发明的工程菌株与出发菌株相比具有显著提高的叶酸、其前体或其中间体的生产能力,其中叶酸、其前体或其中间体的结构如式I所示。
可用于转化为本发明的工程菌株的出发菌株是属于芽孢杆菌属的菌株,特别是枯草芽孢杆菌。野生型出发菌株的叶酸、前体或中间体的合成能力较差(Zhu等,2005),或不具备工业所需量的叶酸、其前体或其中间体的合成能力。本发明的工程菌经过基因改造后,仅将一种Glu残基添加到所产生的叶酸、其前体或其中间体中,从而增强了叶酸从细胞向发酵培养基的分泌表型,叶酸、其前体或其中间体的生产能力显著提高,或与出发菌株相比该能力大大提高。优选地,“显著增加”是指与其出发菌株相比,工程菌株中叶酸、其盐、其前体或其中间体的生产能力增强或增加至少100%,优选至少200-50000%。
此外,可以转化为本发明工程菌株的出发菌株还可以包括如下表3中的菌株。
本发明的工程菌株可以通过以下方法获得:
(a1)敲除宿主细胞中的内源性folC基因;
(b1)培养所述宿主细胞;和
该方法包括以下步骤:
(a2)提供携带外源性folC基因的表达载体;
(b2)将表达载体转入到宿主细胞中;
(c2)培养所述宿主细胞;
其中宿主细胞是出发菌株。
在这里,我们可以有一个部分,即任何由枯草芽孢杆菌菌株产生的叶酸化合物,然后可以转化为不同的衍生物,特别是使用化学步骤并在下面的实施例中描述的叶酸。
药物组合物和施用方式
本发明菌株发酵产物中的叶酸或其前体或其中间体可用于制备药物。本发明的化合物可以施用于哺乳动物,例如人,并且可以口服、直肠、肠道外(静脉内、肌内或皮下)、局部等施用。这些化合物可以单独给药或与其他药学上可接受的化合物联合给药。应当注意,本发明的化合物可以组合给药。
用于口服给药的固体剂型包括胶囊、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中,活性化合物与至少一种常规惰性赋形剂(或载体)混合,例如柠檬酸钠或磷酸二钙,或与以下组分混合:(a)填充剂或增容剂,例如,淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸;(b)粘合剂,例如羟甲基纤维素、藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和阿拉伯树胶;(c)保湿剂,例如甘油;(d)崩解剂,例如琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、海藻酸、某些复合硅酸盐和碳酸钠;(e)慢溶剂,例如石蜡;(f)吸收促进剂,例如季胺化合物;(g)润湿剂,例如鲸蜡醇和单硬脂酸甘油酯;(h)吸附剂,例如高岭土;(i)润滑剂,例如滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠或它们的混合物。在胶囊、片剂和丸剂中,剂型还可以包含缓冲剂。
固体剂型如片剂、糖丸、胶囊、丸剂和颗粒剂可以用包衣和壳如肠溶衣和本领域已知的其他材料制备。它们可以含有遮光剂,并且活性化合物或此类组合物中的化合物的释放可以延迟方式在消化道的一部分中释放。可以使用的嵌入组分的例子是聚合物和蜡质材料。如有必要,活性化合物也可以与一种或多种上述赋形剂形成微胶囊化形式。
用于口服给药的液体剂型包括药学上可接受的乳剂、溶液剂、混悬剂、糖浆剂或酏剂。除活性化合物外,液体剂型还可含有本领域常用的惰性稀释剂,例如水或其他溶剂、增溶剂和乳化剂,例如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1、3-丁二醇、二甲基甲酰胺和油类,尤其是棉籽油、花生油、玉米胚芽油、橄榄油、蓖麻油和芝麻油或这些物质的混合物。
除了这些惰性稀释剂之外,组合物还可以含有助剂,例如润湿剂、乳化剂和悬浮剂、甜味剂和香料。
除了活性化合物外,悬浮液还可以含有悬浮剂,例如乙氧基化异硬脂醇、聚氧乙烯山梨醇和硝酸异山梨酯、微晶纤维素、甲醇铝和琼脂或它们的混合物等。
用于肠道外注射的组合物可包含生理上可接受的无菌水溶液或非水溶液、分散体、悬浮液或乳液,以及用于重构为无菌可注射溶液或分散体的无菌粉末。合适的水性和非水性载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其合适的混合物。
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、推进剂和吸入剂。将活性成分在无菌条件下与生理上可接受的载体和任何防腐剂、缓冲剂或,如果需要的话,推进剂混合。
当使用药物组合物时,将安全有效量的本发明化合物施用于需要治疗的哺乳动物(例如人),其中剂量为药学有效剂量,用于60kg体重的个体,每日给药剂量通常为1-1000mg,优选20-500mg。当然,具体剂量还应考虑给药途径、个体健康状况和其他因素,这些都在熟练医师的技能范围内。
本发明的主要优点包括:
(1)通过本发明的方法进行基因工程改造的菌株在产生的叶酸、其盐、其前体或其中间体上仅添加一个Glu残基,从而增强了叶酸从细胞向发酵培养基的分泌表型,可显著提高叶酸及其前体或其中间体的生产能力;此外,该菌株的特点是叶酸生物合成基因过表达,进一步提高了生产能力;
(2)工程菌株遗传稳定,不易突变;
(3)工程菌株在标准发酵培养基中显示出与其他工业枯草芽孢杆菌菌株相当的生长。
下面结合具体实施例对本发明作进一步说明。应当理解,这些实施例仅用于说明本发明,并不用于限制本发明的范围。以下实施例中未具体说明的实验方法的条件,通常按照Sambrook等人,Molecular Cloning:A Laboratory Manual(New York:Cold SpringHarbor Laboratory Press,1989)中所述的常规条件,或按照微生物学杂志中描述的条件:实验手册(由Pearson Education Press的James Cappuccino和Natalie Sherman编辑)或制造商建议的条件。除非另有说明,百分比和份数均为重量百分比和重量份数。
除非另有说明,实施例中使用的材料均为市售产品。
实施例1:枯草芽孢杆菌基因组中叶酸生物合成基因的鉴定
参与叶酸生物合成途径的基因和酶在文献中是已知的,并且在KEGG数据库(www.genome.jp/kegg/pathway.html)中有详细描述。通过使用BLAST算法研究枯草芽孢杆菌的基因组和蛋白质数据库,获得了枯草芽孢杆菌关键叶酸生物合成基因的核苷酸和蛋白质序列。叶酸生物合成基因和酶的序列被引入作为“查询(query)”,相应的枯草芽孢杆菌序列被确定为“命中(hits)”。叶酸生物合成基因的序列列于如下表2中。
表2.枯草芽孢杆菌中参与叶酸生物合成的基因和酶
实施例2:叶酸生物合成的合成基因的合成,针对枯草芽孢杆菌的优化
氨基酸序列(SEQ ID NO:7、8、9、10和12)用于基因密码子优化(来自IDT集成DNA技术公司的密码子优化工具)以提高枯草芽孢杆菌中的蛋白质表达。合成的DNA片段(分别为SEQ ID NO:13、14、15、16和17)设计为添加RBS序列、用于基因过表达的调节启动子序列(例如p15 SEQ ID NO:38)和进一步组装叶酸操纵子表达盒所需的两端短接头序列。
实施例3:叶酸操纵子的组装
·从枯草芽孢杆菌基因组装的叶酸操纵子
来自枯草芽孢杆菌基因的关键叶酸生物合成基因被合成为DNA片段(SEQ IDNO:13、14、15、16和17),用于组装叶酸操纵子(FOL-OP-BS2)。为了将叶酸操纵子整合到枯草芽孢杆菌基因组中,设计并合成了两个额外的具有lacA同源性和红霉素选择标记(SEQ IDNO:18和19)的DNA片段,用于稳定的基因组整合。
在叶酸操纵子组装的第一步中,使用特定的引物组(引物对SEQ ID NO:26和SEQID NO:27用于片段SEQ ID NO:13;引物对SEQ ID NO::32和SEQ ID NO:28用于片段SEQ IDNO:17;引物对SEQ ID NO:33和SEQ ID NO:29用于片段SEQID NO:15;引物对SEQ ID NO:34和SEQ ID NO:30对于片段SEQ ID NO:16;用于片段SEQ ID NO:14的引物对SEQ ID NO:35和SEQ ID NO:31)对单独的DNA片段进行PCR扩增。
使用Eppendorf循环仪和Phusion聚合酶(Thermo Fisher)扩增片段,缓冲液由制造商提供,添加200μM dNTP、5%DMSO、每种引物0.5μM和大约20ng模板,最终体积为50μl,持续32循环。
使用的程序:98℃2分钟
32个循环(98℃30s、65℃15s、72℃30s)
72℃5分钟
10℃保持
每个片段的PCR在0.8%琼脂糖凝胶上进行,并通过Wizard PCR纯化试剂盒(Promega)中提供的方案从凝胶中纯化。通过重复的限制和连接步骤将片段组装成人工叶酸操纵子。使用了NdeI和AseI限制性位点的组合,以确保成功连接的兼容限制性末端。在每一步连接后,将合并的片段作为新的模板进行下一次PCR扩增。在50μl体积中进行限制,添加5μl FD绿色缓冲液、3μl选定酶和大约1500ngPCR片段。用Wizard SV凝胶和PCR纯化系统(PCR Clean-up system)限制后对片段进行纯化,前两个用于连接。我们使用2,5U T4 DNA连接酶(Thermo Fisher)和制造商提供的缓冲液,并添加5%PEG 4000和两个片段,摩尔比为1:1,最终体积为15μl。在接下来的步骤中,将1μl灭活连接物用作新的50μL PCR中的模板,其中引物为SEQ ID NO:26和SEQ ID NO:28,使用相同的程序(具有更长的延伸时间),并如上所用混合。在0.8%琼脂糖凝胶上进行PCR,从凝胶上切下片段并纯化(cleaned)。用Asel限制性内切酶切割纯化的新片段(SEQ ID NO:13和SEQ IDNO:17的组合),并在与第三个片段(SEQ ID NO:15)连接时使用的额外纯化后,已经用Ndel切割并在之后纯化。在对作为模板的连接物进行新PCR之后,我们还通过相同的方案添加了片段4和5,以制作多达5个叶酸生物合成基因的片段。
在进行总叶酸的培养测量后,将由枯草芽孢杆菌基因组装的构建的叶酸操纵子(如图4所示)用于转化(参见实施例5)以产生菌株FL722(参见实施例13)。
·来自乳酸乳球菌乳酸亚种乳酸乳球菌基因的叶酸操纵子
通过PCR扩增来自乳酸乳球菌乳酸亚种乳酸乳球菌操纵子FOL-OP-LL(SEQ ID NO:49)的异源基因(folA、clpX、ysxL、folB、folE、folP、ylgG和folC),并将分离的基因组DNA用作模板。用于PCR扩增的引物设计用于两个单独的PCR反应,其中第一个PCR反应引物(SEQID NO:45和SEQ ID NO:46)用于特异性扩增来自基因组DNA的基因,而第二个PCR反应引物(SEQ ID NO:47和SEQ ID NO:48)用于在操纵子的两端额外引入限制性位点(NheI和NotI)。将PCR产物亚克隆到低拷贝载体pFOL1中,并在FOL-OP-LL操纵子的起始处添加强组成型启动子P15(SEQ ID NO:38)。为了构建FOL-OP-LL操纵子的整合盒,引入了氯霉素抗性盒和amyE基因座的下游同源性。最后一步,利用SbfI限制性内切酶从克隆载体实现整合盒,用于自连接,实现多拷贝基因组整合。从乳酸乳球菌乳酸亚种乳酸乳球菌基因组装的构建的叶酸操纵子(如图2所示),用于转化以产生菌株FL84,在培养后进行总叶酸的测量(参见实施例13)。
·来自棉阿舒囊霉(Ashbya gossypii)(Eremothecium gossypii)基因的叶酸操纵子
使用两种合成的叶酸生物合成基因fol1-AG(SEQ ID NO:50)和fol2-AG(SEQ IDNO:51)构建了来自已知的产生B2维生素的丝状真菌棉阿舒囊霉(Ashbya gossypii)(Eremothecium gossypii)的表达盒(FOL-OP-AG)。针对枯草芽孢杆菌最佳表达,对基因进行密码子优化并合成为两个单独的DNA片段FOL1-AG(SEQ ID NO:52)和FOL2-AG(SEQ IDNO:53),其中引入额外的调节启动子序列(启动子P15)。首先使用氯霉素抗性盒下游的SpeI/BamHI限制性位点和强组成型启动子P15将FOL1-AG片段亚克隆到低拷贝载体pFOL1中。在第二步中,使用EcoRV限制性位点将FOL2-AG片段亚克隆到amyE基因座同源性上游的低拷贝载体pFOL2中。在下一步中,使用引物(SEQ ID NO:54和SEQ ID NO:55)PCR扩增含有P15-fol2-AG和amyE同源性的DNA片段,并使用BamHI限制位点克隆到氯霉素抗性盒下游的质粒pFOL1和P15-fol1-AG。在最后的步骤中,使用引物(SEQ ID NO:56和SEQ ID NO:57)对组装的整合盒FOL-OP-AG进行PCR扩增,并将PCR产物用于细胞的转化。在进行总叶酸的培养测量后,将构建的由棉阿舒囊霉基因组装的叶酸操纵子(如图3所示)用于转化以产生菌株FL260(参见实施例13)。
实施例4:用于folC替换的遗传构建体的组装
为了用能够在叶酸生物合成中仅连接第一个谷氨酸残基的变体取代能够将多个谷氨酸残基连接到叶酸上的天然叶酰聚谷氨酸合成酶(folC),我们着手生成相应的遗传构建体。folC干扰盒通过使用相应的引物对SEQ ID NO:43和SEQ ID NO:44从gDNAB.subtilis VBB38通过PCR扩增的folC同源末端组装。PCR混合物由制造商提供的Phusion聚合酶(Thermo Fisher)和缓冲液制成,添加5%DMSO、200μM dNTPs和0.5μM的每种引物至最终体积为50μL,共32个循环(退火温度65℃,延伸时间2分钟)。从0.8%琼脂糖凝胶上切下扩增的PCR片段,用Wizard凝聚和PCR纯化系统试剂盒纯化,并在制造商提供的缓冲液A中用T4多核苷酸激酶(Thermo Fisher)磷酸化,添加1mM ATP。
将制备的片段连接到低拷贝质粒pET-29c(Novagen)中,该质粒预先用FspA1和XhoI切割,用DNA聚合酶I、大(Klenow)片段(Thermo Fisher)平末端并用FastAP热敏碱性磷酸酶(Thermo Fisher)去磷酸化。
四环素抗性盒(SEQ ID NO:21)用于破坏folC基因序列。通过用Bsp119l限制性内切酶切割质粒,将四环素抗性盒插入folC序列,用DNA聚合酶l,大(Klenow)片段(ThermoFisher)平末端,使用FastAP去磷酸化并使用T4 DNA连接酶(Thermo Fisher)连接。
此外,来自罗伊氏乳杆菌(folC2-LR)(SEQ ID NO:22)和来自棉阿舒囊霉(folC2-AG)(SEQ ID NO:23)的异源folC2蛋白序列用于设计用于folC2-LR(罗伊氏乳杆菌)(SEQ IDNO:24)和folC2-AG(棉阿舒囊霉)(SEQ ID NO:25)异源基因表达的密码子优化DNA序列。合成DNA片段(IDT集成DNA技术公司)并用于构建两个整合盒(如图5所示)。首先,我们使用DNA聚合酶l、大(Klenow)片段(Thermo Fisher)生成了一个包含Pveg启动子(SEQ ID NO:37)的平末端片段,并将其连接到具有folC同源性的质粒中,之前用Xbal切割并用DNA聚合酶l平端,大(Klenow)片段(Thermo Fisher)平末端。
接下来,用Bcul和FspA1限制性内切酶切割新构建的质粒并使用FastAP去磷酸化。之后,将质粒与folC2-LR中的有序优化序列folC2-AG连接,之前用Bcul和FspA1限制酶切割。在该四环素抗性质粒中,在用FspA1限制质粒并去磷酸化后,将先前用EcoRI限制酶切割并平端的质粒连接起来。构建的质粒用作PCR引物SEQ ID NO:43和SEQ ID NO:44的模板,以产生用于转化的folC干扰/替换盒。
实施例5用于转化的叶酸操纵子构建体的组装
在组装叶酸操纵子(参见实施例3)后,具有叶酸生物合成基因的DNA片段进一步用Xbal限制酶切割,并用引物SEQ ID NO:40和SEQ ID NO:41(62℃,40s)与红霉素抗性盒(SEQID NO:58)的合成DNA片段连接,并用XbaI切割以确保兼容的DNA末端用于连接。连接后,用引物(SEQ ID NO:36和SEQ ID NO:39)对整个片段进行PCR扩增。
在组装的最后一步中,添加了具有lacA同源性和调节启动子区域的片段(SEQ IDNO:18)。用Spel限制酶切割片段并用于连接。将连接混合物用作具有引物(SEQ ID NO:42和SEQ ID NO:39)的PCR模板,我们用它完成人工叶酸操纵子(如图4所示)的组装作为表达盒(SEQ ID NO:20)用于将基因组转化进枯草芽孢杆菌菌株。
实施例6:选择可能的枯草芽孢杆菌宿主菌株用于工程化叶酸生产
不同的芽孢杆菌菌株可用作叶酸生产工程的出发菌株(表3)。芽孢杆菌菌株可以从自然界分离或从培养物保藏中心获得。其中,可以从枯草芽孢杆菌菌株中选择用于叶酸生产的出发菌株,这些菌株已经经历了经典的诱变和选择方法,以过量生产与嘌呤生物合成途径相关的代谢物。例如,可以选择过度生产核黄素、肌苷和鸟苷的菌株。优选经过嘌呤和核黄素途径的随机诱变和毒性代谢抑制剂的菌株,并包含在表3中。
表3.可用于开发叶酸生产的潜在非转基因枯草芽孢杆菌出发菌株。
VKPM B2116菌株是枯草芽孢杆菌168菌株(最常见的枯草芽孢杆菌宿主菌株,基因组约为4Mbp)的杂交菌株,具有来自枯草芽孢杆菌W23菌株的6.4kbp DNA岛。这种结构对于大多数枯草芽孢杆菌工业菌株很常见,是通过用W23(原养型TrpC+)DNA转化168菌株(色氨酸营养缺陷型trpC-)而获得的。它在基因组中有一个6.4kbp的W23岛,与常用的枯草芽孢杆菌SMY菌株相同,它是具有公开可用基因组的枯草芽孢杆菌遗留菌株之一(Ziegler等人,168的起源,W23和其他枯草芽孢杆菌遗留菌株,细菌学杂志,2008,21,6983–6995)。VKPMB2116菌株是SMY菌株的直系后代,通过经典诱变和选择获得。该菌株的另一个名称是枯草芽孢杆菌VNII Genetika 304。该菌株的构建描述在1980年提交的苏联专利SU908092中。通过随后的诱变和代谢抑制剂的选择获得突变。菌株VKPM B2116对玫瑰黄色素(一种维生素B2的有毒类似物)具有抗性,这是由于编码黄素激酶的ribC基因发生突变。该菌株还对8-氮鸟嘌呤(嘌呤碱基的毒性类似物)具有抗性。
实施例7:替换folC并产生用于叶酸生产的最佳宿主菌株
在构建异源folC2(folC2-AG或folC2-LR)基因表达盒后(参见实施例4和图5),我们进行了枯草芽孢杆菌VBB38和枯草芽孢杆菌VBB38Δrib的转化。用引物SEQ ID NO:43和SEQ ID NO:44通过PCR扩增具有天然folC基因破坏同源性的表达盒。转化后,选择对四环素有抗性的菌落,用异源folC2基因(A.gossypii或L.reuteri)替换天然folC基因,用cPCR和获得的PCR产物的测序进行遗传确认。新菌株用于检测总叶酸的产量(参见图18),并比较上清液和细胞生物量之间的总叶酸分布。
实施例8:枯草芽孢杆菌的转化
i)枯草芽孢杆菌天然能力转化
从新鲜的枯草芽孢杆菌平板中接种10mL SpC培养基并培养过夜。将1.3mL的过夜培养物稀释到10mL的新鲜SpC培养基中(9倍稀释)。OD450经测量,预计在0.5左右。培养物在37℃220RPM下生长3小时10分钟。再次测量OD450,预计在1.2-1.6之间。用SpII(饥饿培养基)按1:1稀释培养物。将3.5ml培养物与3.5ml饥饿培养基混合,并添加浓度为50ug/ml的色氨酸。培养物在37℃、220RPM下再生长2小时。孵育后培养物最多可容纳1小时。在2mL微量离心管中将500μl感受态细胞与DNA(5-20μl,取决于浓度)混合,并在37℃下摇动孵育30分钟。加入300μl新鲜LB以回收感受态细胞,并在37℃下再孵育30分钟。微量离心管以3000RPM离心5分钟。将颗粒重悬并铺在具有适当抗生素的LB板上。
培养基:
基于10x T
150mM硫酸铵
800mM K2HPO4
440mM KH2PO4
35mM柠檬酸钠
SpC(最小培养基)
基于100mL 1x T
1mL 50%葡萄糖
1.5mL 1.2%MgSO4
2mL 10%酵母提取物
2.5mL 1%酪蛋白氨基酸
SpII(饥饿培养基)
基于100ml 1x T
1ml 50%葡萄糖
7ml 1.2%MgSO4
1ml 10%酵母提取物
1ml 1%酪蛋白氨基酸
0.5ml 100mM CaCl2
实施例10:使用qPCR测定叶酸操纵子拷贝数
我们使用实时定量PCR(qPCR)技术来确定整合的枯草芽孢杆菌人工叶酸操纵子基因的拷贝数。产生叶酸的枯草芽孢杆菌转化体中人工叶酸操纵子中基因folP、folK、folE、dfrA和KnR(卡那霉素抗性基因)的拷贝数通过(qPCR)和SYBR Green I检测进行估计。通过qPCR对人工枯草芽孢杆菌叶酸操纵子上卡那霉素抗性基因(KnR)的拷贝数和叶酸生物合成基因folP、folK、folE、dfrA的拷贝数进行定量。用SW Wizard基因组DNA纯化试剂盒(Promega)分离枯草芽孢杆菌菌株的基因组DNA。在OD260和OD280分光光度法评估gDNA的浓度和纯度。所有实验中使用的gDNA量等于参考菌株的gDNA量。使用含有基因folP、folK、folE、dfrA和KnR的人工叶酸操纵子的单拷贝的枯草芽孢杆菌作为基因拷贝数相对定量的参考菌株。管家基因DxS是枯草芽孢杆菌基因组中的单拷贝基因,被用作内源性对照基因。叶酸生物合成基因的基因拷贝数的定量使用特定的引物组进行(引物对SEQ ID NO:59和SEQ ID NO:60用于folP基因,引物对SEQ ID NO:61和SEQ ID NO:62用于folK基因,引物对SEQ ID NO:63和SEQ ID NO:64用于folE基因,引物对SEQ ID NO:65和SEQ ID NO:66用于dfrA基因),用于定量连接到叶酸操纵子的卡那霉素抗性标记物(引物对SEQ ID NO:67和SEQ ID NO:68),使用参考DxS基因引物对SEQ ID NO:71和SEQ ID NO:72。在StepOneTM实时荧光定量PCR系统上进行qPCR分析,并使用2-ΔΔCT方法进行定量。
人工BS-FOL-OP菌株中基因的基因拷贝数相对于具有一个基因拷贝的菌株进行量化。以具有一个拷贝数的枯草芽孢杆菌菌株的KnR基因作为参考菌株,对枯草芽孢杆菌转化菌株中人工叶酸操纵子中基因的基因拷贝数进行相对定量。与具有单拷贝基因的枯草芽孢杆菌菌株相比,基因folP、folK、folE、dfrA和KnR基因的qPCR相对定量显示RQ值增加了6倍。叶酸高产菌株FL179和FL722被证实具有叶酸合成操纵子的多拷贝整合。
实施例11:枯草芽孢杆菌菌株的培养
制备来自冷冻的冷冻管的系列稀释液,并用适当的抗生素铺到MB板上,并在37℃下孵育约48小时。为了进一步测试,每个菌株使用来自MB板的至少10-20个单菌落。首先在新鲜的MB板上(使用相同浓度的抗生素)重新修补10-20个单菌落进行测试。
对于营养阶段,使用MC培养基,每个离心管用1个塞子接种(或每个带挡板的锥形烧瓶5个塞子或用于微量滴定板的一小部分贴片)。培养基中加入适当的抗生素。对于微量滴定板,在96深孔中使用500μl培养基,对于离心管,使用5ml培养基(在50ml离心管中),对于锥形烧瓶,使用25ml(在250ml烧瓶中)。培养物在37℃以220RPM孵育18-20小时。
接种到生产培养基(MD)是在营养培养基中18-20小时后。使用10%的接种物(MW为50μl,离心管为0.5ml,锥形烧瓶为2.5ml)。每个菌株在两个等分试样中进行测试。对于微量滴定板,在48深孔中使用500μl培养基,对于离心管,使用5ml培养基,对于带挡板的锥形烧瓶,使用25ml。金属丝用于离心管中以更好地通气,就像用纱布代替锥形烧瓶上的塞子一样。将培养物在37℃以220RPM孵育48小时。根据开发的程序,使用微生物测定法测量24和48小时后的总叶酸滴度。
最好的候选菌株以相同的方式重新测试,并在多次确认后准备在生物反应器中测试。将用于生物反应器测试的选定菌株的100μl冷冻培养物铺展到带有适当抗生素的MB板上,并在37℃下孵育约48小时。用每板2ml无菌20%甘油收集完整的生物质。收集的生物质被分配到100μl等分试样中并在-80℃下冷冻。这被用作生物反应器测试的工作细胞库。
培养基成分:
1)MB(板)
胰蛋白胨 10g/l
酵母提取物 5g/l
NaCl 5g/l
麦芽糖 20g/l
琼脂 20g/l
pH 7.2-7.4
高压蒸汽处理30分钟,121℃
高压蒸汽处理和冷却后加入适当的抗生素。
2)MC(营养培养基)
糖蜜 20g/l
CSL 20g/l
酵母提取物 5g/l
MgSO4*7H2O 0.5g/l
(NH4)2SO4 5g/l
将成分混合在一起并将pH设置为7.2-7.4。然后加入KH2PO4-K2HPO4溶液,最终浓度为KH2PO4 1.5g/l和K2HPO4 3.5g/l。将培养基分配到离心管(5ml/50ml-离心管)或锥形烧瓶(25ml/250ml-带挡板的锥形烧瓶)中,并在121℃下高压蒸汽处理30分钟。高压蒸汽处理后添加无菌葡萄糖,最终浓度为7.5g/l。在接种前添加抗生素。
3)MD(生产培养基)
酵母 20g/l
玉米浆(CSL) 5g/l
MgSO4*7H2O 0.5g/l
对氨基苯甲酸(pABA) 0.5g/L
将成分混合在一起并将pH设置为7.2-7.4。然后加入KH2PO4-K2HPO4溶液,最终浓度为KH2PO4 1.5g/l和K2HPO4 3.5g/l。培养基在121℃高压蒸汽处理30分钟。无菌尿素溶液(20ml原液,终浓度为6g/L),无菌葡萄糖溶液(250ml原液,终浓度为100g/L葡萄糖),无菌pABA溶液(100ml原液,终浓度为0.5g/L),高压蒸汽处理后加入150ml无菌水,得到1L MD+pABA500培养基。接种前加入适当的抗生素。然后将培养基分配到无菌锥形烧瓶中(25ml/250ml-带挡板的锥形烧瓶。
实施例12:定量发酵液中总叶酸的微生物测定
使用海氏肠球菌NRRL B-1295的微生物测定法用于检测枯草芽孢杆菌菌株中产生的总叶酸。微生物测定用于评估枯草芽孢杆菌产生的细胞内(保留在生物质中)和细胞外(释放到培养基中)的总叶酸。对于微生物测定,使用指示生物海氏肠球菌NRRL B-1295,它对叶酸具有营养缺陷型。E.hirae在富含叶酸(乳酸杆菌AOAC肉汤)的生长培养基中在37℃预培养18-24小时。然后在不含叶酸的生长培养基(叶酸测定培养基)中洗涤以去除残留的叶酸。将洗涤过的E.hirae培养物接种到不含叶酸的测定培养基中。微生物测定在96孔微量滴定板中进行。将适当稀释的待测培养基样品和叶酸标准溶液添加到含有指示菌株的生长培养基中,并将板在37℃下孵育20小时。指示生物的生长反应与培养基样品/对照中存在的叶酸的量成正比。通过将一组叶酸标准溶液添加到生长培养基和指示菌株中,为每个测定构建标准曲线。通过测量600nm波长处的光密度(OD)来测量生长。E.hirae对测试样品的生长反应与已知标准溶液的生长反应进行定量比较。如上所述制备和测定含有不同浓度叶酸的稀释系列。通过在已知浓度的叶酸下绘制测量的OD600得到标准曲线。标准曲线用于计算测试样品中总叶酸的量。指示生物E.hirae NRRL B-1295用于检测被测样品中0.05至0.7ng/mL范围内的总叶酸浓度。B.subtilis菌株产生的细胞外和细胞内总叶酸可以通过将适当稀释的测试样品添加到叶酸测定培养基中的指示生物E.hirae中来估计。
实施例13:不同出发菌株和初始folC替换和叶酸操纵子扩增菌株的总叶酸产量分析
其中folC基因被来自A.gossypii(枯草芽孢杆菌菌株FL21)或L.reuteri(枯草芽孢杆菌菌株FL23)的异源folC2基因替换的转化体和具有扩增的叶酸操纵子的转化体在摇床秤(5ml生产培养基MD)测试总叶酸量。发酵后,仔细收集发酵液样品(200μl)以获得均质样品,并在冰冷的提取缓冲液(0.1M磷酸盐缓冲液和1%(w/v)抗坏血酸)中稀释10倍。将样品在14,000rpm和4℃下离心10分钟并过滤灭菌(0.22μm孔径)。对于微生物测定,样品在提取缓冲液中连续稀释并保持在4℃,直到建立微生物测定。表4给出了通过微生物测定法测量的选定菌株的结果。
表4.摇床放大实验中不同枯草芽孢杆菌菌株的总叶酸产量(5ml)
