CN108884483A - 烟酸核糖苷的微生物生产 - Google Patents

烟酸核糖苷的微生物生产 Download PDF

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CN108884483A
CN108884483A CN201780018032.6A CN201780018032A CN108884483A CN 108884483 A CN108884483 A CN 108884483A CN 201780018032 A CN201780018032 A CN 201780018032A CN 108884483 A CN108884483 A CN 108884483A
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亚当·劳伦斯
席琳·维亚罗格
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Abstract

本发明涉及一种通过调节导致产生烟酸核糖苷的途径来产生烟酸核糖苷的新方法、表达载体和宿主细胞。

Description

烟酸核糖苷的微生物生产
本发明涉及一种通过调节导致产生烟酸核糖苷的途径来产生烟酸核糖苷的新方法、表达载体和宿主细胞。
烟酸核糖苷(nicotinic acid riboside，NaR)为维生素B3的吡啶-核苷形式，其用作烟酰胺腺嘌呤二核苷酸或NAD+的前体。人们认为高剂量的烟酸(NA)能够帮助提高高密度脂蛋白胆固醇，并降低低密度脂蛋白胆固醇和游离脂肪酸，但却尚未完全理解其机制。然而，高剂量NA会导致不良的潮红反应。与NA和NR一样，NaR能够在哺乳动物细胞中被转化为NAD+。然而，已阻止了关于NaR对人类和动物营养的潜在益处的研究，这是因为尚未描述产生其的有效且经济的方法。因此，期望识别用于产生烟酸核糖苷(NaR)的新方法。
在20世纪90年代首次阐明了细菌中NAD+的生物合成，且显示出该生物合成依赖于未在真核细胞中发现的两种关键的酶活性：FAD依赖型L-天冬氨酸氧化酶(E.coli NadB,EC1.4.3.16)；以及喹啉合酶(E.coli NadA,EC 2.5.1.72)。L-天冬氨酸氧化酶催化L-天冬氨酸至亚氨基琥珀酸的氧化，其利用分子氧作为电子受体并产生过氧化氢，其中涉及松散结合的黄素腺嘌呤二核苷酸(FAD)辅因子。已知Escherichia coli中的酶受到下游产物NAD+的抑制，但却已产生反馈抗性突变体。含有铁-硫簇的喹啉合酶随后进行亚氨基琥珀酸与磷酸二羟丙酮的缩合和环化，产生喹啉(quinolate)。这两种酶的组合活性将从1摩尔的天冬氨酸和1摩尔的磷酸二羟丙酮产生1摩尔的喹啉。
三种另外的酶活性为NAD+合成的两种经典的从头途径所共有的：喹啉磷酸核糖基转移酶(E.coli NadC,EC 2.4.2.19)；烟酸单核苷酸腺苷转移酶(E.coli NadD,EC2.7.7.18)；以及烟酸单核苷酸腺苷转移酶，即NAD+合成酶(E.coli NadE,EC 6.3.1.5)。喹啉磷酸核糖基转移酶将磷酸核糖基部分从磷酸核糖焦磷酸转移至喹啉氮并催化中间物的后续脱羧以产生烟酸单核苷酸(NaMN)、焦磷酸盐和二氧化碳。烟酸单核苷酸腺苷转移酶(NMNAT)使用三磷酸腺嘌呤(ATP)来腺苷酸化NaMN，产生烟酸二核苷酸(NaAD)和焦磷酸盐。NAD+生物合成中的最后一步是由NAD+合成酶催化的，其利用氨或谷氨酰胺作为氮供体来将NaAD酰胺化成NAD+，将1摩尔的ATP水解成AMP和焦磷酸盐。
除了从头途径之外，还存在多种用于补救NaR、NR、NMN、烟酰胺(Nam)或烟酸(NA)的特征化途径。已经在Saccharomyces cerevisiae中操纵这些补救途径以使得能够产生NR(美国专利号8,114,626)。同时缺失S.cerevisiae基因nrk1、urh1和pnp1使细胞外NR增加～10倍，且额外的nrt1缺失导致进一步增加～4倍至4uM。这些基因分别编码NR激酶(E.coliNadR)、嘌呤核苷磷酸化酶(urh1和pnp1，E.coli DeoD)和NR转运(E.coli pnuC)的活性。