实施例14:使用LC-MS测定叶酸形式和相关化合物的浓度并鉴定10-甲酰基-二氢叶酸和10-甲酰基叶酸作为两种主要产物
除了微生物分析,我们的目标是开发灵敏且通用的分析方法,具有相当短的分析运行时间。该方法必须是与挥发性流动相兼容的LCMS,还必须能够进行UV检测并尽可能多地对叶酸相关分析物进行良好的色谱分离。
仪器和材料:
该方法是在带有PDA检测器的Thermo Accela 1250HPLC仪器上开发的,与支持MS/MS的质谱仪Thermo TSQ Quantum Access MAX相结合,配备hESI源。方法已在ThermoAcclaim RSLC PA2、150x2.1 mm HPLC柱上设置,粒径为2.2μm。PDA检测器设置为282nm,带宽为9nm,扫描速率为80Hz,DAD扫描范围为200-800nm。柱温箱设置为60℃,托盘冷却(traycooling)设置为12℃。进样溶剂为10%甲醇水溶液,清洗和冲洗体积:2000μl。进样量设置为10μl,当需要更高浓度的分析物时,也可以设置为1μl。流动相A是650mM乙酸水溶液,流动相B是甲醇。流动相流速为0.5ml/min,总运行时间为20分钟。方法是使用表5中的梯度程序和表6中描述的MS光谱仪参数。
表5.色谱分析梯度程序
时间/分钟 | %A | %B |
0.00 | 100 | 0 |
2.00 | 100 | 0 |
16.00 | 82 | 18 |
16.01 | 100 | 0 |
20.00 | 100 | 0 |
表6.质谱仪调谐参数(tune parameter)和其他MS/MS相关参数
LCMS检测器连接在DAD检测器之后,在400-600m/z模式下进行扫描,在SIM模式下在其M.W.+1和MS/MS模式下观察分析物(表6)。通过称重并溶解在0.1M NaOH溶液(表7和表8)中制备标准品,并立即放入HPLC仪器。
表7.可用标准
表8.观察到的标准品及其相关的MS/MS方法设置
该方法对高达1000mg/L分析物的MS/MS检测具有线性响应,所有标准品的相关性均高于90%。
实施例15:通过转基因枯草芽孢杆菌生产不同比例的叶酸及其衍生物
其中folC基因被来自A.gossypii(枯草芽孢杆菌菌株FL21)或L.reuteri(枯草芽孢杆菌菌株FL23)的异源folC2基因替换的转化体和具有扩增的叶酸操纵子的转化体在摇床秤(5ml生产培养基MD)测试总叶酸量。
将菌株用适当的抗生素贴在MB板上,并在37℃下培养2天。对于摇瓶实验,将生长的菌株转移到Falcon 50mL锥形离心管(1个塞子/5ml)中的5ml MC(种子)培养基中,并在旋转振荡器上以220RPM和37℃培养16–18小时。使用10%的种子培养物的接种物接种5mL生产培养基(MD+pABA500)。将菌株在旋转摇床上以220RPM和37℃在黑暗中培养48小时。发酵后,仔细收集发酵液样品(200μl)以获得均质样品,并在冰冷的提取缓冲液(0.1M磷酸盐缓冲液和1%(w/v)抗坏血酸)中稀释10倍。将样品在14,000rpm和4℃下离心10分钟并过滤灭菌(0.22μm孔径)。对于不同叶酸种类的定量,如实施例14中所述使用HPLC方法。不同枯草芽孢杆菌菌株的结果显示在表9中,发酵液样品的代表性HPLC色谱图显示在图13中。
表9.摇床放大实验中不同枯草芽孢杆菌菌株的总叶酸产量(5ml)
与野生型菌株枯草芽孢杆菌168相比,具有异源folC-AG和来自枯草芽孢杆菌的过表达叶酸生物合成基因的菌株FL179显示出43297%的10-甲酰基叶酸产量的增加。
实施例16:10-甲酰基二氢叶酸氧化转化为10-甲酰基叶酸
在发酵结束时,肉汤的HPLC分析检测到相对高量(85面积%)的10-甲酰基二氢叶酸(10F-DHF)。此外,我们观察到10-甲酰基二氢叶酸可以氧化转化为10-甲酰基叶酸(见图14)。因此,我们开始开发一种方案,该方案将提供10-甲酰叶酸的定量转化。我们预计随后的去甲酰化步骤将以尽可能高的产量提供叶酸。文献检索揭示了一份报告,描述了在特定pH值的水溶液中空气对四氢叶酸的氧化(Reed1980)。根据该报告,在pH值4、7和10时,主要的氧化产物是对氨基苯甲酰谷氨酸(PABG)和6-甲酰蝶呤。此外,仅在pH=10时检测到7,8-二氢叶酸中间体。我们对发酵液上清液进行了一系列氧化实验,以促进10-甲酰基二氢叶酸快速转化为10-甲酰基叶酸。我们检查了几种氧化试剂,例如O2、H2O2和NaIO4(见图14)。
表10.pH对发酵液上清液中用氧气将10-甲酰基二氢叶酸氧化成10-甲酰基叶酸的影响
使用10mL发酵液上清液在50mL圆底烧瓶中进行实验。pH值由1.0M和0.1M NaOH溶液设定。通过HPLC测量反应进程和结果。HPLC样品在提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)中制备。将所有反应在环境温度(25℃)避光搅拌48小时。
使用1M和0.1M HCl或NaOH调节所需的pH值。较低pH值下的反应较慢并保持相对较高的叶酸总量(表10,条目2-4)。相反,在较高pH值下的反应(表10,条目5-7)提高了10-甲酰基二氢叶酸的消耗,尽管显着显著降低了叶酸的总量。我们预计我们可以使用替代试剂进行氧化,例如过氧化氢或高碘酸钠。
代表性实验程序:
将发酵肉汤以4,500rpm离心并慢慢倒出上清液。将10mL发酵液上清液移液到50mL圆底烧瓶中,该烧瓶配备有搅拌棒、pH计和用于避光的铝箔。滴加氢氧化钠或盐酸(1.0M和0.1M用于微调)以设定pH值并在环境温度(25℃)下剧烈搅拌反应24小时。用来自气球的空气吹扫反应混合物。搅拌48小时后,用9mL提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)稀释1mL每种发酵液,重复两次实验。将悬浮液在涡旋上搅拌,以4,500rpm离心,通过0.22μm过滤器过滤并在HPLC上分析。
表11.过氧化氢浓度对发酵液上清液中10-甲酰基二氢叶酸氧化为10-甲酰基叶酸的影响
使用10mL发酵液上清液在50mL圆底烧瓶中进行实验。过氧化氢以30%的水溶液形式逐滴加入。通过HPLC测量反应进程和结果。HPLC样品在提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)中制备。将所有反应在环境温度(25℃)避光搅拌48小时。
以50-500mg/L的浓度范围添加过氧化氢,一种用于将10-甲酰基二氢叶酸氧化转化为10-甲酰基叶酸的替代氧化剂,从而提供更先进的结果(表11)。在反应的前24小时内,10-甲酰基二氢叶酸的浓度降至其初始值的50%。将反应延长至48小时提供了良好的转化率,从而保持了相对较高的总叶酸总量。
代表性实验程序:
将发酵肉汤以4,500rpm离心并慢慢倒出上清液。将10mL发酵液上清液移液到50mL圆底烧瓶中,该烧瓶配备有搅拌棒、pH计和用于避光的铝箔。将过氧化氢以30%的水溶液形式逐滴加入,并将反应混合物在环境温度(25℃)下剧烈搅拌24-48小时。搅拌48小时后,用9mL提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)稀释1mL每种发酵液,重复试验两次。将悬浮液在涡旋上搅拌,以4,500rpm离心,通过0.22μm过滤器过滤并在HPLC上分析。
表12.高碘酸钠浓度对发酵液上清液中10-甲酰基二氢叶酸氧化为10-甲酰基叶酸的影响
使用10mL发酵液上清液在50mL圆底烧瓶中进行实验。一次性加入高碘酸钠。通过HPLC测量反应进程和结果。HPLC样品在提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)中制备。将所有反应在环境温度(25℃)避光搅拌48小时。
高碘酸钠经常被用作反复无常底物的首选试剂。我们对该试剂的初步实验表明,氧化转化的有效浓度在1-10g/L之间。以两种不同的浓度添加高碘酸钠,5g/L和10g/L。在反应的前24小时内,10-甲酰基二氢叶酸浓度较初始值显著下降(表12)。将反应延长至48小时提供了极好的转化率,从而保持了相对较高的总叶酸总量。
代表性实验程序:
将发酵肉汤以4,500rpm离心并慢慢倒出上清液。将10mL发酵液上清液移液到50mL圆底烧瓶中,该烧瓶配备有搅拌棒、pH计和用于避光的铝箔。一次性加入高碘酸钠并将反应混合物在环境温度(25℃)下剧烈搅拌24小时。搅拌48小时后,用9mL提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)稀释1mL每种发酵液,重复实验2次。将悬浮液在涡旋上搅拌,以4,500rpm离心,通过0.22μm过滤器过滤并在HPLC上分析。
实施例18:在5L生物反应器体积中生产叶酸
在使用适当条件培养和生产叶酸的生物反应器中,叶酸的生产可以大大提高。该过程包括预培养物的制备和主要的补料分批生物工艺。
i)预培养物的制备
用菌株FL179的工作细胞库接种烧瓶中的预培养基(FOL-MC,表13),并在旋转摇床上以37℃和220RPM(2”抛)培养11-14小时。
ii)分批补料生物工艺
叶酸的生产在5L生物反应器中使用FOL-ME培养基进行(表14)。生物反应器启动参数为搅拌=600RPM,通气=1vvm,使用氢氧化铵溶液将pH控制在7。用10%的预培养物接种生物反应器。DO由搅拌和气流控制,以保持空气饱和度在30%以上。当发酵液中的葡萄糖耗尽时,开始加入葡萄糖和CSL混合物(表15)。加料速度需要小心控制,加料速度控制在一个水平,不会导致乙偶姻(不超过10g/L)的积累。如果发酵液中未检测到乙偶姻,则加料速率过低。发酵液中的对氨基苯甲酸(PABA)浓度需要定期测量,并通过分批加入浓缩的PABA原液(50g/L)将其保持在500mg/L以上。生物工艺通常在50小时内完成。叶酸生产生物工艺概况如图17所示。
表13.FOL-MC预培养基
组分 | 量 |
糖蜜 | 20g/L |
玉米浆(CSL) | 20g/L |
酵母 | 5g/L |
(NH4)2SO4 | 5g/L |
MgSO4x7H2O | 0.5g/L |
KH2PO4 | 1.5g/L |
K2HPO4 | 3.5g/L |
葡萄糖 | 7.5g/L |
卡那霉素 | 10mg/L |
四环素 | 10mg/L |
表14.FOL-ME生产培养基
表15.加料溶液(葡萄糖+CSL)
组分 | 量 |
葡萄糖一水合物 | 400g/L |
玉米浆(CSL) | 310g/L |
实施例19:使用qPCR测定叶酸生物合成基因的表达水平
培养生长条件:枯草芽孢杆菌培养物在LB培养基中生长至指数期。将培养物与2倍体积的RNA保护细菌试剂(QIAGEN)混合,以4500rpm离心10分钟并在-80℃下冷冻或立即处理。将细胞沉淀重悬在200μL含有1mg/mL溶菌酶的TE缓冲液中15分钟,以去除细胞壁。根据制造商的方案,使用QIAGEN Rneasy mini试剂盒分离RNA。用分光光度法检查获得的RNA的浓度和质量。用DNase(Ambion试剂盒)处理分离的RNA,并使用RevertAid H Minus第一链cDNA合成试剂盒(Thermo Scientific)逆转录为cDNA。将获得的cDNA稀释,最终的cDNA产量为cca 2.5ng/μL。
获得的cDNA通过qPCR分析(StepOne实时荧光定量PCR系统,Applied Biosystems)和SYBR Green I(Thermo Scientific)检测进行分析。通过实时定量PCR(qPCR)技术对整合的枯草芽孢杆菌人工叶酸操纵子基因folP、folK、folE、dfrA中叶酸操纵子基因的表达进行定量。
内部对照基因用作定量qPCR表达数据标准化的参考,使用来自枯草芽孢杆菌的16S rRNA基因。叶酸生物合成基因的表达使用特定的引物组确定(引物对SEQ ID NO:59和SEQ ID NO:60用于folP基因,引物对SEQ ID NO:61和SEQ ID NO:62用于folK基因,引物对SEQ ID NO:63和SEQ ID NO:64用于folE基因,dfrA基因的引物对SEQ ID NO:65和SEQ IDNO:66),对于选择作为内部对照的16S基因,使用引物对SEQ ID NO:69和SEQ ID NO:70。在StepOneTM实时荧光定量PCR系统上进行qPCR分析,并使用2-ΔΔCT方法进行定量。
在两个单独的基因组位置(amyE和lacA)具有多拷贝合成叶酸操纵子的最佳叶酸生产菌株FL722被证实具有最强的叶酸生物合成基因表达水平。
实施例20:10-甲酰基叶酸向叶酸的化学转化
酸介导的去甲酰化
10-甲酰基叶酸的去甲酰化以0.01mmol规模(5mg)进行。在配备有搅拌棒的2mL微量离心管中称量10-甲酰基叶酸并悬浮在蒸馏水(1mL)中。用酸(50当量,0.5mmol)处理悬浮液并在环境温度下搅拌16小时。随后,用DMSO(800μL)稀释悬浮液(200μL),在涡流搅拌器上均质化并在HPLC上分析。去甲酰化的结果示于表16。
表16.不同酸对10-甲酰叶酸的N-去甲酰化的影响
所有实验均在2mL微量离心管中使用10-甲酰叶酸(5mg,0.01mmol)进行。a转化率通过HPLC测量。b n.d.-没有检测到。由于分析物可能吸附到Dowex 50WX2树脂上,因此在该实验中既没有检测到10-甲酰基叶酸也没有检测到叶酸。c TFA-三氟乙酸。d TCA——三氯乙酸。e PTSA–对甲苯磺酸。
10-甲酰基叶酸与强无机酸的去甲酰化几乎定量地进行到叶酸(表16,条目1和8)。或者,用更强的有机酸进行去甲酰基化可以提供几乎相同效率的叶酸(表16,条目3,4和6)。正如预期的那样,用甲酸和乙酸去甲酰化没有提供转化(表16,条目5和7)。使用Dowex50WX2树脂对去甲酰化的HPLC分析未提供对起始材料或产物的检测,因为分析物可能仍吸附在树脂上并需要洗脱。
发酵液中10-甲酰叶酸的酸介导N-去甲酰化
在之前的实验中,我们已经说明,使用强酸对10-甲酰基叶酸标准品进行去甲酰化可以完全转化为叶酸,如图15所示。在这里,我们将相同的原理应用于更复杂的系统,即发酵液。为了继续对生物样品进行实验,我们选择了盐酸(HCl)作为去甲酰化试剂,因为它比我们研究的其他酸高效且便宜。来自实施例18的发酵液的HPLC分析显示在生物合成过程中形成的其他叶酸中大量的10-甲酰基叶酸(10-甲酰基叶酸46%面积;5-亚氨基甲基四氢叶酸47%面积和5-甲基四氢叶酸7%面积)。发酵液样品用1M HCl处理至不同的pH水平(pH=4、3、2、1和0),并在避光环境温度(25℃)下搅拌24小时。根据我们的HPLC分析,只有在较低的pH值(pH=1和0)下,去甲酰化才提供适量的叶酸。基于这些结果,我们相信酸介导的去甲酰化策略可能适用于叶酸的下游加工。为了在复杂系统(例如发酵液)中开发具有成本效益的甲酰叶酸种类的去甲酰化方案,进一步优化酸量和反应温度是必不可少的。
将来自实施例18的充分搅拌的发酵液移液到六个配备有搅拌棒和pH电极的100mL圆底烧瓶中。在搅拌下滴加盐酸以达到如表17中所述的几个pH值(pH=4、3、2、1、0)。
表17.发酵液3101的酸介导去甲酰化
exp | VFB | VHCl | VTotal | pH |
1 | 50mL | 0.0mL | 50mL | 7.0 |
2 | 50mL | 10.2mL | 60.2mL | 4.0 |
3 | 50mL | 15.6mL | 65.6mL | 3.0 |
4 | 50mL | 21.4mL | 71.4mL | 2.0 |
5 | 50mL | 35.3mL | 85.3mL | 1.0 |
6 | 50mL | 59.0mL | 109.3mL | 0.0 |
通过将烧瓶包裹在铝箔中,将发酵混合物在环境温度(25℃)下搅拌24小时,以屏蔽UV光。尽管没有酸(实验1),但在精确条件下制备了受控样品。搅拌24小时后,用9mL提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)稀释1mL每种发酵液,重复试验2次。将悬浮液涡旋搅拌,以4500rpm离心,通过0.22μm过滤器过滤并在HPLC上分析。表18总结了HPLC结果。根据我们的HPLC分析,只有在较低的pH水平(pH=1和0)下,去甲酰化提供了适量的叶酸。总之,我们开发了一种酸介导的10-甲酰基叶酸的去甲酰化,它是发酵的主要产物。
表18.来自实施例18的发酵液上酸介导的去甲酰化的基于HPLC的结果
exp | pH | 5-FTHF mg/L | 10-FFA mg/L | F SUM mg/L | FA mg/L |
1 | 7.0 | 432 | 487 | 919 | 0 |
2 | 4.0 | 171 | 567 | 738 | 0 |
3 | 3.0 | 97 | 632 | 729 | 0 |
4 | 2.0 | 76 | 529 | 605 | 0 |
5 | 1.0 | 54 | 326 | 549 | 169 |
6 | 0.0 | 37 | 116 | 402 | 249 |
碱基介导的去甲酰化
浏览化学文献,我们发现了一些报告,这些报告描述了叶酸在更高的pH值下表现出更大的稳定性。在这样的pH值下,叶酸表现出更高的溶解度,这简化了合成操作、纯化和下游加工。因此,在使用0.1M NaOH的一系列N-去甲酰化实验中,我们的目标是从10-甲酰基叶酸清洁高效地转化为叶酸(参见图16),这将简化目标产物从发酵液中的分离。使用0.01mmol规模(5mg)对10-甲酰基叶酸的分析标准品进行初步去甲酰化实验。
代表性实验程序:
在配备有搅拌棒和橡胶隔片的10mL圆底烧瓶中称量10-甲酰基叶酸。用0.1M氢氧化钠(50当量,0.5mmol,5mL)处理悬浮液,并在避光环境温度下搅拌24-48小时。随后,用叶酸提取缓冲液(900μL)稀释溶液(100μL),在涡流搅拌器上均质化并在HPLC上分析。在HPLC上对三个时间相关的等分试样进行取样分析。去甲酰化结果列于表19。在24小时后的第一次取样期间,用0.1M NaOH将10-甲酰基叶酸去甲酰化几乎定量地生成叶酸(表19,条目1)。搅拌48小时后,根据HPLC分析,反应进行至完成。在相同条件下长时间搅拌表明,新形成的叶酸即使在144小时(6天)后也没有分解。
表19.在0.1M NaOH存在下10-甲酰基叶酸的N-去甲酰基化为叶酸的时间尺度
使用10-甲酰基叶酸(5mg,0.01mmol)在10mL圆底烧瓶中进行实验。加入过量的0.1M NaOH,50.0当量,5mL。实验开始时,10-FFA的质量浓度约为1000mg/L。通过HPLC测量反应进程。HPLC样品在提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)中制备。
发酵液中10-甲酰叶酸的碱介导N-去甲酰化
在之前的实验中,我们已经说明,使用0.1M NaOH对10-甲酰基叶酸标准品进行去甲酰化可以完全转化为叶酸,如图16所示。在这里,我们将相同的原理应用于更复杂的系统,即发酵液。在去甲酰基化之前对来自实施例18的发酵液的HPLC分析显示大量的10-甲酰基二氢叶酸(10F-DHF;60%面积);和10-甲酰叶酸(10F-FA;40%面积)。用不同v/v比的0.1MNaOH(1:1、1:2、1:3和1:4)处理来自实施例18的发酵液样品(10mL),并在避光环境温度(25℃)下搅拌24小时。根据我们的HPLC分析,发酵液/NaOH v/v 1:1和1:2的实验不会导致去甲酰化,而是导致10-甲酰基二氢叶酸氧化转化为10-甲酰基叶酸,如表20所示(条目2和3)。随后,当相对于发酵液(1:3和1:4)增加NaOH的量时,通过HPLC检测到显著量的叶酸,如表20所示(条目4和5)。有趣的是,较高量的NaOH在某种程度上阻碍了10F-DHF向10F-FA的氧化转化,因为通过HPLC检测到大量的10F-DHF。
代表性实验程序:
将来自实施例18的充分搅拌的发酵液(10mL)移液到配备有搅拌棒和铝箔用于避光的50-100mL圆底烧瓶中。逐滴加入氢氧化钠(0.1M)并将反应在环境温度(25℃)下剧烈搅拌24小时。搅拌24小时后,用9mL提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)稀释1mL每种发酵液,重复试验2次。将悬浮液涡旋搅拌,以4500rpm离心,通过0.22μm过滤器过滤并在HPLC上分析。
表20.添加不同量NaOH对发酵液中10-甲酰叶酸的N-去甲酰化的影响
使用来自实施例18的发酵液(FB3148,10mL)在50-100mL圆底烧瓶中进行实验。基于相对于FB3148(1:1、1:2、1:3和1:4)的体积/体积比添加0.1M NaOH。通过HPLC测量反应进程和结果。HPLC样品在提取缓冲液(含1%(w/v)抗坏血酸的0.1M磷酸盐缓冲液)中制备。将所有反应在环境温度(25℃)避光搅拌24小时。
实施例21:10-甲酰基叶酸的分离
收获后,使用5M NaOH水溶液将含有50g叶酸的发酵液调节至pH=12。将溶液在4℃下以10000rpm离心15分钟。向上清液中加入50g氢氧化钙并在室温下搅拌悬浮液2小时。使所得悬浮液沉降、倾析并在100μl硅藻土(Celite)的帮助下过滤上清液。滤饼用500mL水洗涤并过滤。合并滤液并稀释至最终体积为10升。用1N HCl将澄清的叶酸稀碱溶液的pH值调节至7.0,加热至70℃,然后冷却至室温。接下来,过滤溶液以去除在中性pH下沉淀的杂质。使用1N HCl将澄清的滤液调节至pH=3并在冰上冷却4小时。将悬浮液滤出并重新溶解在8LpH=12的热碱性溶液中(用1M NaOH调节)。向该溶液中加入50克活性炭(1当量/重量的叶酸)并将溶液加热至50℃并搅拌30分钟。过滤悬浮液,滤饼用3L碱化水溶液(pH=12,用NaOH调节)洗涤。合并滤液并使用1N HCl将pH调节至3.0,在连续搅拌期间添加。将所得浆液在冰上冷却24小时或过夜。滤出悬浮液并重新悬浮在1L pH=3的酸化水溶液中(pH用1N HCl调节)。再次过滤悬浮液,然后将所得滤饼冷冻并干燥以获得43克叶酸,其含有10%的水分,并在无水基础上测定90.1%的叶酸。
实施例22叶酸的分离
收获后,使用1M NaOH水溶液将含有30g叶酸的发酵液调节至pH=10。将溶液在4℃下以10000rpm离心15分钟。将所得上清液用1N HCl调节至pH 4.0,加热至70℃,然后冷却至室温。接着,借助100g硅藻土(Celite)过滤溶液。将滤饼重新悬浮在5L pH=10的碱性溶液中(用1M NaOH调节)。向该溶液中加入50克活性炭(1当量/重量的叶酸)并将溶液加热至50℃并搅拌30分钟。过滤悬浮液,滤饼用2L碱化水溶液(pH=12,用NaOH调节)洗涤。合并滤液并使用1N HCl将pH调节至3.0,在连续搅拌期间添加。将所得沉淀在冰上冷却16-24小时或然后滤出并重新悬浮在1L具有pH=3的酸化水溶液中(pH用1N HCl调节)。再次过滤悬浮液并将所得沉淀饼干燥以获得21克10-甲酰基叶酸,测定其含量为92%。
比较例1
确定枯草芽孢杆菌野生型菌株“168”、我们的出发非转基因菌株VBB38(菌株VKPMB2116=B.subtilis VNII Genetika 304)及其转化体的总叶酸产量,其中天然folC基因一步替换为来自A.gossypii(B.subtilis菌株FL21)或L.reuteri(B.subtilis菌株FL23)的异源folC2(FOL3)基因。在摇床规模(5ml生产培养基MD)下测试菌株,并通过使用用于叶酸检测的标准微生物测定法来测定总叶酸。
结果表明,在标准培养条件下(T=37C,在营养丰富的LB培养基中需氧),仅缺失枯草芽孢杆菌天然folC基因而没有同时表达异源folC2基因的敲除突变体不能生长。
文献:
1.Hjortmo S,Patring J,Andlid T.2008生长速度和培养基成分强烈影响酿酒酵母中的叶酸含量.Int J Food Microbiol.123(1-2):93-100.
2.McGuire JJ and Bertino JR.1981.叶酰聚谷氨酸的酶促合成及其功能.MolCell Biochem 38Spec No(Pt 1):19-48.
3.Reed,LS,Archer MC.1980.空气氧化四氢叶酸.J Agric Food Chem.28(4):801-805.
4.Rossi,M.,Raimondi,S.,Costantino,L.,Amaretti,A.,2016.叶酸:化学和微生物生产的相关性。维生素、生物色素和抗氧化剂的工业生物技术.Wiley-VCH Verlag GmbH&Co.KGaA,Weinheim,Germany,pp.103–128.
5.Scaglione and Panzavolta.2014.叶酸和5-甲基四氢叶酸不是一回事.Xenobiotica.44(5):480-488.
6.Serrano-Amatriain C,Ledesma-Amaro R,López-Nicolás R,Ros G,JiménezA,Revuelta JL.2016.通过工程化的Ashbya gossypii生产叶酸.Metab Eng.38:473-482.
7.Sybesma W,Starrenburg M,Kleerebezem M,Mierau I,de Vos WM,HugenholtzJ.2003a.通过乳酸乳球菌的代谢工程增加叶酸的产量.Appl Environ Microbiol.69(6):3069-3076.
8.Sybesma,W.,Starrenburg,M.,Tijsseling,L.,Hoefnagel,M.H.N.,Hugenholtz,J.,2003b.培养条件对乳酸菌产生叶酸的影响.Applied and EnvironmentalMicrobiology.69(8):4542–4548.
9.Sybesma W,Van Den Born E,Starrenburg M,Mierau I,Kleerebezem M,DeVos WM,Hugenholtz J.2003c.乳酸乳球菌代谢工程对叶酸多谷氨酰尾长的控制调控.ApplEnviron Microbiol.69(12):7101-7107.
10.Zeigler DR,Prágai Z,Rodriguez S,Chevreux B,Muffler A,Albert T,BaiR,Wyss M,Perkins JB.2008.168、W23和其他枯草芽孢杆菌遗留菌株的起源,Journal ofBacteriology.190(21):6983–6995
11.Zhu T,Pan Z,Domagalski N,Koepsel R,Ataai MM,Domach MM.2005.用于增强叶酸全合成的枯草芽孢杆菌工程.Appl Environ Microbiol.71(11):7122-7129.12.Walkey CJ,Kitts DD,Liu Y,van Vuuren HJJ.2015.Bioengineering yeast toenhance folate levels in wine.Process Biochem 50(2):205-210.
在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 赤峰制药股份有限公司
<120> 叶酸生产菌株及其制备和应用
<130> P2020-0689
<150> PCT/CN2019/102317
<151> 2019-08-23
<160> 86
<170> PatentIn version 3.5
<210> 1
<211> 573
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<213> Bacillus subtilis
<400> 1
atgaaagaag ttaataaaga gcaaatcgaa caagctgttc gtcaaatttt agaagcgatc 60
ggagaagacc cgaatagaga agggcttctt gatactccga aaagagtcgc aaagatgtat 120
gccgaagtat tctccggctt gaatgaagat ccaaaagaac atttccagac tatcttcggt 180
gaaaaccatg aggagcttgt tcttgtaaaa gatatagcgt ttcattctat gtgtgagcat 240
caccttgttc ccttttatgg aaaagcacat gttgcatata tcccgcgagg cggaaaggtc 300
acaggactca gcaaactggc acgtgccgtt gaagccgttg caaagcgccc gcagcttcag 360
gaacgcatca cttctacaat tgcagaaagc atcgtagaaa cgcttgatcc gcatggcgta 420
atggtagtgg ttgaagcgga acacatgtgc atgacgatgc gcggtgtaag aaaaccgggt 480
gcgaaaactg tgacttcagc agtcagaggc gtttttaaag atgatgccgc tgcccgtgca 540
gaagtattgg aacatattaa acgccaggac taa 573
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gctggacaga cagacgacct tgagcaaacg atcaactatg ctgagctcta tcacgtatgt 180
aaagatatcg tggaagggga gcctgtgaaa ttggtggaaa cgctggcgga acgtattgct 240
ggcactgttc tcggaaaatt tcagcctgtt cagcaatgta cggtgaaagt gattaagcca 300
gacccgccaa ttcccggaca ctataaatca gtagcaattg aaattacgag aaaaaagtca 360
tga 363
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atttacgaaa ctgacccggt cggatacgaa gatcaagctc aatttttgaa tatggctgtt 180
gaaatcaaga catcattgaa cccttttgaa ctccttgaac tgacgcagca gatagaaaat 240
gaattaggca gaacaaggga agtaagatgg gggccgcgga cggcagacct tgacattttg 300
ttatttaatc gtgaaaatat tgaaacagag caactaattg ttccgcatcc gagaatgtat 360
gagcgtttgt ttgtccttgc gccgcttgcg gaaatttgcc agcaggttga aaaagaggct 420
acaagcgccg aaacagacca agaaggtgta agagtatgga agcagaaatc tggggtagac 480
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ggcggaaaat atgacagctt ggacaaggcg ctgctgcacg cgaaagagat gatcgatgat 180
ggtgcccata tcattgatat tggaggggaa tcgacaaggc ctggcgctga gtgcgtatct 240
gaggatgagg agatgtccag agtcattccg gtgattgagc ggattacgaa agagcttggt 300
gttcctattt ctgtagacac gtacaaggct tctgtcgcag atgaagcagt gaaagccggt 360
gcatccatta tcaatgatat ttggggagcc aaacatgatc cgaagatggc ttccgttgca 420
gctgaacata atgttccaat tgtactcatg cataaccgcc ctgaaagaaa ctacaatgac 480
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gtagacgaga agaacattat tcttgatcct ggtatcggtt tcgcgaaaac ctatcacgat 600
aacttggcag tgatgaacaa actagagatt ttcagcggat tgggatatcc ggttcttctg 660
gcaacctccc gaaaaagatt catcggacgt gttctggatc ttccgcctga ggagcgggct 720
gagggcacag gcgcgactgt gtgtctcggc attcaaaaag gctgtgacat tgtcagggtc 780
catgatgtaa agcaaattgc cagaatggcg aaaatgatgg acgcgatgct gaataaggga 840
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ttatttcaac ctttttcgaa tgtcagaaat aaagtaaaga gatccggtaa tcagcacaat 60
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gcttttattg ctgatttcac ttgcatcata gagatctttt gcaagggaag cacgcgggaa 180
atcaaaagaa gcaaaatgaa tcgcatgagc aatggtttcc agtcttttaa tcatgttctg 240
atacggtttg tcctttaacg cgctaaacac cacagaaatg cgtgaattgg cgaaacgctg 300
cttcatcgtt tccgccagct tttcaacacc ttcttcgtta tgcgcaccgt ctaaatatac 360
cggaggatgt tcctgaacaa gctctaaccg tcccggccaa gcagccttca caagcccgct 420
ccttaacgct tcgtcactga tatgggcgat attctcctta ttgagccact ccgcagccaa 480
aatggacaaa gcagcatttt gtctttgatg ggtgccaatc agagatgtcc gaatatcttc 540
atagcatttc tcttccgttt tgaatgaaaa ctgttctcct gcaggcagag cctcttcatt 600
gaaaataaca catgcatcat gcaatgactg gaacggcgca gcatgccgtt cggcttcatg 660
gcggatgacc tgtaaagctt ccggctgggt aactgctgta acgattggaa taccttcttt 720
aataatgccg gccttttctc ctgcaatttc ttcaatggtg tttcccaaaa tgttcatatg 780
atcgtgtccg atgcttgtaa tcacagttaa gagcggttca accacattag tagaatcgaa 840
tctaccgccc agacctgttt caaaaataac aaaatcgacc ttatgaaact ctgcaaaata 900
taaaaatgca caagctgtca taatttcaaa ttctgtcggc tgtccgtatt ccgtttgatc 960
aagggcttca acgtgcggtt tcatttgatt gacgagtgct gtccattcct catctgaaat 1020
cggtatcccg tttacgctga tccgttcatt aaacgtaata atataaggcg atgtaaatgt 1080
tccaaccgta tatccggctt cctgcagcat agaacggata aaagcgacag ttgatccttt 1140
accgtttgtt cctgcgacgt ggaacgctcg gatttttttt tcaggatgtc ctaaccgcgc 1200
catcagctgt ttcattcgac caagtccggg cttcaccccg aatttcagcc tcccgtgaat 1260
ccagctgcgc gcatcttgat atgcagtaaa caa 1293
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<213> Bacillus subtilis
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atgatttcat tcatttttgc gatggatgcc aacaggctta tcggcaaaga caatgatttg 60
ccgtggcatt tgcccaatga tcttgcatac tttaagaaaa taacatcggg ccattcaatc 120
attatgggcc ggaaaacatt tgaatcgatc ggacgtccgc ttccaaatcg gaaaaatatt 180
gtcgttacct cagcgccgga ttcagaattt cagggatgca cggttgtcag ttcattaaag 240
gatgtactgg acatttgttc aggccctgaa gaatgctttg tgatcggagg ggctcagctc 300
tatacggacc tgttccctta tgcggacaga ctgtatatga cgaaaattca tcacgagttt 360
gagggtgacc gtcactttcc tgaatttgat gaatccaatt ggaagctggt ttcttctgag 420
caggggacca aagacgaaaa aaacccgtat gattacgaat ttctaatgta tgaaaaaaag 480
aattcttcta aagcgggagg attttaa 507
<210> 7
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Met Lys Glu Val Asn Lys Glu Gln Ile Glu Gln Ala Val Arg Gln Ile
1 5 10 15
Leu Glu Ala Ile Gly Glu Asp Pro Asn Arg Glu Gly Leu Leu Asp Thr
20 25 30
Pro Lys Arg Val Ala Lys Met Tyr Ala Glu Val Phe Ser Gly Leu Asn
35 40 45
Glu Asp Pro Lys Glu His Phe Gln Thr Ile Phe Gly Glu Asn His Glu
50 55 60
Glu Leu Val Leu Val Lys Asp Ile Ala Phe His Ser Met Cys Glu His
65 70 75 80
His Leu Val Pro Phe Tyr Gly Lys Ala His Val Ala Tyr Ile Pro Arg
85 90 95
Gly Gly Lys Val Thr Gly Leu Ser Lys Leu Ala Arg Ala Val Glu Ala
100 105 110
Val Ala Lys Arg Pro Gln Leu Gln Glu Arg Ile Thr Ser Thr Ile Ala
115 120 125
Glu Ser Ile Val Glu Thr Leu Asp Pro His Gly Val Met Val Val Val
130 135 140
Glu Ala Glu His Met Cys Met Thr Met Arg Gly Val Arg Lys Pro Gly
145 150 155 160
Ala Lys Thr Val Thr Ser Ala Val Arg Gly Val Phe Lys Asp Asp Ala
165 170 175
Ala Ala Arg Ala Glu Val Leu Glu His Ile Lys Arg Gln Asp
180 185 190
<210> 8
<211> 120
<212> PRT
<213> Bacillus subtilis
<400> 8
Met Asp Lys Val Tyr Val Glu Gly Met Glu Phe Tyr Gly Tyr His Gly
1 5 10 15
Val Phe Thr Glu Glu Asn Lys Leu Gly Gln Arg Phe Lys Val Asp Leu
20 25 30
Thr Ala Glu Leu Asp Leu Ser Lys Ala Gly Gln Thr Asp Asp Leu Glu
35 40 45
Gln Thr Ile Asn Tyr Ala Glu Leu Tyr His Val Cys Lys Asp Ile Val
50 55 60
Glu Gly Glu Pro Val Lys Leu Val Glu Thr Leu Ala Glu Arg Ile Ala
65 70 75 80
Gly Thr Val Leu Gly Lys Phe Gln Pro Val Gln Gln Cys Thr Val Lys
85 90 95
Val Ile Lys Pro Asp Pro Pro Ile Pro Gly His Tyr Lys Ser Val Ala
100 105 110
Ile Glu Ile Thr Arg Lys Lys Ser
115 120
<210> 9
<211> 167
<212> PRT
<213> Bacillus subtilis
<400> 9
Met Asn Asn Ile Ala Tyr Ile Ala Leu Gly Ser Asn Ile Gly Asp Arg
1 5 10 15
Glu Thr Tyr Leu Arg Gln Ala Val Ala Leu Leu His Gln His Ala Ala
20 25 30
Val Thr Val Thr Lys Val Ser Ser Ile Tyr Glu Thr Asp Pro Val Gly
35 40 45
Tyr Glu Asp Gln Ala Gln Phe Leu Asn Met Ala Val Glu Ile Lys Thr
50 55 60
Ser Leu Asn Pro Phe Glu Leu Leu Glu Leu Thr Gln Gln Ile Glu Asn
65 70 75 80
Glu Leu Gly Arg Thr Arg Glu Val Arg Trp Gly Pro Arg Thr Ala Asp
85 90 95
Leu Asp Ile Leu Leu Phe Asn Arg Glu Asn Ile Glu Thr Glu Gln Leu
100 105 110
Ile Val Pro His Pro Arg Met Tyr Glu Arg Leu Phe Val Leu Ala Pro
115 120 125
Leu Ala Glu Ile Cys Gln Gln Val Glu Lys Glu Ala Thr Ser Ala Glu
130 135 140
Thr Asp Gln Glu Gly Val Arg Val Trp Lys Gln Lys Ser Gly Val Asp
145 150 155 160
Glu Phe Val His Ser Glu Ser
165
<210> 10
<211> 285
<212> PRT
<213> Bacillus subtilis
<400> 10
Met Ala Gln His Thr Ile Asp Gln Thr Gln Val Ile His Thr Lys Pro
1 5 10 15
Ser Ala Leu Ser Tyr Lys Glu Lys Thr Leu Val Met Gly Ile Leu Asn
20 25 30
Val Thr Pro Asp Ser Phe Ser Asp Gly Gly Lys Tyr Asp Ser Leu Asp
35 40 45
Lys Ala Leu Leu His Ala Lys Glu Met Ile Asp Asp Gly Ala His Ile
50 55 60
Ile Asp Ile Gly Gly Glu Ser Thr Arg Pro Gly Ala Glu Cys Val Ser
65 70 75 80
Glu Asp Glu Glu Met Ser Arg Val Ile Pro Val Ile Glu Arg Ile Thr
85 90 95
Lys Glu Leu Gly Val Pro Ile Ser Val Asp Thr Tyr Lys Ala Ser Val
100 105 110
Ala Asp Glu Ala Val Lys Ala Gly Ala Ser Ile Ile Asn Asp Ile Trp
115 120 125
Gly Ala Lys His Asp Pro Lys Met Ala Ser Val Ala Ala Glu His Asn
130 135 140
Val Pro Ile Val Leu Met His Asn Arg Pro Glu Arg Asn Tyr Asn Asp
145 150 155 160
Leu Leu Pro Asp Met Leu Ser Asp Leu Met Glu Ser Val Lys Ile Ala
165 170 175
Val Glu Ala Gly Val Asp Glu Lys Asn Ile Ile Leu Asp Pro Gly Ile
180 185 190
Gly Phe Ala Lys Thr Tyr His Asp Asn Leu Ala Val Met Asn Lys Leu
195 200 205
Glu Ile Phe Ser Gly Leu Gly Tyr Pro Val Leu Leu Ala Thr Ser Arg
210 215 220
Lys Arg Phe Ile Gly Arg Val Leu Asp Leu Pro Pro Glu Glu Arg Ala
225 230 235 240
Glu Gly Thr Gly Ala Thr Val Cys Leu Gly Ile Gln Lys Gly Cys Asp
245 250 255
Ile Val Arg Val His Asp Val Lys Gln Ile Ala Arg Met Ala Lys Met
260 265 270
Met Asp Ala Met Leu Asn Lys Gly Gly Val His His Gly
275 280 285
<210> 11
<211> 430
<212> PRT
<213> Bacillus subtilis
<400> 11
Met Phe Thr Ala Tyr Gln Asp Ala Arg Ser Trp Ile His Gly Arg Leu
1 5 10 15
Lys Phe Gly Val Lys Pro Gly Leu Gly Arg Met Lys Gln Leu Met Ala
20 25 30
Arg Leu Gly His Pro Glu Lys Lys Ile Arg Ala Phe His Val Ala Gly
35 40 45
Thr Asn Gly Lys Gly Ser Thr Val Ala Phe Ile Arg Ser Met Leu Gln
50 55 60
Glu Ala Gly Tyr Thr Val Gly Thr Phe Thr Ser Pro Tyr Ile Ile Thr
65 70 75 80
Phe Asn Glu Arg Ile Ser Val Asn Gly Ile Pro Ile Ser Asp Glu Glu
85 90 95
Trp Thr Ala Leu Val Asn Gln Met Lys Pro His Val Glu Ala Leu Asp
100 105 110
Gln Thr Glu Tyr Gly Gln Pro Thr Glu Phe Glu Ile Met Thr Ala Cys
115 120 125
Ala Phe Leu Tyr Phe Ala Glu Phe His Lys Val Asp Phe Val Ile Phe
130 135 140
Glu Thr Gly Leu Gly Gly Arg Phe Asp Ser Thr Asn Val Val Glu Pro
145 150 155 160
Leu Leu Thr Val Ile Thr Ser Ile Gly His Asp His Met Asn Ile Leu
165 170 175
Gly Asn Thr Ile Glu Glu Ile Ala Gly Glu Lys Ala Gly Ile Ile Lys
180 185 190
Glu Gly Ile Pro Ile Val Thr Ala Val Thr Gln Pro Glu Ala Leu Gln
195 200 205
Val Ile Arg His Glu Ala Glu Arg His Ala Ala Pro Phe Gln Ser Leu
210 215 220
His Asp Ala Cys Val Ile Phe Asn Glu Glu Ala Leu Pro Ala Gly Glu
225 230 235 240
Gln Phe Ser Phe Lys Thr Glu Glu Lys Cys Tyr Glu Asp Ile Arg Thr
245 250 255
Ser Leu Ile Gly Thr His Gln Arg Gln Asn Ala Ala Leu Ser Ile Leu
260 265 270
Ala Ala Glu Trp Leu Asn Lys Glu Asn Ile Ala His Ile Ser Asp Glu
275 280 285
Ala Leu Arg Ser Gly Leu Val Lys Ala Ala Trp Pro Gly Arg Leu Glu
290 295 300
Leu Val Gln Glu His Pro Pro Val Tyr Leu Asp Gly Ala His Asn Glu
305 310 315 320
Glu Gly Val Glu Lys Leu Ala Glu Thr Met Lys Gln Arg Phe Ala Asn
325 330 335
Ser Arg Ile Ser Val Val Phe Ser Ala Leu Lys Asp Lys Pro Tyr Gln
340 345 350
Asn Met Ile Lys Arg Leu Glu Thr Ile Ala His Ala Ile His Phe Ala
355 360 365
Ser Phe Asp Phe Pro Arg Ala Ser Leu Ala Lys Asp Leu Tyr Asp Ala
370 375 380
Ser Glu Ile Ser Asn Lys Ser Trp Ser Glu Asp Pro Asp Asp Val Ile
385 390 395 400
Lys Phe Ile Glu Ser Lys Lys Gly Ser Asn Glu Ile Val Leu Ile Thr
405 410 415
Gly Ser Leu Tyr Phe Ile Ser Asp Ile Arg Lys Arg Leu Lys
420 425 430
<210> 12
<211> 168
<212> PRT
<213> Bacillus subtilis
<400> 12
Met Ile Ser Phe Ile Phe Ala Met Asp Ala Asn Arg Leu Ile Gly Lys
1 5 10 15
Asp Asn Asp Leu Pro Trp His Leu Pro Asn Asp Leu Ala Tyr Phe Lys
20 25 30
Lys Ile Thr Ser Gly His Ser Ile Ile Met Gly Arg Lys Thr Phe Glu
35 40 45
Ser Ile Gly Arg Pro Leu Pro Asn Arg Lys Asn Ile Val Val Thr Ser
50 55 60
Ala Pro Asp Ser Glu Phe Gln Gly Cys Thr Val Val Ser Ser Leu Lys
65 70 75 80
Asp Val Leu Asp Ile Cys Ser Gly Pro Glu Glu Cys Phe Val Ile Gly
85 90 95
Gly Ala Gln Leu Tyr Thr Asp Leu Phe Pro Tyr Ala Asp Arg Leu Tyr
100 105 110
Met Thr Lys Ile His His Glu Phe Glu Gly Asp Arg His Phe Pro Glu
115 120 125
Phe Asp Glu Ser Asn Trp Lys Leu Val Ser Ser Glu Gln Gly Thr Lys
130 135 140
Asp Glu Lys Asn Pro Tyr Asp Tyr Glu Phe Leu Met Tyr Glu Lys Lys
145 150 155 160
Asn Ser Ser Lys Ala Gly Gly Phe
165
<210> 13
<211> 683
<212> DNA
<213> Bacillus subtilis
<400> 13
cgcagcatac gcagcgaaat cagcatcacc ggagaatccc aacgaagcca actagtatga 60
aagaagtcaa taaagaacaa attgaacagg cagtgagaca gattcttgaa gcaattggag 120
aagatccgaa cagagagggc ttacttgata caccgaaaag agttgctaaa atgtatgcgg 180
aagtcttttc aggcttaaat gaagatccga aagagcattt tcagacaatt ttcggagaaa 240
accatgaaga gctggtcctt gtgaaagata ttgcgtttca ctcaatgtgc gaacatcacc 300
tggtgccgtt ttacggcaag gcacacgttg cgtatattcc tagaggcgga aaagttacag 360
gcttgtcaaa attagcacgc gcagttgaag ctgttgcaaa aagaccgcaa ttacaggaac 420
gcattacatc tacaattgcg gaatcaattg tcgagacatt agaccctcat ggcgttatgg 480
ttgtcgttga agctgaacac atgtgcatga caatgcgcgg cgtcagaaaa cctggcgcaa 540
aaacagtcac atcagcagtc agaggcgtgt ttaaagatga tgcggcagct cgtgcggaag 600
tcctggaaca tattaaacgc caggactgaa aaagagggga gggaaacatt aatgacgacc 660
tggctaacga gtctcgccga tct 683
<210> 14
<211> 454
<212> DNA
<213> Bacillus subtilis
<400> 14
ttctttttgc gccaggtagc catagctggt catatgatgg ataaagttta tgtggaagga 60
atggaatttt atggctatca tggcgtcttc acagaagaga acaaattggg acaacgcttc 120
aaagtagatc tgacagcaga actggattta tcaaaagcag gacaaacaga cgaccttgaa 180
cagacaatta actatgcaga gctttaccat gtctgtaaag acattgtcga aggcgagccg 240