Nad基因的表达通常是通过转录抑制物在细菌中进行共调节的。在E.coli中，nadA、nadB和pncB的转录受到NadR蛋白的抑制，该NadR蛋白也具有有助于补救途径的催化活性。NadR通过在NAD+存在下结合至保守基序来阻断转录。在Bacillus subtilis中，一种名为YrxA的不同蛋白质通过在NA存在下阻断两个背驰转录的操纵子nadB-nadA-nadC和nifS-yrxA来执行类似的作用(Rossolillo,2005,J.Bacteriol.,187(20),7155-7160)。
因此，一直期望找到用于增加烟酸的产生的更有效的方式。
本发明人现已惊奇地发现了一种用于显著增加烟酸核糖苷的产生的新方法以及所创建的用于这种方法中的宿主细胞和表达载体。
本发明涉及一种能够产生烟酸核糖苷(NaR)的经遗传修饰的细菌，其中细菌包括选自以下所组成的组的至少一种修饰：a)通过抑制nadA、nadB、nadC基因或其组合的转录来阻断或降低具有抑制NAD+生物合成作用的蛋白质的活性；b)添加或增加编码L-天冬氨酸氧化酶、喹啉合酶、喹啉磷酸核糖基转移酶的基因或其组合的转录；以及c)阻断或降低用作烟酸单核苷酸腺苷转移酶的蛋白质的活性；其中，与没有任何该修饰的细菌相比，具有至少一种该修饰的细菌产生了增加量的NaR。
可选地，细菌还可以包括选自以下所组成的组的至少一种修饰：d)阻断或降低用作核苷磷酸化酶的蛋白质的活性；e)阻断或降低用作烟酸核糖苷激酶的蛋白质的活性；f)阻断或降低用作烟酸核糖苷转运蛋白的蛋白质的活性；g)阻断或降低用作烟酸磷酸核糖基转移酶的蛋白质的活性；h)添加或增加用作烟酰胺单核苷酸酰胺水解酶的蛋白质的活性；以及i)添加或增加用作烟酸单核苷酸水解酶的蛋白质的活性。
在一些实施方式中，NAD+生物合成的负调节物为包含SEQ ID NO：1、2或3中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有抑制NAD+生物合成的活性。
在一些实施方式中，喹啉合酶是包含SEQ ID NO：23、24或25中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有将亚氨基琥珀酸和磷酸二羟丙酮转化成喹啉和磷酸盐的活性。
在一些实施方式中，L-天冬氨酸氧化酶是包含SEQ ID NO：26或27中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有在FAD依赖型反应中将天冬氨酸转化成亚氨基琥珀酸的活性。
在一些实施方式中，喹啉磷酸核糖基转移酶是包含SEQ ID NO：28、29或30中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有将喹啉和磷酸核糖焦磷酸转化成烟酰胺单核苷酸和二氧化碳的活性。
在一些实施方式中，烟酸单核苷酸腺苷转移酶蛋白是包含SEQ ID NO：4、5或6中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有将烟酸单核苷酸转化成烟酸腺嘌呤二核苷酸的烟酸单核苷酸腺苷转移酶活性。
在一些实施方式中，烟酸核糖苷磷酸化酶是包含SEQ ID NO：7、8、18或19中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有将烟酸核糖苷转化成烟酸和磷酸核糖的核苷裂解活性。
在一些实施方式中，烟酸核糖苷转运蛋白是包含SEQ ID NO：9、10或11中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有输入烟酸核糖苷的烟酸核糖苷转运活性。
在一些实施方式中，烟酸单核苷酸水解酶是包含SEQ ID NO：12、13或14中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有将烟酸单核苷酸转化成烟酸核糖苷的烟酸单核苷酸水解酶活性。
在一些实施方式中，烟酸磷酸核糖基转移酶是包含SEQ ID NO：15、16或17中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有将烟酸、5-磷酸-核糖1-二磷酸和三磷酸腺苷转化为烟酸单核苷酸、二磷酸腺苷、二磷酸盐和磷酸盐的烟酸磷酸核糖基转移酶活性。
在一些实施方式中，烟酸核糖苷激酶是包含SEQ ID NO：1的氨基酸序列的多肽，其中该多肽具有将烟酸核糖苷转化成烟酸单核苷酸的烟酸核糖苷激酶活性。