gtcaaattgg tagagaccct tgctgagcgg atagctggca cagttttagg taaatttcag 300
ccggttcaac aatgtacggt gaaagttatt aaaccagatc cgccgattcc tggccactat 360
aaatcagtag caattgaaat tacgagaaaa aagtcataaa ttaattctag agtcgatccc 420
cgggttcgcc agcaatgact accggcagcc cgcc 454
<210> 15
<211> 595
<212> DNA
<213> Bacillus subtilis
<400> 15
ggcggggctt cttttatcga atccagcgtg acatatgatg aacaacattg cgtacattgc 60
gcttggctct aatattggag atagagaaac gtatctgcgc caggccgttg cgttactgca 120
tcaacatgct gcggtcacag ttacaaaagt cagctcaatt tatgaaacag atccggtcgg 180
ctatgaagac caagcccagt ttttaaatat ggcggttgaa attaaaacaa gcctgaatcc 240
gtttgaactt ctggaactga cacagcaaat cgaaaacgaa ctgggccgca cacgcgaagt 300
tagatggggc ccgagaacag cggatttaga cattctgctg tttaacagag aaaacattga 360
aacagagcag ttaattgtcc cgcatcctcg catgtatgaa cgcctgtttg ttcttgcgcc 420
gcttgcggaa atttgccagc aggtcgagaa agaagcgaca agcgcggaaa cggatcaaga 480
aggagttaga gtttggaaac aaaaatcagg cgttgacgaa tttgtacata gcgaaagctg 540
aaaaagaggg gagggaaaca ttaatgaccg accctcatgg aaacccttcc tggcg 595
<210> 16
<211> 948
<212> DNA
<213> Bacillus subtilis
<400> 16
gaccgaccct catggaaacc cttcctggcg catatgatgg cgcagcacac aatagatcaa 60
acacaagtca ttcatacgaa accgagcgcg ctttcatata aagaaaaaac actggtcatg 120
ggcattctta acgttacacc tgattctttt agcgatggtg gaaaatatga cagcttggac 180
aaggcgcttc tgcatgccaa agaaatgatc gacgacggcg cgcacattat tgacatagga 240
ggcgagagca caagaccggg agctgaatgc gtcagcgaag acgaagaaat gtctcgggtc 300
attccggtca ttgaacgcat cacaaaggaa ctcggcgtcc cgatttcagt ggatacatat 360
aaagcatctg tggcagacga agcagtcaaa gcgggcgcat ctattatcaa tgacatttgg 420
ggagcgaaac atgatccgaa gatggcaagc gtcgcagcgg aacataacgt tccaattgtc 480
ctgatgcaca atcggccaga acggaattat aacgaccttc ttccggatat gctgagcgac 540
cttatggaat cagtcaaaat tgcggttgag gcgggcgtgg atgagaaaaa tattatttta 600
gatccgggca tcggcttcgc gaagacatac catgataatc ttgcagtgat gaataagtta 660
gagatcttca gcggacttgg ctatcctgtc ctgctggcta catctcgtaa aagatttatc 720
ggaagagttc ttgatttacc gcctgaagag agagcagagg gcacaggagc gacagtctgc 780
ttgggcattc agaaaggatg cgacatagtg cgtgttcatg atgtcaagca aattgccaga 840
atggcgaaaa tgatggacgc gatgctgaat aagggagggg tgcaccatgg atgaaaaaga 900
ggggagggaa acattaattt ctttttgcgc caggtagcca tagctggt 948
<210> 17
<211> 598
<212> DNA
<213> Bacillus subtilis
<400> 17
gacgacctgg ctaacgagtc tcgccgatct catatgatga tttcatttat tttcgcaatg 60
gacgcgaata gactgatagg caaagacaat gatctgccgt ggcatttacc gaatgacctg 120
gcttatttta aaaaaattac aagcggccat agcatcatta tgggacgtaa aacatttgag 180
tcaattggca gacctcttcc gaacagaaaa aacattgttg tcacatctgc gccggattca 240
gaatttcagg gctgcacagt cgtctcaagc cttaaagacg ttcttgatat ttgcagcgga 300
ccggaagagt gttttgtcat tggcggagcg caattataca cagatctttt tccgtacgcg 360
gatagactgt atatgacaaa aatccaccat gaatttgaag gcgacagaca ctttcctgaa 420
tttgacgaga gcaactggaa actcgtgtct agcgaacagg gcacgaagga tgagaaaaat 480
ccgtatgact atgaatttct tatgtatgaa aagaaaaaca gcagcaaagc gggaggcttt 540
tgaaaaagag gggagggaaa cattaatggc ggggcttctt ttatcgaatc cagcgtga 598
<210> 18
<211> 1460
<212> DNA
<213> 人工序列(artificial sequence)
<400> 18
gccttttaat cccggcaaca gcttaatcag tacatccatc attccgaagc atccgacatt 60
cgatcattac aaggaattat ttgcgggcaa ggaaagcctt caatatgtgc agtggtatgt 120
caactctatg aagatcagcc tgtttacaat ggcagggtct ttgctctgtg tgacgtttac 180
ggcctatgcg ttttcgcgct ttcggtttaa agggaggaaa tacgctttaa cgctcttttt 240
attgctgcag atgattcctc agttttcagc tttaattgcc ttgtttgtgc tggcgcaaat 300
cttgggaatg atcaatagcc actggctgct aatcttgctt tatatcggcg gcctgatccc 360
gatgaatacg tatttgatga aagggtacat ggattccatt ccgatggatt tagacgaaag 420
cgccaagatt gacggagcca gcagcaccag aatcttcttc cagatcattc tgccattatc 480
aaaaccgatg gcggcagtcg tggccatgaa cggctttacc ggtccgctcg gagattttgt 540
gctgtcctca accatattga gaacgcctga atcatataca ttgcccgtcg gtctattcaa 600
tttagtgaat gatgtcatgg gggccagcta tacgacattt gcggccggag ccctgcttat 660
cagcataccg gttgccgtca tctttattat gctgcaaaag aattttgtgt ccggattaac 720
cgcaggcgga acgaagggct aagagaacaa ggaggagaat gtgatgtcaa agcttgaaaa 780
aacgcacgta acaaaagcaa aatttatgct ccatggggga gactacaacc ccgatcagtg 840
gctggatcgg cccgatattt tagctgacga tatcaaactg atgaagcttt ctcatacgaa 900
tacgttttct gtcggcattt ttgcatggag cgcacttgag ccggaggagg gcgtatatca 960
atttgaatgg ctggatgata tttttgagcg gattcacagt ataggcggcc gggtcatatt 1020
agcaacgccg agcggagccc gtccggcctg gctgtcgcaa acctatccgg aagttttgcg 1080
cgtcaatgcc tcccgcgtca aacagctgca cggcggaagg cacaaccact gcctcacatc 1140
taaagtctac cgagaaaaaa cacggcacat caaccgctac gggtgcgcat gatcgtatgg 1200
ttcactgtcc accaaccaaa actgtgctca gtaccgccaa tatttctccc ttggggggta 1260
caaagaggtg tccctagaag agatccacgc tgtgtaaaaa ttttacaaaa aggtattgac 1320
tttccctaca gggtgtgtaa taatttaatt acaggcgggg gcaaccccgc tcagtaccta 1380
gagcgtaaaa gaggggaggg aaacactagt tggcttcgtt gggattctcc ggtgatgctg 1440
atttcgctgc gtatgctgcg 1460
<210> 19
<211> 1038
<212> DNA
<213> 人工序列(artificial sequence)
<400> 19
tctagaaatt aagaaggagg gattcgtcat gttggtattc caaatgcgtt atgtagataa 60
aacatctact gttttgaaac agactaaaaa cagtgattac gcagataaat aaatacgtta 120
gattaattcc taccagtgac taatcttatg actttttaaa cagataacta aaattacaaa 180
caaatcgttt aacttctgta tttgtttata gatgtaatca cttcaggagt gattacatga 240
acaaaaatat aaaatattct caaaactttt taacgagtga aaaagtactc aaccaaataa 300
taaaacaatt gaatttaaaa gaaaccgata ccgtttacga aattggaaca ggtaaagggc 360
atttaacgac gaaactggct aaaataagta aacaggtaac gtctattgaa ttagacagtc 420
atctattcaa cttatcgtca gaaaaattaa aactgaacat tcgtgtcact ttaattcacc 480
aagatattct acagtttcaa ttccctaaca aacagaggta taaaattgtt gggaatattc 540
cttaccattt aagcacacaa attattaaaa aagtggtttt tgaaagccat gcgtctgaca 600
tctatctgat tgttgaagaa ggattctaca agcgtacctt ggatattcac cgaacactag 660
ggttgctctt gcacactcaa gtctcgattc agcaattgct taagctgcca gcggaatgct 720
ttcatcctaa accaaaagta aacagtgtct taataaaact tacccgccat accacagatg 780
ttccagataa atattggaag ctatatacgt actttgtttc aaaatgggtc aatcgagaat 840
atcgtcaact gtttactaaa aatcagtttc atcaagcaat gaaacacgcc aaagtaaaca 900
atttaagtac cgttacttat gagcaagtat tgtctatttt taatagttat ctattattta 960
acgggaggaa ataattctat gagtcgcttt tgtaaatttg gaaagttaca cgttactaaa 1020
gggaatgtag atggatcc 1038
<210> 20
<211> 5390
<212> DNA
<213> 人工序列(artificial sequence)
<400> 20
tcccggcaac agcttaatca gtacatccat cattccgaag catccgacat tcgatcatta 60
caaggaatta tttgcgggca aggaaagcct tcaatatgtg cagtggtatg tcaactctat 120
gaagatcagc ctgtttacaa tggcagggtc tttgctctgt gtgacgttta cggcctatgc 180
gttttcgcgc tttcggttta aagggaggaa atacgcttta acgctctttt tattgctgca 240
gatgattcct cagttttcag ctttaattgc cttgtttgtg ctggcgcaaa tcttgggaat 300
gatcaatagc cactggctgc taatcttgct ttatatcggc ggcctgatcc cgatgaatac 360
gtatttgatg aaagggtaca tggattccat tccgatggat ttagacgaaa gcgccaagat 420
tgacggagcc agcagcacca gaatcttctt ccagatcatt ctgccattat caaaaccgat 480
ggcggcagtc gtggccatga acggctttac cggtccgctc ggagattttg tgctgtcctc 540
aaccatattg agaacgcctg aatcatatac attgcccgtc ggtctattca atttagtgaa 600
tgatgtcatg ggggccagct atacgacatt tgcggccgga gccctgctta tcagcatacc 660
ggttgccgtc atctttatta tgctgcaaaa gaattttgtg tccggattaa ccgcaggcgg 720
aacgaagggc taagagaaca aggaggagaa tgtgatgtca aagcttgaaa aaacgcacgt 780
aacaaaagca aaatttatgc tccatggggg agactacaac cccgatcagt ggctggatcg 840
gcccgatatt ttagctgacg atatcaaact gatgaagctt tctcatacga atacgttttc 900
tgtcggcatt tttgcatgga gcgcacttga gccggaggag ggcgtatatc aatttgaatg 960
gctggatgat atttttgagc ggattcacag tataggcggc cgggtcatat tagcaacgcc 1020
gagcggagcc cgtccggcct ggctgtcgca aacctatccg gaagttttgc gcgtcaatgc 1080
ctcccgcgtc aaacagctgc acggcggaag gcacaaccac tgcctcacat ctaaagtcta 1140
ccgagaaaaa acacggcaca tcaaccgcta cgggtgcgca tgatcgtatg gttcactgtc 1200
caccaaccaa aactgtgctc agtaccgcca atatttctcc cttggggggt acaaagaggt 1260
gtccctagaa gagatccacg ctgtgtaaaa attttacaaa aaggtattga ctttccctac 1320
agggtgtgta ataatttaat tacaggcggg ggcaaccccg ctcagtacct agagcgtaaa 1380
agaggggagg gaaacactag ttggcttcgt tgggattctc cggtgatgct gatttcgctg 1440
cgtatgctgc gatgaaagaa gtcaataaag aacaaattga acaggcagtg agacagattc 1500
ttgaagcaat tggagaagat ccgaacagag agggcttact tgatacaccg aaaagagttg 1560
ctaaaatgta tgcggaagtc ttttcaggct taaatgaaga tccgaaagag cattttcaga 1620
caattttcgg agaaaaccat gaagagctgg tccttgtgaa agatattgcg tttcactcaa 1680
tgtgcgaaca tcacctggtg ccgttttacg gcaaggcaca cgttgcgtat attcctagag 1740
gcggaaaagt tacaggcttg tcaaaattag cacgcgcagt tgaagctgtt gcaaaaagac 1800
cgcaattaca ggaacgcatt acatctacaa ttgcggaatc aattgtcgag acattagacc 1860
ctcatggcgt tatggttgtc gttgaagctg aacacatgtg catgacaatg cgcggcgtca 1920
gaaaacctgg cgcaaaaaca gtcacatcag cagtcagagg cgtgtttaaa gatgatgcgg 1980
cagctcgtgc ggaagtcctg gaacatatta aacgccagga ctgaaaaaga ggggagggaa 2040
acattatgat gatttcattt attttcgcaa tggacgcgaa tagactgata ggcaaagaca 2100
atgatctgcc gtggcattta ccgaatgacc tggcttattt taaaaaaatt acaagcggcc 2160
atagcatcat tatgggacgt aaaacatttg agtcaattgg cagacctctt ccgaacagaa 2220
aaaacattgt tgtcacatct gcgccggatt cagaatttca gggctgcaca gtcgtctcaa 2280
gccttaaaga cgttcttgat atttgcagcg gaccggaaga gtgttttgtc attggcggag 2340
cgcaattata cacagatctt tttccgtacg cggatagact gtatatgaca aaaatccacc 2400
atgaatttga aggcgacaga cactttcctg aatttgacga gagcaactgg aaactcgtgt 2460
ctagcgaaca gggcacgaag gatgagaaaa atccgtatga ctatgaattt cttatgtatg 2520
aaaagaaaaa cagcagcaaa gcgggaggct tttgaaaaag aggggaggga aacattatga 2580
tgaacaacat tgcgtacatt gcgcttggct ctaatattgg agatagagaa acgtatctgc 2640
gccaggccgt tgcgttactg catcaacatg ctgcggtcac agttacaaaa gtcagctcaa 2700
tttatgaaac agatccggtc ggctatgaag accaagccca gtttttaaat atggcggttg 2760
aaattaaaac aagcctgaat ccgtttgaac ttctggaact gacacagcaa atcgaaaacg 2820
aactgggccg cacacgcgaa gttagatggg gcccgagaac agcggattta gacattctgc 2880
tgtttaacag agaaaacatt gaaacagagc agttaattgt cccgcatcct cgcatgtatg 2940
aacgcctgtt tgttcttgcg ccgcttgcgg aaatttgcca gcaggtcgag aaagaagcga 3000
caagcgcgga aacggatcaa gaaggagtta gagtttggaa acaaaaatca ggcgttgacg 3060
aatttgtaca tagcgaaagc tgaaaaagag gggagggaaa cattatgatg gcgcagcaca 3120
caatagatca aacacaagtc attcatacga aaccgagcgc gctttcatat aaagaaaaaa 3180
cactggtcat gggcattctt aacgttacac ctgattcttt tagcgatggt ggaaaatatg 3240
acagcttgga caaggcgctt ctgcatgcca aagaaatgat cgacgacggc gcgcacatta 3300
ttgacatagg aggcgagagc acaagaccgg gagctgaatg cgtcagcgaa gacgaagaaa 3360
tgtctcgggt cattccggtc attgaacgca tcacaaagga actcggcgtc ccgatttcag 3420
tggatacata taaagcatct gtggcagacg aagcagtcaa agcgggcgca tctattatca 3480
atgacatttg gggagcgaaa catgatccga agatggcaag cgtcgcagcg gaacataacg 3540
ttccaattgt cctgatgcac aatcggccag aacggaatta taacgacctt cttccggata 3600
tgctgagcga ccttatggaa tcagtcaaaa ttgcggttga ggcgggcgtg gatgagaaaa 3660
atattatttt agatccgggc atcggcttcg cgaagacata ccatgataat cttgcagtga 3720
tgaataagtt agagatcttc agcggacttg gctatcctgt cctgctggct acatctcgta 3780
aaagatttat cggaagagtt cttgatttac cgcctgaaga gagagcagag ggcacaggag 3840
cgacagtctg cttgggcatt cagaaaggat gcgacatagt gcgtgttcat gatgtcaagc 3900
aaattgccag aatggcgaaa atgatggacg cgatgctgaa taagggaggg gtgcaccatg 3960
gatgaaaaag aggggaggga aacattatga tggataaagt ttatgtggaa ggaatggaat 4020
tttatggcta tcatggcgtc ttcacagaag agaacaaatt gggacaacgc ttcaaagtag 4080
atctgacagc agaactggat ttatcaaaag caggacaaac agacgacctt gaacagacaa 4140
ttaactatgc agagctttac catgtctgta aagacattgt cgaaggcgag ccggtcaaat 4200
tggtagagac ccttgctgag cggatagctg gcacagtttt aggtaaattt cagccggttc 4260
aacaatgtac ggtgaaagtt attaaaccag atccgccgat tcctggccac tataaatcag 4320
tagcaattga aattacgaga aaaaagtcat aaattaattc tagaaattaa gaaggaggga 4380
ttcgtcatgt tggtattcca aatgcgttat gtagataaaa catctactgt tttgaaacag 4440
actaaaaaca gtgattacgc agataaataa atacgttaga ttaattccta ccagtgacta 4500
atcttatgac tttttaaaca gataactaaa attacaaaca aatcgtttaa cttctgtatt 4560
tgtttataga tgtaatcact tcaggagtga ttacatgaac aaaaatataa aatattctca 4620
aaacttttta acgagtgaaa aagtactcaa ccaaataata aaacaattga atttaaaaga 4680
aaccgatacc gtttacgaaa ttggaacagg taaagggcat ttaacgacga aactggctaa 4740
aataagtaaa caggtaacgt ctattgaatt agacagtcat ctattcaact tatcgtcaga 4800
aaaattaaaa ctgaacattc gtgtcacttt aattcaccaa gatattctac agtttcaatt 4860
ccctaacaaa cagaggtata aaattgttgg gaatattcct taccatttaa gcacacaaat 4920
tattaaaaaa gtggtttttg aaagccatgc gtctgacatc tatctgattg ttgaagaagg 4980
attctacaag cgtaccttgg atattcaccg aacactaggg ttgctcttgc acactcaagt 5040
ctcgattcag caattgctta agctgccagc ggaatgcttt catcctaaac caaaagtaaa 5100
cagtgtctta ataaaactta cccgccatac cacagatgtt ccagataaat attggaagct 5160
atatacgtac tttgtttcaa aatgggtcaa tcgagaatat cgtcaactgt ttactaaaaa 5220
tcagtttcat caagcaatga aacacgccaa agtaaacaat ttaagtaccg ttacttatga 5280
gcaagtattg tctattttta atagttatct attatttaac gggaggaaat aattctatga 5340
gtcgcttttg taaatttgga aagttacacg ttactaaagg gaatgtagat 5390
<210> 21
<211> 2124
<212> DNA
<213> 人工序列(artificial sequence)
<400> 21
aattcttact gcagatagtg tacgtaaaaa gattaaatta ttgcttggtg aaaaaagtct 60
tgcaatggtg caggttgttc tcaatgtcga aaatatgtat ttgtatttaa cgcacgagag 120
caaggacgct attgctaaga agaaacatgt ttatgataag gctgatataa agctaatcaa 180
taattttgat attgaccgtt atgtgacgtt agatgtcgag gaaaagaccg aacttttcaa 240
tgtggttgta tcgcttattc gtgcgtacac tctccaaaat atttttgatt tgtatgattt 300
cattgacgaa aatggagaaa cttatgggtt gactataaat ttggttaacg aagttattgc 360
agggaaaact ggttttatga aattgttgtt tgacggagct tatcaacgta gtaagcgtgg 420
aacaaagaac gaagagagat aaaaagttga tctttgtgaa aactacagaa agtaaagaat 480
gaaaagagta atgctaacat agcattacgg attttatgac cgatgatgaa gaaaagaatt 540
tgaaacttag tttatatgtg gtaaaatgtt ttaatcaagt ttaggaggaa ttaattatga 600
agtgtaatta atgtaacagg gttcaattaa aagagggaag cgtatcatta accctataaa 660
ctacgtctgc cctcattatt ggagggtgaa atgtgaatac atcctattca caatcgaatt 720
tacgacacaa ccaaatttta atttggcttt gcattttatc tttttttagc gtattaaatg 780
aaatggtttt gaacgtctca ttacctgata ttgcaaatga ttttaataaa ccacctgcga 840
gtacaaactg ggtgaacaca gcctttatgt taaccttttc cattggaaca gctgtatatg 900
gaaagctatc tgatcaatta ggcatcaaaa ggttactcct atttggaatt ataataaatt 960
gtttcgggtc ggtaattggg tttgttggcc attctttctt ttccttactt attatggctc 1020
gttttattca aggggctggt gcagctgcat ttccagcact cgtaatggtt gtagttgcgc 1080
gctatattcc aaaggaaaat aggggtaaag catttggtct tattggatcg atagtagcca 1140
tgggagaagg agtcggtcca gcgattggtg gaatgatagc ccattatatt cattggtcct 1200
atcttctact cattcctatg ataacaatta tcactgttcc gtttcttatg aaattattaa 1260
agaaagaagt aaggataaaa ggtcattttg atatcaaagg aattatacta atgtctgtag 1320
gcattgtatt ttttatgttg tttacaacat catatagcat ttcttttctt atcgttagcg 1380
tgctgtcatt cctgatattt gtaaaacata tcaggaaagt aacagatcct tttgttgatc 1440
ccggattagg gaaaaatata ccttttatga ttggagttct ttgtggggga attatatttg 1500
gaacagtagc agggtttgtc tctatggttc cttatatgat gaaagatgtt caccagctaa 1560
gtactgccga aatcggaagt gtaattattt tccctggaac aatgagtgtc attattttcg 1620
gctacattgg tgggatactt gttgatagaa gaggtccttt atacgtgtta aacatcggag 1680
ttacatttct ttctgttagc tttttaactg cttcctttct tttagaaaca acatcatggt 1740
tcatgacaat tataatcgta tttgttttag gtgggctttc gttcaccaaa acagttatat 1800
caacaattgt ttcaagtagc ttgaaacagc aggaagctgg tgctggaatg agtttgctta 1860
actttaccag ctttttatca gagggaacag gtattgcaat tgtaggtggt ttattatcca 1920
tacccttact tgatcaaagg ttgttaccta tggaagttga tcagtcaact tatctgtata 1980
gtaatttgtt attacttttt tcaggaatca ttgtcattag ttggctggtt accttgaatg 2040
tatataaaca ttctcaaagg gatttctaaa tcgttaaggg atcaactttg ggagagagtt 2100
caaaattgat cctttctgca gaag 2124
<210> 22
<211> 419
<212> PRT
<213> Lactobacillus reuteri
<400> 22
Met Arg Thr Tyr Glu Gln Ile Asn Ala Gly Phe Asn Arg Gln Met Leu
1 5 10 15
Gly Gly Gln Arg Asp Arg Val Lys Phe Leu Arg Arg Ile Leu Thr Arg
20 25 30
Leu Gly Asn Pro Asp Gln Arg Phe Lys Ile Ile His Ile Ala Gly Thr
35 40 45
Asn Gly Lys Gly Ser Thr Gly Thr Met Leu Glu Gln Gly Leu Gln Asn
50 55 60
Ala Gly Tyr Arg Val Gly Tyr Phe Ser Ser Pro Ala Leu Val Asp Asp
65 70 75 80
Arg Glu Gln Ile Lys Val Asn Asp His Leu Ile Ser Lys Lys Asp Phe
85 90 95
Ala Met Thr Tyr Gln Lys Ile Thr Glu His Leu Pro Ala Asp Leu Leu
100 105 110
Pro Asp Asp Ile Thr Ile Phe Glu Trp Trp Thr Leu Ile Met Leu Gln
115 120 125
Tyr Phe Ala Asp Gln Lys Val Asp Trp Ala Val Ile Glu Cys Gly Leu
130 135 140
Gly Gly Gln Asp Asp Ala Thr Asn Ile Ile Ser Ala Pro Phe Ile Ser
145 150 155 160
Val Ile Thr His Ile Ala Leu Asp His Thr Arg Ile Leu Gly Pro Thr
165 170 175
Ile Ala Lys Ile Ala Gln Ala Lys Ala Gly Ile Ile Lys Thr Gly Thr
180 185 190
Lys Gln Val Phe Leu Ala Pro His Gln Glu Lys Asp Ala Leu Thr Ile
195 200 205
Ile Arg Glu Lys Ala Gln Gln Gln Lys Val Gly Leu Thr Gln Ala Asp
210 215 220
Ala Gln Ser Ile Val Asp Gly Lys Ala Ile Leu Lys Val Asn His Lys
225 230 235 240
Ile Tyr Lys Val Pro Phe Asn Leu Leu Gly Thr Phe Gln Ser Glu Asn
245 250 255
Leu Gly Thr Val Val Ser Val Phe Asn Phe Leu Tyr Gln Arg Arg Leu
260 265 270
Val Thr Ser Trp Gln Pro Leu Leu Ser Thr Leu Ala Thr Val Lys Ile
275 280 285
Ala Gly Arg Met Gln Lys Ile Ala Asp His Pro Pro Ile Ile Leu Asp
290 295 300
Gly Ala His Asn Pro Asp Ala Ala Lys Gln Leu Thr Lys Thr Ile Ser
305 310 315 320
Lys Leu Pro His Asn Lys Val Ile Met Val Leu Gly Phe Leu Ala Asp
325 330 335
Lys Asn Ile Ser Gln Met Val Lys Ile Tyr Gln Gln Met Ala Asp Glu
340 345 350
Ile Ile Ile Thr Thr Pro Asp His Pro Thr Arg Ala Leu Asp Ala Ser
355 360 365
Ala Leu Lys Ser Val Leu Pro Gln Ala Ile Ile Ala Asn Asn Pro Arg
370 375 380
Gln Gly Leu Val Val Ala Lys Lys Ile Ala Glu Pro Asn Asp Leu Ile
385 390 395 400
Ile Val Thr Gly Ser Phe Tyr Thr Ile Lys Asp Ile Glu Ala Asn Leu
405 410 415
Asp Glu Lys
<210> 23
<211> 406
<212> PRT
<213> Ashbya gossypii
<400> 23
Met Glu Leu Gly Leu Gly Arg Ile Thr Gln Val Leu Arg Gln Leu His
1 5 10 15
Ser Pro His Glu Arg Met Arg Val Leu His Val Ala Gly Thr Asn Gly
20 25 30
Lys Gly Ser Val Cys Ala Tyr Leu Ala Ala Val Leu Arg Ala Gly Gly
35 40 45
Glu Arg Val Gly Arg Phe Thr Ser Pro His Leu Val His Pro Arg Asp
50 55 60
Ala Ile Thr Val Asp Gly Glu Val Ile Gly Ala Ala Thr Tyr Ala Ala
65 70 75 80
Leu Lys Ala Glu Val Val Ala Ala Gly Thr Cys Thr Glu Phe Glu Ala
85 90 95
Gln Thr Ala Val Ala Leu Thr His Phe Ala Arg Leu Glu Cys Thr Trp
100 105 110
Cys Val Val Glu Val Gly Val Gly Gly Arg Leu Asp Ala Thr Asn Val
115 120 125
Val Pro Gly Gly Arg Lys Leu Cys Ala Ile Thr Lys Val Gly Leu Asp
130 135 140
His Gln Ala Leu Leu Gly Gly Thr Leu Ala Val Val Ala Arg Glu Lys
145 150 155 160
Ala Gly Ile Val Val Pro Gly Val Arg Phe Val Ala Val Asp Gly Thr
165 170 175
Asn Ala Pro Ser Val Leu Ala Glu Val Arg Ala Ala Ala Ala Lys Val
180 185 190
Gly Ala Glu Val His Glu Thr Gly Gly Ala Pro Val Cys Thr Val Ser
195 200 205
Trp Gly Ala Val Ala Ala Ser Ala Leu Pro Leu Ala Gly Ala Tyr Gln
210 215 220
Val Gln Asn Ala Gly Val Ala Leu Ala Leu Leu Asp His Leu Gln Gln
225 230 235 240
Leu Gly Glu Ile Ser Val Ser His Ala Ala Leu Glu Arg Gly Leu Lys
245 250 255
Ala Val Glu Trp Pro Gly Arg Leu Gln Gln Val Glu Tyr Asp Leu Gly
260 265 270
Gly Val His Val Pro Leu Leu Phe Asp Gly Ala His Asn Pro Cys Ala
275 280 285
Ala Glu Glu Leu Ala Arg Phe Leu Asn Glu Arg Tyr Arg Gly Pro Gly
290 295 300
Gly Ser Pro Leu Ile Tyr Val Leu Ala Val Thr Cys Gly Lys Glu Ile
305 310 315 320
Asp Ala Leu Leu Ala Pro Leu Leu Lys Pro His Asp Arg Val Phe Ala
325 330 335
Thr Ser Phe Gly Ala Val Glu Ser Met Pro Trp Val Ala Ala Met Ala
340 345 350
Ser Glu Asp Val Ala Ala Ala Ala Arg Arg Tyr Thr Ala His Val Ser
355 360 365
Ala Val Ala Asp Pro Leu Asp Ala Leu Arg Ala Ala Ala Ala Ala Arg
370 375 380
Gly Asp Ala Asn Leu Val Val Cys Gly Ser Leu Tyr Leu Val Gly Glu
385 390 395 400
Leu Leu Arg Arg Glu His
405
<210> 24
<211> 1399
<212> DNA
<213> Lactobacillus reuteri
<400> 24
ttttactagt atgagaacat acgaacaaat taatgcagga tttaatcgcc agatgctggg 60
cggccagaga gacagagtca agttccttag acgcatcctt acgagacttg gaaaccctga 120
tcagcgcttt aaaattattc atatcgcggg aacgaacggc aaaggatcaa caggcactat 180
gttagaacag ggccttcaga atgcgggata ccgcgtcggc tactttagct ctcctgcgct 240
ggttgatgat cgcgaacaaa ttaaagtcaa tgatcacctt atcagcaaga aagattttgc 300
gatgacctat cagaaaatta cggagcatct gcctgctgac cttctgcctg atgatattac 360
aatctttgag tggtggacgt taatcatgct tcaatacttt gcggatcaaa aggttgactg 420
ggcggtgatt gaatgtggtc ttggcggcca agacgatgcg acaaacatca tctcagcgcc 480
gttcatttca gtcattaccc atatcgctct tgaccacacc cgtatcctgg gccctacaat 540
tgcgaagatt gcgcaagcta aggcaggcat tataaagaca gggactaaac aggttttcct 600
ggcaccacat caagagaagg atgcgttaac aatcattcgc gaaaaagcgc aacagcaaaa 660
ggtcggactg acgcaggcag atgcacagag cattgtggac ggaaaagcta ttttaaaagt 720
gaatcacaag atttacaagg tcccttttaa tctgctgggc acatttcagt cagaaaacct 780
gggaacggtt gttagcgtct ttaactttct gtatcagcgc cgtcttgtca cgtcatggca 840
accgttactt agcacactgg caacagttaa aattgcagga agaatgcaaa aaatcgcgga 900
tcatcctccg atcattcttg atggcgcaca taatccggat gctgcaaagc agcttacaaa 960
gacaattagc aaactcccac ataataaagt cataatggtg ttaggcttcc ttgctgacaa 1020
aaacatttca cagatggtca agatttacca acagatggcg gatgaaatta tcattacaac 1080
gcctgaccat cctacaagag cgctggacgc ctcagccctt aaatcagtct taccgcaagc 1140
aattattgcg aataatcctc gtcagggact ggttgttgct aagaaaattg cagagccgaa 1200
cgatcttatc atcgtcacgg gcagcttcta cacaatcaag gatattgagg caaatttaga 1260
tgagaaataa gcagaggctg tgatcagtct ctgctttttt ttctgcgttc tatttctttt 1320
tcacgttcac ggatgacgtc agtccgatcc cgcaaacggt gtttgtcgat aagaaatatg 1380
aattcgcgtg cgcattgga 1399
<210> 25
<211> 1360
<212> DNA
<213> Ashbya gossypii
<400> 25
ttttactagt atggagttag gcttaggccg catcacacaa gtgctgagac aattacatag 60
ccctcatgaa agaatgcgtg tcttacatgt tgcaggaaca aatggcaaag gaagcgtctg 120
tgcgtattta gcggctgttt taagagcggg cggagaaaga gttggcagat ttacaagccc 180
tcacttagtt catccgcgcg atgctatcac agtcgacggc gaagttattg gagcggcgac 240
atatgctgca cttaaagctg aagtcgttgc ggcaggcaca tgcacggagt ttgaagcaca 300
aacggcggtt gcgcttacgc attttgcaag acttgaatgc acatggtgtg tcgtcgaagt 360
gggcgtcggc ggcagattag acgctacaaa tgtcgtccct ggcggacgca aactgtgtgc 420
aattacaaag gttggattag atcatcaggc gttacttggc ggaacactgg ctgttgttgc 480
aagagagaag gccggcattg tggttccggg agtgcgcttt gtcgctgtcg acggcacgaa 540
cgcaccttca gttctggcgg aggttcgggc ggctgcagcg aaagttggcg cagaggtcca 600
tgagacagga ggcgcgccgg tttgcacagt cagctggggt gcggttgctg caagcgcact 660
tccgttagcg ggagcttacc aggtacaaaa cgcgggcgtt gcacttgcac tgcttgatca 720
tcttcaacaa ctgggagaga tctcagtcag ccatgcagca ctggaaagag gactgaaagc 780
agtcgaatgg cctggcagac ttcaacaagt tgagtatgac cttggaggcg tccatgtccc 840
gctgttattt gacggagcac acaatccgtg tgcagcggaa gagcttgcaa gattcttaaa 900
cgagagatac cgcggaccgg gaggatctcc gctgatctat gtgctggctg tcacgtgtgg 960
caaagagatc gacgcacttc ttgcacctct tctgaaaccg cacgatagag tcttcgcaac 1020
cagctttggc gcggttgagt ctatgccgtg ggtcgcagcg atggcaagcg aggatgtcgc 1080
agcggcggcg agacgctaca cagcccacgt ttcagcggtt gcggacccgc tggacgcgtt 1140
acgcgccgca gcggcagcac gcggcgatgc taatctggtc gtctgcggat cattatatct 1200
tgtcggcgaa cttctgcgcc gcgaacatta agcagaggct gtgatcagtc tctgcttttt 1260
tttctgcgtt ctatttcttt ttcacgttca cggatgacgt cagtccgatc ccgcaaacgg 1320
tgtttgtcga taagaaatat gaattcgcgt gcgcattgga 1360
<210> 26
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 26
gcagcgaaat cagcatcacc 20
<210> 27
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 27
gactcgttag ccaggtcgtc 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 28
tcgataaaag aagccccgcc 20
<210> 29
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 29
ggtttccatg agggtcggtc 20
<210> 30
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 30
gctacctggc gcaaaaagaa 20
<210> 31
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 31
cggtagtcat tgctggcgaa 20
<210> 32
<211> 19
<212> DNA
<213> 人工序列(artificial sequence)
<400> 32
acgacctggc taacgagtc 19
<210> 33
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 33
ggcggggctt cttttatcga 20
<210> 34
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 34
gaccgaccct catggaaacc 20
<210> 35
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 35
ttctttttgc gccaggtagc 20
<210> 36
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 36
ggagaatccc aacgaagcca 20
<210> 37
<211> 152
<212> DNA
<213> 人工序列(artificial sequence)
<400> 37
gcatcactat ctgcagtaaa atcggaattc aattttgtca aaataatttt attgacaacg 60
tcttattaac gttgatataa tttaaatttt atttgacaaa aatgggctcg tgttgtacaa 120
taaatgttac tagagtaaag gaggaaacta gt 152
<210> 38
<211> 228
<212> DNA
<213> 人工序列(artificial sequence)
<400> 38
gtgcgcatga tcgtatggtt cactgtccac caaccaaaac tgtgctcagt accgccaata 60
tttctccctt ggggggtaca aagaggtgtc cctagaagag atccacgctg tgtaaaaatt 120
ttacaaaaag gtattgactt tccctacagg gtgtgtaata atttaattac aggcgggggc 180
aaccccgctc agtacctaga gcgtaaaaga ggggagggaa acactagt 228
<210> 39
<211> 25
<212> DNA
<213> 人工序列(artificial sequence)
<400> 39
atctacattc cctttagtaa cgtgt 25
<210> 40
<211> 32
<212> DNA
<213> 人工序列(artificial sequence)
<400> 40
aaatctagaa attaagaagg agggattcgt ca 32
<210> 41
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<400> 41
aaaggatcca tctacattcc ctttagtaac gtgt 34
<210> 42
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 42
tcccggcaac agcttaatca 20
<210> 43
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 43
ggagccgatt ctctgcgtta 20
<210> 44
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 44