在一些实施方式中，烟酰胺单核苷酸酰胺水解酶是包含SEQ ID NO：20、21或22中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有将烟酰胺单核苷酸转化成烟酸单核苷酸的烟酰胺单核苷酸酰胺水解酶活性。
在一些实施方式中，经遗传修饰的细菌可以是E.coli、B.subtilis、C.glutamicum、A.baylyi或R.eutropha。
本发明还涉及一种产生NaR的方法，其包括：在有效产生NaR的条件下培养细菌细胞并从培养基回收NaR，从而产生NaR，其中宿主微生物包括选自以下所组成的组的至少一种修饰：a)通过抑制nadA、nadB、nadC基因或其组合的转录来阻断或降低具有抑制NAD+生物合成作用的蛋白质的活性；b)添加或增加编码L-天冬氨酸氧化酶、喹啉合酶、喹啉磷酸核糖基转移酶的基因或其组合的转录；以及c)阻断或降低用作烟酸单核苷酸腺苷转移酶的蛋白质的活性。可选地，细菌还可以包括选自以下所组成的组的至少一种修饰：d)阻断或降低用作烟酸核糖苷磷酸化酶的蛋白质的活性；e)阻断或降低用作烟酸核糖苷激酶的蛋白质的活性；f)阻断或降低用作烟酸核糖苷转运蛋白的蛋白质的活性；g)阻断或降低用作烟酸磷酸核糖基转移酶的蛋白质的活性；h)添加或增加用作烟酰胺单核苷酸酰胺水解酶的蛋白质的活性；以及i)添加或增加用作烟酸单核苷酸水解酶的蛋白质的活性。
本发明还涉及从上述经遗传修饰的细菌中的任一个获得的烟酸核糖苷化合物。
本发明还涉及一种组合物，其包含从上述经遗传修饰的细菌获得的烟酸核糖苷化合物。
本发明还涉及一种食品或饲料，其包含从上述经遗传修饰的细菌获得的烟酸核糖苷化合物。
除非本文另有定义外，否则本文使用的科学和技术术语将具有本领域普通技术人员通常所理解的含义。
术语“喹啉合酶”表示能够将亚氨基琥珀酸和磷酸二羟丙酮转化成喹啉和磷酸盐的酶，参见图1。本发明中使用的喹啉合酶能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。喹啉合酶蛋白的实例包括具有氨基酸序列SEQ ID NO：23、24或25的多肽。编码喹啉合成活性的基因是在例如，登记号ACX40525(E.coli)、NP_390663(B.subtilis)和CAF19774(C.glutamicum)下提供的。如所定义的喹啉合酶包括上述喹啉合酶的功能变体。
术语“L-天冬氨酸氧化酶”表示能够在FAD依赖型反应中将天冬氨酸转化成亚氨基琥珀酸的酶。本发明中使用的L-天冬氨酸氧化酶能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。核苷水解酶蛋白的实例包括具有氨基酸序列SEQ ID NO：26或27的多肽。编码L-天冬氨酸氧化酶活性的基因是在例如，登记号ACX38768(E.coli)和NP_390665(B.subtilis)下提供的。如所定义的L-天冬氨酸氧化酶包括上述L-天冬氨酸氧化酶的功能变体。
术语“喹啉磷酸核糖基转移酶”表示能够将喹啉和磷酸核糖焦磷酸转化成烟酰胺单核苷酸和二氧化碳的酶。本发明中使用的喹啉磷酸核糖基转移酶能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。核苷水解酶蛋白的实例包括具有氨基酸序列SEQID NO：28、29或30的多肽。编码喹啉磷酸核糖基转移酶活性的基因是在例如，登记号ACX41108(E.coli)、NP_390664(B.subtilis)和CAF19773(C.glutamicum)下提供的。如所定义的喹啉磷酸核糖基转移酶包括上述喹啉磷酸核糖基转移酶的功能变体。
术语“NAD+生物合成的负调节物”表示能够通过抑制喹啉合酶(NadA)、FAD依赖型L-天冬氨酸氧化酶(NadB)、喹啉磷酸核糖基转移酶(NadC)或其组合的转录来抑制NAD+生物合成活性的酶。本发明中描述的NAD+生物合成蛋白的负调节物能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。NAD+生物合成蛋白的负调节物的实例包括具有氨基酸序列SEQ ID NO：1、2或3的多肽。编码NAD+生物合成活性的负调节物的基因是在例如，登记号NP_418807(E.coli)、P39667(B.subtilis)和BAF54131(C.glutamicum)下提供的。如所定义的NAD+生物合成的负调节物包括上述NAD+生物合成的负调节物的功能变体。