aaaatgctcc ctgcggctat 20
<210> 45
<211> 21
<212> DNA
<213> 人工序列(artificial sequence)
<400> 45
caatgagagg ggttgctatg a 21
<210> 46
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 46
tcgaacggtc aagcacgtta 20
<210> 47
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<400> 47
tttgctagca tgataattgg aatatgggca gaag 34
<210> 48
<211> 34
<212> DNA
<213> 人工序列(artificial sequence)
<400> 48
tttgcggccg ccctcctcgt catttcttca aaag 34
<210> 49
<211> 6975
<212> DNA
<213> Lactococcus lactis
<400> 49
atgataattg gaatatgggc agaagatgaa gcaggtctta tcggtgaagc tgataaaatg 60
ccttggtctt tacctgctga acaacaacat tttaaagaaa caaccatgaa tcaagtgatt 120
ttgatgggac gaaaaacgtt tgaaggcatg aataaacgtg tattgccagg gagaataagt 180
attattttaa ctcgcgatga aacttatcaa tcagataatg aaaaagtgct catcatgcac 240
agccctaagg aagttctaga ttggtaccat aagcaaaata aagacttatt tatcacagga 300
ggagctgaaa ttttagccct ttttgaatct gaacttgaat tgctctatcg aacagttgtt 360
catgaaaaat ttaaaggaga tacttatttt ccaagtacat ttgactttgg aagatttaag 420
ctagtctctg aaaaatttca cgataaagat gagcggaatt cttatacttt tacaattaaa 480
aaatatgaaa aagtgaaaca accatgacaa aatcaatttt tgggcttttc acagctctcc 540
tttgttggat tagcattgtc atcgctattc aatgctttag aaaaaaacgt tggggtctgg 600
gagtattgtt tttactcaat gcttttacga acctcgtaaa tacaattcac gctttttctg 660
gaactttatt ttaaaaaata aaaaaagtgc cttttaagta agccaataac acttactttt 720
tatgttagtg aaatcaggaa taaaataact atgtcaaata cacaaaatcc aaatatacat 780
tgttctttct gtggaaagag tcaagatgat gtaaaaaaat tgattgccgg ttcagacgtt 840
tatatttgta atgaatgtat tgaactttca actcgaatct tagaagaaga attaagagaa 900
gaacaagatt cagaaatgct tgaagttaaa acacctaaag aaatgtttga ccatttaaat 960
gaatatgtga taggtcaaga aaaagcaaaa cgtgcacttg cagttgccgt ttataatcat 1020
tacaaacgaa ttaattttgc agcaagtaaa attgctgaag atattgaact acaaaaatca 1080
aatattctat taatcggacc taccggttct ggtaagactt ttctcgctca aactttagcg 1140
aaatcactca acgttccatt tgcgattgca gatgcgacaa gtttaactga agctggttat 1200
gttggagaag acgttgaaaa tattctctta aaacttttac aagcgagtga tttcaatatt 1260
gaacgtgctg aacgtggaat tatctatatc gatgaaattg ataaaattgc taaaaaatct 1320
gaaaatgtat caattactcg tgacgtttcc ggggaaggtg ttcaacaagc ccttttgaaa 1380
attattgaag gaacggtagc tagtgttcca ccacaaggtg gacgtaaaca tcctaatcaa 1440
gaaatgattc aaattgatac caaaaatatc ttatttattg ttggtggagc ttttgacggg 1500
attgaagaaa ttgtcaaaca acgtttaggt gaaaaaatta ttggttttgg tgccaataat 1560
aaaaaattaa atgacgatga ttcttatatg caagaaatta ttgccgagga cattcaaaaa 1620
ttcggattaa ttcctgaatt tattggtcgt ctgccaattg ttgctgcttt ggaacgtttg 1680
accgaagagg atttgattca aattttgaca gaacctaaaa atgctttgat taaacaatat 1740
aaacaactcc ttttatttga taatgttgaa cttgaatttg aagatgaagc cctcatggca 1800
attgctagaa aagcaattga gcgcaaaaca ggagcgcgtg gacttcgttc aattattgaa 1860
gaagtaatga tggatatcat gtttgaagtt ccaagtcatg aagaaattac aaaagttatt 1920
attaatgaag cagttgttga cggaaaagct gagccacaaa tgattcgaga ggccaagaaa 1980
aaatgaccat aaatacaaat aatctgacaa taacaatttc agcagcatca aaaaaacaat 2040
atccagaaaa tgattggcca gaaattgcct tagctgggcg ttcaaatgtc ggtaaatcaa 2100
gttttattaa tactttactt aatcgtaaaa actttgccag aacttctggt caacctggta 2160
aaacacagtt gctcaatttt tataatattg atgatcaact tcatttcgtt gacgtacctg 2220
gttacggcta cgctcgtgtt tctaaaaagg aacgcgaaaa atggggtaaa atgattgagg 2280
aatatttgac aacaagagaa aatttaaaag cagttgtcag cttagttgat attcgtcatg 2340
aaccctcaga agatgatttg atgatgtatg agtttttgaa atactaccat attccagtga 2400
ttttagttgc gaccaaagcc gataaagttc cacgtggtaa gtggaataaa catgaatcta 2460
ttatcaaaaa agcaatgaaa tttgatagta cagatgattt tattatcttt tcttctactg 2520
ataagacagg atttgaagaa gcttgggaag cgattttaag atatctctga aaatagtgct 2580
atgaagagat tcatagcctt ttctacactt aaaaagagga aatatgtaca aaataaaact 2640
taataatata aaatttaggg cacatattgg tgttctgcca gaagaaaaag ttctcggaca 2700
aaatctcgaa attgatttaa tcgtggaaac aaattttgat ttttcaggaa aagacgaatt 2760
agatgaaact ttgtcttatg ttgatttcta tgaggcaaca aaagcagttg tagaatcttc 2820
aaaagctgat ttaattgaac atgttgcctt tgaaattatt caagcagtaa aggctacttc 2880
agagcgtata tcaacggttg aagtccatct tagaaaatta gccgtaccga ttgaaggaat 2940
ttttgattca gctgaaattg agatgagagg ctaaagctgg tttttaagat aaatatttta 3000
aagagataga agagaaacaa aatcataaaa gattatgtct aaatggagga cttatgcaaa 3060
caacttactt aagcatggga agtaatattg gtgaccgtca gtattattta catgaagcca 3120
ttcgtttatt gggaaaacac cctaaaatta tgattgaaaa agtatcaaat ttttatgaaa 3180
gtactccagt cggcggcgtc aaacaagatg attttactaa tttggcatta aaggtggcaa 3240
cgctacttga acctttggaa ttattatctt ttattcatga agttgagtta tctttgaacc 3300
gtgagcgaaa aattcattgg gggccaagaa caattgatat tgatattatt ttctatgacg 3360
acttagaaat gcaagtagaa aacttggtta ttccacataa agaagctttt aatcgtcttt 3420
ttgtcttgaa acctattttt gaacttattg ataaagactt taaatattat gcgtcaatag 3480
aaaaagcaat agccgaactt tcagtaagtg aacaagagct ccatgtaata aaagaagaaa 3540
aaacaccgag aaatcgtatt gaagatgccg ttaaagagat tctctttgca gtaggtgaaa 3600
atccaaatcg agaaggatta cttgaaactc cagcaagagt agctaaaatg tatgaagaaa 3660
ttctttcgtc acaacgctta agcaagttta atgagtataa actttttgaa attgattctt 3720
ctaaaacgga ttcaatcgtg ttgattaaag atattccttt ttattcaatg tgtgagcatc 3780
atatgttacc attttttggg aaagctcatg ttgcatatat tccagctgat ggaaaaatta 3840
ttggcttgtc aaaaattccc cgtttagttg attatgtttc gcgcaaactc tcggttcaag 3900
aaaatatcac tcatgatatt ggagatattt tgactgatat tttgaatcct aaaggagtgg 3960
cagttcttgt tgaaggacgt catatgtgcg ttgaaatgcg tggagtaaaa aaagtaaatt 4020
ctattactaa aacttcttat tttttaggtg aatttaaaga aaataatgaa aaaagaatgg 4080
aatttttaga aagtctttta tgaaaatctt agaacttaat caagaatctt tttctcttaa 4140
aaatattatc ctaaaatttg atgagttaaa tcacaatgaa atgatttctc ttcaaaaaaa 4200
actttatcga aatggtagtt tgacaagact ggctccagac tccttgttag tagttttaac 4260
aattgatgac ttagcaaaat tgattaatct ttttgaaaat gatgaagata aaaaaatgct 4320
tgaagtgatt tataagcgtc atcaaatcat ttggtcaggt aaaaatttca attttgattt 4380
aactagaaag tcaattgtct attcaatcgt caatgttaca ccagactctt tttatgatgg 4440
aaatccagat aatttaaacc tctctcatat tttaaaaaga gtagaagctg atttagaaaa 4500
tggagcttct gttcttgagc tgggagggaa atcatcgaaa ccaggatatg acgatattag 4560
cccagaagag gaatggaaca gactgaaaga acctattctt gagttgaaaa aaaactttcc 4620
taaagcgatt tttgctgtcg atacggatga agcttatgtc atggaacgag ttttagacgc 4680
tggggttgat attattaacg atattgatgg ttttgataca aatgataaat taaaagtggt 4740
agaaaagtat caaccggctt tagttgctat gaataatggg cgagctggtt ttagttatgc 4800
tgataatgtt tatgaagaac ttccattatt ttttgaaaat aaaaaagaag agttacttca 4860
acttggttta aaagctgagc aaatcgttat tgatcctgga gttggttttt ttaatggaga 4920
ttcaggttca gatagtcttg agcgggttaa agcaactgaa attttaagca gaataggttt 4980
acctcttatg attgcaatct ctcgtaagtc atttatggga aaactcttca atgcccaagg 5040
agatgagcgg cttttttcaa gccttgtcct agaagcgcaa atggttgctg atgggggacg 5100
gattttgcgt gttcatgatg ttaaggagac taaacgttta ctcgatgcaa ttgaaattta 5160
taaggaattt taaaaatgaa tgaagaccta attgctgaaa ttcaagcttt atctgctatt 5220
ggaagtgaag aaaaattttc cgagattatt cgattattga aaaattcgac tttagagctt 5280
cgggggaaaa agaatccaga tttacaattg tcagcaagtg cattagtttt taaaaaacat 5340
aaactatttt ttattgaaca cccttatcaa aaggagcttt tgcttccagc aggtcatgtt 5400
gaactaggag aaaagccatt ggaaactgcg attcgtgagt tccatgaaga aacaggtttt 5460
tcagcgtcag aatcaggcaa gttggtagat gttaacttga ttaatattcc ttacaacaaa 5520
attaagaatg agaaagaaca tcaacacatt gattttcgtt ttctattgga actaaaagaa 5580
aaagaagcag gccttgctga attgcctttt ttccttcttg atagaactga agctcctgat 5640
gaatttaaaa aatattatca atacaaaaga taaagtagaa aaggtcacaa aatgtctata 5700
gaagaagcat tggaatggat acattcacgt ttaaaattta atattcgccc aggcctaagt 5760
cgtgtttcgg cccttttaga attgcttggt catccagaag agtctttgtc aatgattcac 5820
gttgctggaa caaatggaaa aggctccaca gtcgctttca cacgctcaat ctttatgcag 5880
gcaggtctga aggttgcttc tttcacaagt cctttcatca ccacttttgg tgagcggatg 5940
tcgattaatg cactcccgat tgctgatgat aaattaattt attatgtaga aatgatccaa 6000
ccacttgttg ctgaacttga taaagatgct gaactgactg gaattaccga atttgaaatt 6060
atcacggcaa tggcttttaa atatttctct gatgagcagg ttgatttagc ggttattgaa 6120
gttggtttag gtggacttct tgattcaaca aatgtgatta aacctgttgt ttctggaatt 6180
acaacaattg gtttagatca tattgatatt cttggttcga ccattgaaga aatcgcagct 6240
caaaaggctg gaattattaa accaggaatt ccagtagttg ttggaaatat tgaattaaaa 6300
gcacttcggg ttatatggga agtggctaga aaaaatacag cgcgtgttta tcaatttcca 6360
tatgattatc gtacggaagt ggaagaacac gaacatttta atttcttttc tggtcaagaa 6420
gcaatattgg atattgaaaa atctttagtt ggcttacatc aaatagaaaa tgctggtatg 6480
gctattgaac tttctctggt ttatgcaagt aaggttggga ttgaattgac tgaggatgtg 6540
attcgctctg gaattcgtga ggctttttgg ccagctcgta tggaaaaatt gggtgaaaaa 6600
ccactcattt tactggatgg tgctcataat gttcatgcga tgaatcgttt gcttgaaaat 6660
cttagctctg agtttccaga taaaaaaatt acaatcattt tttcagccat taccacaaaa 6720
gatattagtc aaatgataaa aatgcttcaa actgtgaaaa attcgcatct gattttgaca 6780
acttttgatt atccaaaagc tttgaatttg ggagattttc aaagattgga agaagaaggg 6840
gttgaattgg ctccaagttg ggaattagct ttagttcgtg cgcaaaaaaa tttagctgaa 6900
gatgatttgt tattagttac aggctctctc tatttctcat ctcaagttcg tgagtttttg 6960
aaaaaagaga agtaa 6975
<210> 50
<211> 2463
<212> DNA
<213> Ashbya gossypii
<400> 50
atgcagtccc ttggattcaa gtgtttgctg tctcgcagga gcctgagcag gatatcaatc 60
tgtacaagag gaatgagtag tgctaacggt ggacgaagta atgatactgt gcatatacag 120
agacaggcac tgaaagttgt tgctgggctt gacggatggg gtcaattgca ggcgcaggat 180
gtgaaattga ccatgaatat gaacacagat tttcgtgctt cctcgcagac ggatgatctg 240
aagtactcct tgaattatgc ggtgatttca cgtggggtgc ataggttcgt tgagggctgt 300
ggacggtacc gctctcttgg tcacttggcc agggaggtaa agaagttttc catgaatgag 360
tatccgggta tccaaactat agaggtgggt gcggaggcgg acgcggccca tttgcgatgc 420
ggaagtctgg gcgtcgtggt gaacagcgat gggcatcgtc ctgatgagat tttgctttct 480
ggaatgaagc ttctgacact aataggggtg ttcacttttg aacggcgtcg gaagcagtac 540
gttgacttga agctgtcatt tccgtggccg aaggaggctg gtgaatttcc ggattgccag 600
gaattattgg acgatgttgt gagctatgta gagagagcga attttaaaac ggcagagtct 660
cttgctgaga gtgtagctca cgttgttacc ttgagagagt attttcagct gcatcgtggg 720
ttaccggtaa aagtcaaggt aattaagctt aatgccatta ctgagactga gggagttggt 780
gtgagctgtg taagaagtgc ggatgaattt acggggaaac cgcccttctg ggaagatatt 840
ccaaacgatc gagcagacgt gtttaacctt cctgtattcc agcagccaca tgcatctgtc 900
agtgagtgga atcgtgtgtt tctggcgttt ggatctaata taggggatag gtttgctcac 960
attgagcgaa gcttacgtct acttgcggaa gatcctaaag ttaaactact tcgctcgtcg 1020
tctctgttcg agagtgaacc aatgtacttt aaggagcagt ccccgtttat gaatggcgtt 1080
gtagaagtgc agacacggta tagcccgcac gagttactag agctatgcaa aaggatagaa 1140
tatgaacatt taaaacgtgt caaagagttt gataacggcc ctcgcagcat tgatttagat 1200
attttattgt accaaaatgc aaactttgag catgtggtac tgaactccga ggatttagtt 1260
attcctcatc caaggatgtt ggagagatcg tttgttttag agcctctctg tgaattgttg 1320
gctttccatg aagtgcaccc catttcggct gaatctgtcc aaagtcacct aaaagaattg 1380
taccgtaagg ggaataagga agacattctt gttaaacttg tacctttgcc gggtattccg 1440
tcaaatatac ctacaacgcg atttctgaag tttagacggg agtatgagga ggatcaatcg 1500
acaagcgaat tggttcttag gaccaagtca aatacatatg tcatgggcat cgtgaatgtg 1560
acacctgatt ctttttctga tggatctcct atgtggaatg atgttaatca tttcctctta 1620
aaagtacaaa ggatgatcct tgacgttttg aagttacatg aaaacgttat cattgatatt 1680
ggaggctgtt cgactaggcc tggtagtcag caaccatcag tggaagaaga acttagtcgt 1740
actattcccc taataacagc gatcaggggt tgcagagatt tttcgcaaga gaatgtgatc 1800
atatctatag acacttacag aagtgctgtt gctgaaaagg ccataacagc aggggctgat 1860
attgtgaacg atatttcagg aggtagtttt gatacaaata tgtttaaggt tatcagcgcg 1920
tatccgaatg ttggttatgt gctatcacac ataaggggag atatgactac catgacgagc 1980
ctgaataagt atgatgatac agttggtttg gatggcgttg aagaattcat ttacggtaag 2040
aaacagcact cagaacggac taaggtgatc cggaacattt gtagggaact tgcggagcga 2100
taccagcttg cccttgctag cggaattaag cgctggcaga ttattttgga tccgggtatt 2160
ggttttgcga agaatgctaa acagaactta gatatcatca agcatacccc gtcaattaag 2220
ggttatagtt gtgtgacaca tggacaattt gtaaattttg ccaaccttcc tgtgttgctt 2280
gggccttcca ggaagaactt tattgggact ataattcaag aggcacaggt cgagcgaagg 2340
gactttgcaa cggggactat tgtaggctcc tgtgttggtt atgatgcgga tatcatcagg 2400
gtacatgatg taactaactg tagcaaaagt gctaggttag cggatgagct ttataggaaa 2460
tag 2463
<210> 51
<211> 732
<212> DNA
<213> Ashbya gossypii
<400> 51
atgtgtcagg ggggcagtaa aggactagtt aggcaggaca cgcccctaaa gacgaggcct 60
gtctcgccat atacgctcca ggccccagtt gaggcggacg gactgtcctg gccgagtgca 120
ggggcacgtg tgcgggtcga ggagggcacg gaggaagagg cggcacgcgc agcccggata 180
gctgatgcag tcaagacgat tttgacggag ctgggcgaag acgtgacgcg ggagggcctg 240
ctggacaccc cgcaacggta cgccaaagcg atgctgtact tcaccaaggg ctaccaagac 300
aatattttga acgatgtgat caataatgct gtgtttgacg aagatcatga cgagatggta 360
attgtgcggg atattgagat ccattcgctg tgcgagcacc acctggtacc cttcttcggg 420