术语“烟酸单核苷酸腺苷转移酶”表示能够催化烟酸单核苷酸至烟酸腺嘌呤二核苷酸的转化的酶。该酶在E.coli中被称为NadD。本发明中描述的烟酸单核苷酸腺苷转移酶蛋白能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。烟酸单核苷酸腺苷转移酶蛋白的实例包括具有氨基酸序列SEQ ID NO：4、5或6的多肽。编码烟酸单核苷酸腺苷转移酶活性的基因是在例如，登记号NP_415172(E.coli)、NP_390442(B.subtilis)和CAF21017(C.glutamicum)下提供的。如所定义的烟酸单核苷酸腺苷转移酶包括上述烟酸单核苷酸腺苷转移酶的功能变体。
术语“烟酸核糖苷磷酸化酶”表示能够催化烟酸核糖苷至烟酸和磷酸核糖的转化的酶。该酶在E.coli中被称为PncB。本发明中描述的烟酸核糖苷磷酸化酶蛋白能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。烟酸核糖苷磷酸化酶蛋白的实例包括具有氨基酸序列SEQ ID NO：7、8、18或19的多肽。编码烟酸核糖苷磷酸化酶活性的基因是在例如，登记号NP_418801(E.coli)、NP_389844(B.subtilis)、NP_390230(B.Subtilis)和NP_391819(B.subtilis)下提供的。如所定义的烟酸核糖苷磷酸化酶包括上述烟酸核糖苷磷酸化酶的功能变体。
术语“烟酸核糖苷转运蛋白”表示能够催化烟酸核糖苷的转运以将烟酸核糖苷从周质输入至细胞质中的蛋白质。该酶在E.coli中被称为PnuC。本发明中描述的烟酸核糖苷转运蛋白能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。烟酸核糖苷转运蛋白的实例包括具有氨基酸序列SEQ ID NO：9、10或11的多肽。编码NaR转运活性的基因是在例如，登记号CAG67923(A.baylyi)、NP_599316(C.glutamicum)和NP_415272(E.coli)下提供的。如所定义的烟酸核糖苷转运蛋白包括上述烟酸核糖苷转运蛋白的功能变体。
术语“烟酸单核苷酸水解酶”表示能够催化烟酸单核苷酸至烟酸核糖苷的水解的酶。该酶在E.coli中被称为UshA。本发明中使用的核苷水解酶能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。核苷水解酶蛋白的实例包括具有氨基酸序列SEQ IDNO：12、13或14的多肽。编码核苷水解酶活性的基因是在例如，登记号NP_415013(E.coli),NP_388665(B.subtilis)和CAF18899(C.glutamicum)下提供的。如所定义的烟酸单核苷酸水解酶包括上述烟酸单核苷酸水解酶的功能变体。
术语“烟酸磷酸核糖基转移酶”表示能够催化烟酸、5-磷酸-核糖1-二磷酸和三磷酸腺苷至烟酸单核苷酸、二磷酸腺苷、二磷酸盐和磷酸盐的转化的酶。该酶还催化了逆反应。本发明中描述的烟酸磷酸核糖基转移酶蛋白能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。烟酸磷酸核糖基转移酶蛋白的实例包括具有氨基酸序列SEQID NO：15、16或17的多肽。编码烟酸磷酸核糖基转移酶活性的基因是在例如，登记号NP_415451(E.coli)、NP_391053(B.subtilis)和CAF21180(C.glutamicum).下提供的。如所定义的烟酸磷酸核糖基转移酶包括上述烟酸磷酸核糖基转移酶的功能变体。
术语“烟酰胺单核苷酸酰胺水解酶”表示能够催化烟酰胺单核苷酸至烟酸单核苷酸的转化的酶。该酶在E.coli中被称为PncC。本发明中描述的烟酰胺单核苷酸酰胺水解酶能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。烟酰胺单核苷酸酰胺水解酶蛋白的实例包括具有氨基酸序列SEQ ID NO：20、21或22的多肽。编码烟酰胺单核苷酸酰胺水解酶活性的基因是在例如，登记号NP_417180(E.coli)、AAB00568(B.subtilis)和CAF20304(C.glutamicum)下提供的。如所定义的烟酰胺单核苷酸酰胺水解酶包括上述烟酰胺单核苷酸酰胺水解酶的功能变体。
术语“烟酸核糖苷激酶”表示能够催化烟酸核糖苷至烟酸单核苷酸的转化的酶。本发明中描述的烟酸核糖苷激酶蛋白能够来自多种生物，诸如E.coli、B.subtilis、C.glutamicum等。烟酸核糖苷激酶蛋白的实例包括具有氨基酸序列SEQ ID NO：1的多肽。编码烟酸核糖苷激酶活性的基因是在例如，登记号NP_418807(E.coli)下提供的。如所定义的烟酸核糖苷激酶包括上述烟酸核糖苷激酶的功能变体。
序列同源性：两个氨基酸序列之间或两个核苷酸序列之间的相关性是由参数“序列同源性”描述的。
出于本公开的目的，在两个氨基酸序列之间的序列同源性程度是使用如在EMBOSS软件包的Needle程序(EMBOSS：欧洲分子生物学开放软件套件，Rice等，2000，TrendsGenet.16:276-277)，优选为版本3.0.0或更高版本中实施的Needleman-Wunsch算法(Needleman and Wunsch,1970,J.Mol.Biol.48:443-453)确定的。所使用的可选参数为缺口开放罚分10，缺口延伸罚分0.5和EBLOSUM62(BLOSUM62的EMBOSS版本)替代矩阵。标记为“最长同源性”的Needle输出(使用-nobrief选项获得的)被用作同源性百分比且计算如下：
(相同的残基×100)/(对齐长度-对齐中的缺口总数)
出于本公开的目的，在两个脱氧核糖核苷酸序列之间的序列同源性程度是使用如在EMBOSS软件包的Needle程序(EMBOSS：欧洲分子生物学开放软件套件，Rice等，2000，同上)，优选为版本3.0.0或更高版本中实施的Needleman-Wunsch算法(Needleman andWunsch,1970，同上)确定的。所使用的可选参数为缺口开放罚分10，缺口延伸罚分0.5和EDNAFULL(NCBI NUC4.4的EMBOSS版本)替代矩阵。标记为“最长同源性”的Needle输出(使用-nobrief选项获得的)被用作同源性百分比且计算如下：
(相同的脱氧核糖核苷酸×100)/(对齐长度-对齐中的缺口总数)
核酸构建体：术语“核酸构建体”是指单链或双链的核酸分子，其是从天然存在的基因分离的或进行修饰以含有不存在于自然界中或是合成的核酸片段。当核酸构建体含有表达本公开的编码序列所需的控制序列时，术语核酸构建体与术语“表达盒”同义。
控制序列：术语“控制序列”是指表达编码本公开的多肽的多核苷酸所必需的所有组分。每个控制序列对于编码多肽的多核苷酸来说可以是天然的或外源的，或对彼此而言是天然的或外源的。这种控制序列包括，但不限于，前导、多腺苷酸化序列、前肽序列、启动子、信号肽序列和转录终止子。至少，控制序列包括启动子以及转录和翻译终止信号。控制序列可以具有用于引入特定限制性位点的接头序列，该特定限制性位点便于进行控制序列与编码多肽的多核苷酸的编码区的连接。
可操作性连接的：术语“可操作性连接的”是指一种构型，其中控制序列被置于相对于多核苷酸的编码序列的适当位置上，以使得控制序列指导编码序列的表达。
表达：术语“表达”包括在多肽的产生中所涉及的任何步骤，包括但不限于，转录、转录后修饰、翻译、翻译后修饰和分泌。
表达载体：术语“表达载体”是指线性或环状DNA分子，其包括编码多肽的多核苷酸且可操作性连接至为其表达而提供的额外的核苷酸。
宿主细胞：术语“宿主细胞”是指具有包括编码本公开的多肽序列中的任一个的多核苷酸的核酸构建体或表达载体的易于发生转化、转染、转导等的任何细菌细胞类型。术语“宿主细胞”包含由于在复制期间发生的突变而与亲本细胞不同的亲本细胞的任何后代。
本发明提供了用于产生烟酸核糖苷的具有经遗传修饰的特性的细菌菌株。
已通过补救途径的修饰来在酵母中描述了烟酰胺核糖苷的产生。令人惊讶的是，在细菌中修饰NAD+生物合成和补救途径的结合导致了烟酸核糖苷的产生。
因此，在本发明的第一个实施方式中，期望引入一种或多种遗传修饰，其导致了在宿主细胞内的烟酸单核苷酸的产生率增加。修饰可以包括基因表达的缺失或减少，该基因抑制从头NAD+生物合成途径的基因nadA、nadB和/或nadC中的全部或一些的转录。参见图2。该修饰还可以或替代地包括增加由例如，nadB(E.coli、B.subtilis)、nadA(E.coli、B.subtilis、C.glutamicum)或nadC(E.coli、B.subtilis、C.glutamicum)编码的L-天冬氨酸氧化酶基因、喹啉合酶基因、喹啉磷酸核糖焦磷酸基因或其组合的表达。该修饰还可以或替代地包括对nadB基因的修饰，其使得基因对下游代谢物NAD+产生的抑制具有抗性。