aaggtgcata ttggctacat acctcggagg agagtcctcg ggttgtcgaa gctcgcccgg 480
ctagcggaaa tgtacgcgcg caggctgcag gtgcaggagc ggctgacgaa gcagattgcg 540
atggcattgc aggatatact gcgccctaga ggagtagccg ttgtggtgga ggccacgcat 600
atgtgcatgg tgtcacgggg ggtccagaag tccgggtcct caactgtcac ctcgtgtatg 660
ctgggctgct tcagagacat gcacaagacc cgggaagaat tcttgaacct cttgagaaat 720
agaagtgtat ag 732
<210> 52
<211> 2484
<212> DNA
<213> 人工序列(artificial sequence)
<400> 52
ttttactagt atgcaatcac tgggctttaa atgtcttctg agcagaagaa gcctgagccg 60
cattagcatc tgcacgagag gaatgagctc agcgaatgga ggaagaagca atgatacagt 120
tcatattcag cgccaggcac ttaaggtcgt tgcgggcctt gatggctggg gacagctgca 180
ggcgcaagac gttaagctga caatgaacat gaacacagac tttcgtgcgt caagccaaac 240
agatgacctt aaatacagcc ttaattacgc tgtgattagc cgtggagtcc accgttttgt 300
cgagggatgc ggaagatacc gtagcctggg acatctggcg agagaggtca aaaagttttc 360
aatgaatgag taccctggca ttcagaccat tgaggtgggt gccgaggcgg acgcggcaca 420
cctgagatgc ggatctttag gcgttgttgt gaatagcgat ggacacagac ctgatgagat 480
cttattgtca ggcatgaaac ttctgacgct gattggagtc tttacattcg agcgtcgcag 540
aaagcaatac gtcgatctga aactgagctt cccgtggcct aaagaagcag gagagttccc 600
ggattgtcag gaacttctgg atgacgttgt gagctacgtc gagagagcga acttcaaaac 660
ggcagagtct ctggcggagt ctgtggcaca cgtggtcaca cttcgcgaat attttcaact 720
tcatcgtggc ttgcctgtca aagtgaaagt cattaagctg aacgcgatca cagaaacgga 780
gggcgtcgga gttagctgtg tcagatctgc cgatgaattt acaggcaagc ctccattttg 840
ggaagacatc ccgaacgata gagcggacgt ctttaattta cctgtgttcc agcaacctca 900
tgcaagcgtt tcagagtgga atagagtgtt tctggcgttt ggctccaaca ttggagatag 960
attcgcgcat atcgagagat ctttacgtct gcttgctgaa gatcctaaag tcaaactgct 1020
tagaagcagc agccttttcg aatctgagcc tatgtatttc aaggagcagt ccccgtttat 1080
gaacggagtc gttgaggtcc aaacgagata ttcaccgcat gaacttttag agttgtgcaa 1140
acgtatcgaa tatgaacacc tgaaacgtgt taaagagttt gataatggcc cgcgttcaat 1200
tgacctggat atcttactgt accagaacgc gaactttgag catgtggtcc ttaattccga 1260
agacctggta attccgcatc ctagaatgct ggaacgcagc ttcgtgctgg agcctttatg 1320
cgagctgctt gcgtttcacg aggttcaccc tatatcagcc gagtcagtgc agagccatct 1380
gaaagaatta tacagaaaag gcaataaaga ggacatttta gtcaagttag tccctctgcc 1440
tggaatccct tctaatattc cgacgacgag atttcttaaa tttagacgcg aatatgaaga 1500
ggaccagtct acatcagaat tagtcctgcg tacgaaaagc aacacatacg ttatgggaat 1560
tgtcaatgtt acgcctgact catttagcga cggctcacct atgtggaacg acgtcaatca 1620
tttccttctg aaggtgcaac gcatgatcct ggatgtcctg aaactgcatg agaatgtcat 1680
tattgatatc ggaggctgct ctacaagacc tggctctcag caaccgagcg ttgaagaaga 1740
gttatcacgc acgattcctc ttattacagc tattcgcggc tgcagagatt tttcacaaga 1800
gaatgttatt atctcaattg acacataccg gtcagcggtc gctgagaaag caattacggc 1860
aggagcggat attgttaatg atatttctgg cggatctttc gatacaaata tgtttaaagt 1920
tatttcagcg tatcctaatg tcggctacgt tctgtcccat atccgtggcg atatgacaac 1980
gatgacgtca ctgaacaaat atgatgacac agtcggctta gatggcgttg aggaatttat 2040
ctatggcaaa aaacaacatt cagaacgtac aaaagtcatc cgtaacatct gtcgcgaact 2100
tgcagaacgc taccagcttg cacttgcttc aggcattaaa cgctggcaaa ttatccttga 2160
tcctggcatt ggcttcgcta aaaatgctaa acaaaacctg gatattatta aacacacgcc 2220
gagcattaaa ggatactcat gtgtgacgca tggacaattt gtgaatttcg cgaatttacc 2280
ggtactgctg ggcccgtctc gcaagaattt catcggcaca attattcagg aggcgcaagt 2340
agaacgcaga gatttcgcaa caggcacgat tgtgggctca tgtgtcggct atgacgctga 2400
tattatccgc gttcacgatg tcacgaattg tagcaagagt gcacgcctgg cggatgaact 2460
gtatcgcaaa taaggatcca tttt 2484
<210> 53
<211> 1090
<212> DNA
<213> 人工序列(artificial sequence)
<400> 53
tattggatcc tatggttcac tgtccaccaa ccaaaactgt gctcagtacc gccaatattt 60
ctcccttggg gggtacaaag aggtgtccct agaagagatc cacgctgtgt aaaaatttta 120
caaaaaggta ttgactttcc ctacagggtg tgtaataatt taattacagg cgggggcaac 180
cccgctcagt acctagagcg taaaagaggg gagggaaaca ctagtatgtg tcaaggcgga 240
agcaaaggac tggttagaca agacacaccg ctgaaaacaa gacctgtctc accttataca 300
ctgcaagcac ctgtcgaagc agacggatta agctggccga gcgcgggcgc gagagttaga 360
gtggaagagg gaacggagga agaagcagcg cgcgcggcta gaattgcgga tgcagtcaaa 420
acaatattaa cagagctggg cgaagacgtg acaagagaag gtcttctgga cacaccgcag 480
cggtatgcga aagctatgct gtactttacg aagggatacc aagacaacat cctgaacgat 540
gtcattaaca atgcggtttt tgacgaggat catgatgaga tggttatcgt tcgcgacata 600
gagatacaca gcctgtgtga gcatcacctg gtcccatttt tcggcaaggt ccacataggc 660
tacattccga gaagacgtgt cctgggactt tctaaactgg cgcgcttagc tgaaatgtac 720
gcacgcagac tccaggtcca agaacgttta accaaacaga tcgcaatggc actgcaagat 780
atccttcgcc ctagaggcgt ggcagtcgtt gttgaggcta cgcacatgtg catggtctct 840
cgcggagtgc aaaagagcgg atcatcaacg gtaacatcat gtatgctggg atgtttcaga 900
gacatgcaca agacgagaga ggaatttctt aatttactta gaaacagaag cgtttaagca 960
gaggctgtga tcagtctctg cttttttttc tgcgttctat ttctttttca cgttcacgga 1020
tgacgtcagt ccgatcccgc aaacggtgtt tgtcgataag aaatattacg taatatggcc 1080
tcgagtttta 1090
<210> 54
<211> 25
<212> DNA
<213> 人工序列(artificial sequence)
<400> 54
tattggatcc tatggttcac tgtcc 25
<210> 55
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 55
gcggtagtgg tgcttacgat 20
<210> 56
<211> 23
<212> DNA
<213> 人工序列(artificial sequence)
<400> 56
tgcagggtct ttattcttca act 23
<210> 57
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 57
gcggtagtgg tgcttacgat 20
<210> 58
<211> 1038
<212> DNA
<213> 人工序列(artificial sequence)
<400> 58
tctagaaatt aagaaggagg gattcgtcat gttggtattc caaatgcgtt atgtagataa 60
aacatctact gttttgaaac agactaaaaa cagtgattac gcagataaat aaatacgtta 120
gattaattcc taccagtgac taatcttatg actttttaaa cagataacta aaattacaaa 180
caaatcgttt aacttctgta tttgtttata gatgtaatca cttcaggagt gattacatga 240
acaaaaatat aaaatattct caaaactttt taacgagtga aaaagtactc aaccaaataa 300
taaaacaatt gaatttaaaa gaaaccgata ccgtttacga aattggaaca ggtaaagggc 360
atttaacgac gaaactggct aaaataagta aacaggtaac gtctattgaa ttagacagtc 420
atctattcaa cttatcgtca gaaaaattaa aactgaacat tcgtgtcact ttaattcacc 480
aagatattct acagtttcaa ttccctaaca aacagaggta taaaattgtt gggaatattc 540
cttaccattt aagcacacaa attattaaaa aagtggtttt tgaaagccat gcgtctgaca 600
tctatctgat tgttgaagaa ggattctaca agcgtacctt ggatattcac cgaacactag 660
ggttgctctt gcacactcaa gtctcgattc agcaattgct taagctgcca gcggaatgct 720
ttcatcctaa accaaaagta aacagtgtct taataaaact tacccgccat accacagatg 780
ttccagataa atattggaag ctatatacgt actttgtttc aaaatgggtc aatcgagaat 840
atcgtcaact gtttactaaa aatcagtttc atcaagcaat gaaacacgcc aaagtaaaca 900
atttaagtac cgttacttat gagcaagtat tgtctatttt taatagttat ctattattta 960
acgggaggaa ataattctat gagtcgcttt tgtaaatttg gaaagttaca cgttactaaa 1020
gggaatgtag atggatcc 1038
<210> 59
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 59
taggaggcga gagcacaaga 20
<210> 60
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 60
gccgagttcc tttgtgatgc 20
<210> 61
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 61
gcccgagaac agcggattta 20
<210> 62
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 62
cgcaagaaca aacaggcgtt 20
<210> 63
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 63
tggcgttatg gttgtcgttg 20
<210> 64
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 64
taaacacgcc tctgactgct 20
<210> 65
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 65
ggcggagcgc aattatacac 20
<210> 66
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 66
caggaaagtg tctgtcgcct 20
<210> 67
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 67
gattggccgc ttacacatgg 20
<210> 68
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 68
aacgtttggg cttctaccga 20
<210> 69
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 69
cagctcgtgt cgtgagatgt 20
<210> 70
<211> 20
<212> DNA
<213> 人工序列(artificial sequence)
<400> 70
agagtgccca actgaatgct 20
<210> 71
<211> 21
<212> DNA
<213> 人工序列(artificial sequence)
<400> 71
gccctgcata aggaatttaa c 21
<210> 72
<211> 23
<212> DNA
<213> 人工序列(artificial sequence)
<400> 72
agcttatgga catacgactg atg 23
<210> 73
<211> 406
<212> PRT
<213> Ashbya gossypii
<400> 73
Met Glu Leu Gly Leu Gly Arg Ile Thr Gln Val Leu Arg Gln Leu His
1 5 10 15
Ser Pro His Glu Arg Met Arg Val Leu His Val Ala Gly Thr Asn Gly
20 25 30
Lys Gly Ser Val Cys Ala Tyr Leu Ala Ala Val Leu Arg Ala Gly Gly
35 40 45
Glu Arg Val Gly Arg Phe Thr Ser Pro His Leu Val His Pro Arg Asp
50 55 60
Ala Ile Thr Val Asp Gly Glu Val Ile Gly Ala Ala Thr Tyr Ala Ala
65 70 75 80
Leu Lys Ala Glu Val Val Ala Ala Gly Thr Cys Thr Glu Phe Glu Ala
85 90 95
Gln Thr Ala Val Ala Leu Thr His Phe Ala Arg Leu Glu Cys Thr Trp
100 105 110
Cys Val Val Glu Val Gly Val Gly Gly Arg Leu Asp Ala Thr Asn Val
115 120 125
Val Pro Gly Gly Arg Lys Leu Cys Ala Ile Thr Lys Val Gly Leu Asp
130 135 140
His Gln Ala Leu Leu Gly Gly Thr Leu Ala Val Val Ala Arg Glu Lys
145 150 155 160
Ala Gly Ile Val Val Pro Gly Val Arg Phe Val Ala Val Asp Gly Thr
165 170 175
Asn Ala Pro Ser Val Leu Ala Glu Val Arg Ala Ala Ala Ala Lys Val
180 185 190
Gly Ala Glu Val His Glu Thr Gly Gly Ala Pro Val Cys Thr Val Ser
195 200 205
Trp Gly Ala Val Ala Ala Ser Ala Leu Pro Leu Ala Gly Ala Tyr Gln
210 215 220
Val Gln Asn Ala Gly Val Ala Leu Ala Leu Leu Asp His Leu Gln Gln
225 230 235 240
Leu Gly Glu Ile Ser Val Ser His Ala Ala Leu Glu Arg Gly Leu Lys
245 250 255
Ala Val Glu Trp Pro Gly Arg Leu Gln Gln Val Glu Tyr Asp Leu Gly
260 265 270
Gly Val His Val Pro Leu Leu Phe Asp Gly Ala His Asn Pro Cys Ala
275 280 285
Ala Glu Glu Leu Ala Arg Phe Leu Asn Glu Arg Tyr Arg Gly Pro Gly
290 295 300
Gly Ser Pro Leu Ile Tyr Val Leu Ala Val Thr Cys Gly Lys Glu Ile
305 310 315 320
Asp Ala Leu Leu Ala Pro Leu Leu Lys Pro His Asp Arg Val Phe Ala
325 330 335
Thr Ser Phe Gly Ala Val Glu Ser Met Pro Trp Val Ala Ala Met Ala
340 345 350
Ser Glu Asp Val Ala Ala Ala Ala Arg Arg Tyr Thr Ala His Val Ser
355 360 365
Ala Val Ala Asp Pro Leu Asp Ala Leu Arg Ala Ala Ala Ala Ala Arg
370 375 380
Gly Asp Ala Asn Leu Val Val Cys Gly Ser Leu Tyr Leu Val Gly Glu
385 390 395 400
Leu Leu Arg Arg Glu His
405
<210> 74
<211> 1221
<212> DNA
<213> Ashbya gossypii
<400> 74
atggagttag gcttaggccg catcacacaa gtgctgagac aattacatag ccctcatgaa 60
agaatgcgtg tcttacatgt tgcaggaaca aatggcaaag gaagcgtctg tgcgtattta 120
gcggctgttt taagagcggg cggagaaaga gttggcagat ttacaagccc tcacttagtt 180
catccgcgcg atgctatcac agtcgacggc gaagttattg gagcggcgac atatgctgca 240
cttaaagctg aagtcgttgc ggcaggcaca tgcacggagt ttgaagcaca aacggcggtt 300
gcgcttacgc attttgcaag acttgaatgc acatggtgtg tcgtcgaagt gggcgtcggc 360
ggcagattag acgctacaaa tgtcgtccct ggcggacgca aactgtgtgc aattacaaag 420
gttggattag atcatcaggc gttacttggc ggaacactgg ctgttgttgc aagagagaag 480
gccggcattg tggttccggg agtgcgcttt gtcgctgtcg acggcacgaa cgcaccttca 540
gttctggcgg aggttcgggc ggctgcagcg aaagttggcg cagaggtcca tgagacagga 600
ggcgcgccgg tttgcacagt cagctggggt gcggttgctg caagcgcact tccgttagcg 660
ggagcttacc aggtacaaaa cgcgggcgtt gcacttgcac tgcttgatca tcttcaacaa 720
ctgggagaga tctcagtcag ccatgcagca ctggaaagag gactgaaagc agtcgaatgg 780
cctggcagac ttcaacaagt tgagtatgac cttggaggcg tccatgtccc gctgttattt 840
gacggagcac acaatccgtg tgcagcggaa gagcttgcaa gattcttaaa cgagagatac 900
cgcggaccgg gaggatctcc gctgatctat gtgctggctg tcacgtgtgg caaagagatc 960
gacgcacttc ttgcacctct tctgaaaccg cacgatagag tcttcgcaac cagctttggc 1020
gcggttgagt ctatgccgtg ggtcgcagcg atggcaagcg aggatgtcgc agcggcggcg 1080
agacgctaca cagcccacgt ttcagcggtt gcggacccgc tggacgcgtt acgcgccgca 1140
gcggcagcac gcggcgatgc taatctggtc gtctgcggat cattatatct tgtcggcgaa 1200
cttctgcgcc gcgaacatta a 1221
<210> 75
<211> 419
<212> PRT
<213> Lactobacillus reuteri
<400> 75
Met Arg Thr Tyr Glu Gln Ile Asn Ala Gly Phe Asn Arg Gln Met Leu
1 5 10 15
Gly Gly Gln Arg Asp Arg Val Lys Phe Leu Arg Arg Ile Leu Thr Arg
20 25 30
Leu Gly Asn Pro Asp Gln Arg Phe Lys Ile Ile His Ile Ala Gly Thr
35 40 45
Asn Gly Lys Gly Ser Thr Gly Thr Met Leu Glu Gln Gly Leu Gln Asn
50 55 60
Ala Gly Tyr Arg Val Gly Tyr Phe Ser Ser Pro Ala Leu Val Asp Asp
65 70 75 80
Arg Glu Gln Ile Lys Val Asn Asp His Leu Ile Ser Lys Lys Asp Phe
85 90 95
Ala Met Thr Tyr Gln Lys Ile Thr Glu His Leu Pro Ala Asp Leu Leu
100 105 110
Pro Asp Asp Ile Thr Ile Phe Glu Trp Trp Thr Leu Ile Met Leu Gln
115 120 125
Tyr Phe Ala Asp Gln Lys Val Asp Trp Ala Val Ile Glu Cys Gly Leu
130 135 140
Gly Gly Gln Asp Asp Ala Thr Asn Ile Ile Ser Ala Pro Phe Ile Ser
145 150 155 160
Val Ile Thr His Ile Ala Leu Asp His Thr Arg Ile Leu Gly Pro Thr
165 170 175
Ile Ala Lys Ile Ala Gln Ala Lys Ala Gly Ile Ile Lys Thr Gly Thr
180 185 190
Lys Gln Val Phe Leu Ala Pro His Gln Glu Lys Asp Ala Leu Thr Ile
195 200 205
Ile Arg Glu Lys Ala Gln Gln Gln Lys Val Gly Leu Thr Gln Ala Asp
210 215 220
Ala Gln Ser Ile Val Asp Gly Lys Ala Ile Leu Lys Val Asn His Lys
225 230 235 240
Ile Tyr Lys Val Pro Phe Asn Leu Leu Gly Thr Phe Gln Ser Glu Asn
245 250 255
Leu Gly Thr Val Val Ser Val Phe Asn Phe Leu Tyr Gln Arg Arg Leu
260 265 270
Val Thr Ser Trp Gln Pro Leu Leu Ser Thr Leu Ala Thr Val Lys Ile
275 280 285
Ala Gly Arg Met Gln Lys Ile Ala Asp His Pro Pro Ile Ile Leu Asp
290 295 300
Gly Ala His Asn Pro Asp Ala Ala Lys Gln Leu Thr Lys Thr Ile Ser
305 310 315 320
Lys Leu Pro His Asn Lys Val Ile Met Val Leu Gly Phe Leu Ala Asp
325 330 335
Lys Asn Ile Ser Gln Met Val Lys Ile Tyr Gln Gln Met Ala Asp Glu
340 345 350
Ile Ile Ile Thr Thr Pro Asp His Pro Thr Arg Ala Leu Asp Ala Ser
355 360 365
Ala Leu Lys Ser Val Leu Pro Gln Ala Ile Ile Ala Asn Asn Pro Arg
370 375 380
Gln Gly Leu Val Val Ala Lys Lys Ile Ala Glu Pro Asn Asp Leu Ile
385 390 395 400
Ile Val Thr Gly Ser Phe Tyr Thr Ile Lys Asp Ile Glu Ala Asn Leu
405 410 415
Asp Glu Lys
<210> 76
<211> 1260
<212> DNA
<213> Lactobacillus reuteri
<400> 76
atgagaacat acgaacaaat taatgcagga tttaatcgcc agatgctggg cggccagaga 60
gacagagtca agttccttag acgcatcctt acgagacttg gaaaccctga tcagcgcttt 120
aaaattattc atatcgcggg aacgaacggc aaaggatcaa caggcactat gttagaacag 180
ggccttcaga atgcgggata ccgcgtcggc tactttagct ctcctgcgct ggttgatgat 240
cgcgaacaaa ttaaagtcaa tgatcacctt atcagcaaga aagattttgc gatgacctat 300
cagaaaatta cggagcatct gcctgctgac cttctgcctg atgatattac aatctttgag 360
tggtggacgt taatcatgct tcaatacttt gcggatcaaa aggttgactg ggcggtgatt 420
gaatgtggtc ttggcggcca agacgatgcg acaaacatca tctcagcgcc gttcatttca 480
gtcattaccc atatcgctct tgaccacacc cgtatcctgg gccctacaat tgcgaagatt 540
gcgcaagcta aggcaggcat tataaagaca gggactaaac aggttttcct ggcaccacat 600
caagagaagg atgcgttaac aatcattcgc gaaaaagcgc aacagcaaaa ggtcggactg 660
acgcaggcag atgcacagag cattgtggac ggaaaagcta ttttaaaagt gaatcacaag 720
atttacaagg tcccttttaa tctgctgggc acatttcagt cagaaaacct gggaacggtt 780
gttagcgtct ttaactttct gtatcagcgc cgtcttgtca cgtcatggca accgttactt 840
agcacactgg caacagttaa aattgcagga agaatgcaaa aaatcgcgga tcatcctccg 900
atcattcttg atggcgcaca taatccggat gctgcaaagc agcttacaaa gacaattagc 960
aaactcccac ataataaagt cataatggtg ttaggcttcc ttgctgacaa aaacatttca 1020
cagatggtca agatttacca acagatggcg gatgaaatta tcattacaac gcctgaccat 1080
cctacaagag cgctggacgc ctcagccctt aaatcagtct taccgcaagc aattattgcg 1140
aataatcctc gtcagggact ggttgttgct aagaaaattg cagagccgaa cgatcttatc 1200
atcgtcacgg gcagcttcta cacaatcaag gatattgagg caaatttaga tgagaaataa 1260
<210> 77
<211> 190
<212> PRT
<213> Bacillus subtilis
<400> 77
Met Lys Glu Val Asn Lys Glu Gln Ile Glu Gln Ala Val Arg Gln Ile
1 5 10 15
Leu Glu Ala Ile Gly Glu Asp Pro Asn Arg Glu Gly Leu Leu Asp Thr
20 25 30
Pro Lys Arg Val Ala Lys Met Tyr Ala Glu Val Phe Ser Gly Leu Asn
35 40 45
Glu Asp Pro Lys Glu His Phe Gln Thr Ile Phe Gly Glu Asn His Glu
50 55 60
Glu Leu Val Leu Val Lys Asp Ile Ala Phe His Ser Met Cys Glu His
65 70 75 80
His Leu Val Pro Phe Tyr Gly Lys Ala His Val Ala Tyr Ile Pro Arg
85 90 95
Gly Gly Lys Val Thr Gly Leu Ser Lys Leu Ala Arg Ala Val Glu Ala
100 105 110
Val Ala Lys Arg Pro Gln Leu Gln Glu Arg Ile Thr Ser Thr Ile Ala
115 120 125
Glu Ser Ile Val Glu Thr Leu Asp Pro His Gly Val Met Val Val Val
130 135 140
Glu Ala Glu His Met Cys Met Thr Met Arg Gly Val Arg Lys Pro Gly
145 150 155 160
Ala Lys Thr Val Thr Ser Ala Val Arg Gly Val Phe Lys Asp Asp Ala
165 170 175
Ala Ala Arg Ala Glu Val Leu Glu His Ile Lys Arg Gln Asp
180 185 190
<210> 78
<211> 573
<212> DNA
<213> Bacillus subtilis
<400> 78
atgaaagaag tcaataaaga acaaattgaa caggcagtga gacagattct tgaagcaatt 60
ggagaagatc cgaacagaga gggcttactt gatacaccga aaagagttgc taaaatgtat 120
gcggaagtct tttcaggctt aaatgaagat ccgaaagagc attttcagac aattttcgga 180
gaaaaccatg aagagctggt ccttgtgaaa gatattgcgt ttcactcaat gtgcgaacat 240
cacctggtgc cgttttacgg caaggcacac gttgcgtata ttcctagagg cggaaaagtt 300
acaggcttgt caaaattagc acgcgcagtt gaagctgttg caaaaagacc gcaattacag 360
gaacgcatta catctacaat tgcggaatca attgtcgaga cattagaccc tcatggcgtt 420
atggttgtcg ttgaagctga acacatgtgc atgacaatgc gcggcgtcag aaaacctggc 480
gcaaaaacag tcacatcagc agtcagaggc gtgtttaaag atgatgcggc agctcgtgcg 540
gaagtcctgg aacatattaa acgccaggac tga 573
<210> 79
<211> 120
<212> PRT
<213> Bacillus subtilis
<400> 79
Met Asp Lys Val Tyr Val Glu Gly Met Glu Phe Tyr Gly Tyr His Gly
1 5 10 15
Val Phe Thr Glu Glu Asn Lys Leu Gly Gln Arg Phe Lys Val Asp Leu
20 25 30
Thr Ala Glu Leu Asp Leu Ser Lys Ala Gly Gln Thr Asp Asp Leu Glu
35 40 45
Gln Thr Ile Asn Tyr Ala Glu Leu Tyr His Val Cys Lys Asp Ile Val
50 55 60
Glu Gly Glu Pro Val Lys Leu Val Glu Thr Leu Ala Glu Arg Ile Ala
65 70 75 80
Gly Thr Val Leu Gly Lys Phe Gln Pro Val Gln Gln Cys Thr Val Lys
85 90 95
Val Ile Lys Pro Asp Pro Pro Ile Pro Gly His Tyr Lys Ser Val Ala
100 105 110
Ile Glu Ile Thr Arg Lys Lys Ser
115 120
<210> 80
<211> 363
<212> DNA
<213> Bacillus subtilis
<400> 80
atggataaag tttatgtgga aggaatggaa ttttatggct atcatggcgt cttcacagaa 60
gagaacaaat tgggacaacg cttcaaagta gatctgacag cagaactgga tttatcaaaa 120
gcaggacaaa cagacgacct tgaacagaca attaactatg cagagcttta ccatgtctgt 180
aaagacattg tcgaaggcga gccggtcaaa ttggtagaga cccttgctga gcggatagct 240
ggcacagttt taggtaaatt tcagccggtt caacaatgta cggtgaaagt tattaaacca 300
gatccgccga ttcctggcca ctataaatca gtagcaattg aaattacgag aaaaaagtca 360
taa 363
<210> 81
<211> 167
<212> PRT
<213> Bacillus subtilis
<400> 81
Met Asn Asn Ile Ala Tyr Ile Ala Leu Gly Ser Asn Ile Gly Asp Arg
1 5 10 15
Glu Thr Tyr Leu Arg Gln Ala Val Ala Leu Leu His Gln His Ala Ala
20 25 30
Val Thr Val Thr Lys Val Ser Ser Ile Tyr Glu Thr Asp Pro Val Gly
35 40 45
Tyr Glu Asp Gln Ala Gln Phe Leu Asn Met Ala Val Glu Ile Lys Thr
50 55 60
Ser Leu Asn Pro Phe Glu Leu Leu Glu Leu Thr Gln Gln Ile Glu Asn
65 70 75 80
Glu Leu Gly Arg Thr Arg Glu Val Arg Trp Gly Pro Arg Thr Ala Asp
85 90 95
Leu Asp Ile Leu Leu Phe Asn Arg Glu Asn Ile Glu Thr Glu Gln Leu
100 105 110
Ile Val Pro His Pro Arg Met Tyr Glu Arg Leu Phe Val Leu Ala Pro
115 120 125
Leu Ala Glu Ile Cys Gln Gln Val Glu Lys Glu Ala Thr Ser Ala Glu
130 135 140
Thr Asp Gln Glu Gly Val Arg Val Trp Lys Gln Lys Ser Gly Val Asp
145 150 155 160
Glu Phe Val His Ser Glu Ser
165
<210> 82
<211> 504
<212> DNA
<213> Bacillus subtilis
<400> 82
atgaacaaca ttgcgtacat tgcgcttggc tctaatattg gagatagaga aacgtatctg 60
cgccaggccg ttgcgttact gcatcaacat gctgcggtca cagttacaaa agtcagctca 120
atttatgaaa cagatccggt cggctatgaa gaccaagccc agtttttaaa tatggcggtt 180
gaaattaaaa caagcctgaa tccgtttgaa cttctggaac tgacacagca aatcgaaaac 240
gaactgggcc gcacacgcga agttagatgg ggcccgagaa cagcggattt agacattctg 300
ctgtttaaca gagaaaacat tgaaacagag cagttaattg tcccgcatcc tcgcatgtat 360
gaacgcctgt ttgttcttgc gccgcttgcg gaaatttgcc agcaggtcga gaaagaagcg 420
acaagcgcgg aaacggatca agaaggagtt agagtttgga aacaaaaatc aggcgttgac 480
gaatttgtac atagcgaaag ctga 504
<210> 83
<211> 285
<212> PRT
<213> Bacillus subtilis
<400> 83
Met Ala Gln His Thr Ile Asp Gln Thr Gln Val Ile His Thr Lys Pro
1 5 10 15
Ser Ala Leu Ser Tyr Lys Glu Lys Thr Leu Val Met Gly Ile Leu Asn
20 25 30
Val Thr Pro Asp Ser Phe Ser Asp Gly Gly Lys Tyr Asp Ser Leu Asp
35 40 45
Lys Ala Leu Leu His Ala Lys Glu Met Ile Asp Asp Gly Ala His Ile
50 55 60
Ile Asp Ile Gly Gly Glu Ser Thr Arg Pro Gly Ala Glu Cys Val Ser
65 70 75 80
Glu Asp Glu Glu Met Ser Arg Val Ile Pro Val Ile Glu Arg Ile Thr
85 90 95
Lys Glu Leu Gly Val Pro Ile Ser Val Asp Thr Tyr Lys Ala Ser Val
100 105 110
Ala Asp Glu Ala Val Lys Ala Gly Ala Ser Ile Ile Asn Asp Ile Trp
115 120 125
Gly Ala Lys His Asp Pro Lys Met Ala Ser Val Ala Ala Glu His Asn
130 135 140
Val Pro Ile Val Leu Met His Asn Arg Pro Glu Arg Asn Tyr Asn Asp
145 150 155 160
Leu Leu Pro Asp Met Leu Ser Asp Leu Met Glu Ser Val Lys Ile Ala
165 170 175
Val Glu Ala Gly Val Asp Glu Lys Asn Ile Ile Leu Asp Pro Gly Ile
180 185 190
Gly Phe Ala Lys Thr Tyr His Asp Asn Leu Ala Val Met Asn Lys Leu
195 200 205
Glu Ile Phe Ser Gly Leu Gly Tyr Pro Val Leu Leu Ala Thr Ser Arg
210 215 220
Lys Arg Phe Ile Gly Arg Val Leu Asp Leu Pro Pro Glu Glu Arg Ala
225 230 235 240
Glu Gly Thr Gly Ala Thr Val Cys Leu Gly Ile Gln Lys Gly Cys Asp
245 250 255
Ile Val Arg Val His Asp Val Lys Gln Ile Ala Arg Met Ala Lys Met
260 265 270
Met Asp Ala Met Leu Asn Lys Gly Gly Val His His Gly
275 280 285
<210> 84
<211> 858
<212> DNA
<213> Bacillus subtilis
<400> 84
atggcgcagc acacaataga tcaaacacaa gtcattcata cgaaaccgag cgcgctttca 60
tataaagaaa aaacactggt catgggcatt cttaacgtta cacctgattc ttttagcgat 120
ggtggaaaat atgacagctt ggacaaggcg cttctgcatg ccaaagaaat gatcgacgac 180
ggcgcgcaca ttattgacat aggaggcgag agcacaagac cgggagctga atgcgtcagc 240
gaagacgaag aaatgtctcg ggtcattccg gtcattgaac gcatcacaaa ggaactcggc 300
gtcccgattt cagtggatac atataaagca tctgtggcag acgaagcagt caaagcgggc 360
gcatctatta tcaatgacat ttggggagcg aaacatgatc cgaagatggc aagcgtcgca 420
gcggaacata acgttccaat tgtcctgatg cacaatcggc cagaacggaa ttataacgac 480
cttcttccgg atatgctgag cgaccttatg gaatcagtca aaattgcggt tgaggcgggc 540
gtggatgaga aaaatattat tttagatccg ggcatcggct tcgcgaagac ataccatgat 600
aatcttgcag tgatgaataa gttagagatc ttcagcggac ttggctatcc tgtcctgctg 660
gctacatctc gtaaaagatt tatcggaaga gttcttgatt taccgcctga agagagagca 720
gagggcacag gagcgacagt ctgcttgggc attcagaaag gatgcgacat agtgcgtgtt 780
catgatgtca agcaaattgc cagaatggcg aaaatgatgg acgcgatgct gaataaggga 840
ggggtgcacc atggatga 858
<210> 85
<211> 168
<212> PRT
<213> Bacillus subtilis
<400> 85
Met Ile Ser Phe Ile Phe Ala Met Asp Ala Asn Arg Leu Ile Gly Lys
1 5 10 15
Asp Asn Asp Leu Pro Trp His Leu Pro Asn Asp Leu Ala Tyr Phe Lys
20 25 30
Lys Ile Thr Ser Gly His Ser Ile Ile Met Gly Arg Lys Thr Phe Glu
35 40 45
Ser Ile Gly Arg Pro Leu Pro Asn Arg Lys Asn Ile Val Val Thr Ser
50 55 60
Ala Pro Asp Ser Glu Phe Gln Gly Cys Thr Val Val Ser Ser Leu Lys
65 70 75 80
Asp Val Leu Asp Ile Cys Ser Gly Pro Glu Glu Cys Phe Val Ile Gly
85 90 95
Gly Ala Gln Leu Tyr Thr Asp Leu Phe Pro Tyr Ala Asp Arg Leu Tyr
100 105 110
Met Thr Lys Ile His His Glu Phe Glu Gly Asp Arg His Phe Pro Glu
115 120 125
Phe Asp Glu Ser Asn Trp Lys Leu Val Ser Ser Glu Gln Gly Thr Lys
130 135 140
Asp Glu Lys Asn Pro Tyr Asp Tyr Glu Phe Leu Met Tyr Glu Lys Lys
145 150 155 160
Asn Ser Ser Lys Ala Gly Gly Phe
165
<210> 86
<211> 507
<212> DNA
<213> Bacillus subtilis
<400> 86
atgatttcat ttattttcgc aatggacgcg aatagactga taggcaaaga caatgatctg 60
ccgtggcatt taccgaatga cctggcttat tttaaaaaaa ttacaagcgg ccatagcatc 120
attatgggac gtaaaacatt tgagtcaatt ggcagacctc ttccgaacag aaaaaacatt 180
gttgtcacat ctgcgccgga ttcagaattt cagggctgca cagtcgtctc aagccttaaa 240
gacgttcttg atatttgcag cggaccggaa gagtgttttg tcattggcgg agcgcaatta 300
tacacagatc tttttccgta cgcggataga ctgtatatga caaaaatcca ccatgaattt 360
gaaggcgaca gacactttcc tgaatttgac gagagcaact ggaaactcgt gtctagcgaa 420
cagggcacga aggatgagaa aaatccgtat gactatgaat ttcttatgta tgaaaagaaa 480
aacagcagca aagcgggagg cttttga 507
Claims (9)
1.一种用于合成叶酸、其盐、其前体或其中间体的基因工程菌株,其中所述工程菌株中内源性folC基因的表达水平降低,并且引入外源性folC基因并且所述工程菌株与其出发菌株相比具有显著提高的叶酸、其前体或其中间体的生产能力,叶酸、其盐、其前体或其中间体的结构式如式I所示:
其中,当a为单键时,a’为无或当a’为单键时,a为无;
当b是单键时,b′是无或当b′是单键时,b是无;
R1选自下组:-H、-CH3(5-甲基)、-CHO(5-甲酰基)、-CH=或=CH-(5,10-亚甲基)、-CH2-(5,10-亚甲基)、-CH=NH(5-亚胺甲基)、或其组合;
R2选自下组:-H、-CHO(10-甲酰基)、-CH=、=CH-(5,10-亚甲基)、-CH2-(5,10-亚甲基)、或其组合,所述外源性folC基因来源于棉囊阿舒霉或罗伊氏乳杆菌,所述外源性folC基因的序列如SEQ ID NO.:24或25所示;所述工程菌株的出发菌株为枯草芽孢杆菌;
并且还引入或上调叶酸生物合成基因,所述叶酸生物合成基因为folE/mtrA、folB、folK、folP/sul和folA/dfrA,所述叶酸生物合成基因的序列分别如SEQ ID NO.13-17所示,叶酸生物合成基因来自枯草芽孢杆菌。
2.如权利要求1所述的基因工程菌株,其特征在于,外源性folC基因的表达产物包含选自下组的多肽或其衍生多肽:二氢叶酸合酶。
3.如权利要求2所述的基因工程菌株,其特征在于,二氢叶酸合酶的氨基酸序列如SEQID NO.:22或23所示。
4.一种叶酸、其盐、其前体或其中间体的制备方法,其特征在于,包括以下步骤:
(i)提供权利要求1所述的工程菌株;
(ii)培养步骤(i)所述的工程菌株,从而获得含有叶酸、其盐、其前体或其中间体的一种或多种化合物的发酵产物;
(iii)任选地,对步骤(ii)中得到的发酵产物进行分离纯化,进一步获得叶酸、其盐、其前体或其中间体的一种或多种化合物;
(iv)任选地,将步骤(ii)或(iii)中得到的产物经酸性或碱性条件进一步得到叶酸、其盐、其前体或其中间体的不同化合物;
其中叶酸、其盐、其前体或其中间体的结构式如式I所示:
并且R1、R2、a、a′、b、b′如权利要求1中所定义。
5.如权利要求4所述的方法,其特征在于,叶酸、其盐、其前体或其中间体是叶酸。
6.一种制备叶酸、其前体或其中间体的方法,其特征在于,包括以下步骤:
(i)提供权利要求1所述的工程菌株;
(ii)培养步骤(i)所述的工程菌株,从而得到含叶酸的发酵产物;
(iii)任选地,对步骤(ii)中得到的发酵产物进行分离纯化,进一步得到叶酸、其前体或其中间体。
7.如权利要求6所述的方法,其特征在于,所述方法还包括在步骤(ii)的培养过程中添加对氨基苯甲酸(PABA)的步骤。
8.一种权利要求1所述的工程菌株的制备方法,其特征在于,包括以下步骤:
(a)降低出发菌株中内源性folC基因的表达水平,并引入所述外源性folC基因;
所述方法还包括在出发菌株中引入或上调叶酸生物合成基因的步骤(b),所述叶酸生物合成基因为folE/mtrA、folB、folK、folP/sul和folA/dfrA,所述叶酸生物合成基因的序列分别如SEQ ID NO.13-17所示,叶酸生物合成基因来自枯草芽孢杆菌;从而获得权利要求1所述的工程菌株。
9.一种权利要求1所述的工程菌株的用途,其用作用于发酵生产叶酸、其盐、其前体或其中间体的工程菌株;
其中叶酸、其盐、其前体或其中间体的结构式如式I所示:
并且R1、R2、a、a′、b、b′如权利要求1中所定义。
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