在E.coli、B.subtilis、C.glutamicum和其他种的细菌中，NaMN通过烟酸单核苷酸腺苷转移酶(NMNAT,EC 2.7.7.18)的作用被转化成烟酸腺嘌呤二核苷酸(NaAD)。NMNAT活性的降低导致细胞内NaMN水平增加，这导致NaMN输出增加以及至NaR的去磷酸化。因此，在第二个实施方式中，将一种或多种遗传修饰引入宿主细胞以降低NMNAT活性。在某些优选实施方式中，通过改变具有NMNAT活性的多肽的氨基酸序列来实现NMNAT活性的降低。例如，在某些实施方式中，修饰可以包括将在B.subtilis nadD基因(SEQ ID NO:6)的位置10处编码的苏氨酸或在位置39处编码的天冬酰胺改变成另一种氨基酸。在其他实施方式中，修饰可以包括将在E.coli nadD基因(SEQ ID NO:7)的位置11处编码的苏氨酸或在位置40处编码的天冬酰胺改变成另一种氨基酸。在其他实施方式中，修饰可以包括将在C.glutamicum nadD基因(SEQ ID NO:8)的位置25处编码的苏氨酸改变成另一种氨基酸。修饰还可以包括对编码NMNAT的开放阅读框的区域5’或3’的修饰，以使得基因的转录和/或翻译以较低的效率发生。
在一些实施方式中，NAD+生物合成的负调节物为包含SEQ ID NO：1、2或3中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有抑制NAD+生物合成的活性。
在一些实施方式中，喹啉合酶是包含SEQ ID NO：23、24或25中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有由亚氨基琥珀酸和磷酸二羟丙酮形成喹啉的活性。
在一些实施方式中，L-天冬氨酸氧化酶是包含SEQ ID NO：26或27中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有由天冬氨酸形成亚氨基琥珀酸的活性。
在一些实施方式中，喹啉磷酸核糖基转移酶是包含SEQ ID NO：28、29或30中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有由喹啉和磷酸核糖焦磷酸形成烟酸单核苷酸的活性。
在一些实施方式中，烟酸单核苷酸腺苷转移酶蛋白是包含SEQ ID NO：4、5或6中的任一个的氨基酸序列的多肽或该多肽的变体，其中该多肽具有将烟酸单核苷酸转化成烟酸腺嘌呤二核苷酸的烟酸单核苷酸腺苷转移酶活性。
本发明还包括一种经遗传修饰的细菌菌株，其在烟酸核糖苷输入和补救途径中有缺陷。参见图4。预计细菌中NaR补救途径的破坏会导致细胞外NaR的累积，这是因为这种菌株无法在细胞内或在细胞外将NaR输入细胞质中，且也无法将NaR磷酸化成NaMN，或将NaR降解为烟酸(NA)和磷酸核糖。四种酶活性对于工程菌NaR的产生来说是特别重要的。烟酸核糖苷激酶对细胞内NaR的磷酸化使该化合物再循环回NAD+生物合成途径中。通过缺失或降低编码NR激酶活性的基因，诸如E.coli中的nadR的表达来去除该活性将通过防止将其转化成NaMN来增加细胞内的NaR池。通过核苷磷酸化酶活性将NaR降解为NA和磷酸核糖去除了产物，且缺失或降低编码该活性的基因，例如，在E.coli中的deoD或B.subtilis中的pdp的表达将增加产生形成率。通过烟酸磷酸核糖基转移酶活性将烟酸和5-磷酸-α-D-核糖1-二磷酸连接到烟酸单核苷酸。该反应在生理条件下是可逆的，且可以用于将源于水解的NaMN重新路由至副产物烟酸中。编码该活性的基因，例如，在E.coli中的pncB、在B.subtilis中的yueK或在C.glutamicum中的cg2774的表达减少或缺失将通过阻止该降解反应来增加NaR的形成。转运蛋白负责细胞外NaR的输入，且编码该活性的基因，例如，在E.coli中的pnuC或在B.subtilis中的nupC的表达缺失或减少将导致细胞外NaR的产生率的增加。
因此，在本发明的第三个实施方式中，期望减少或阻断烟酰胺核糖苷输入和补救途径且从而使宿主细胞保留已产生的烟酰胺核糖苷。在某些实施方式中，本发明的细菌菌株具有下列特性中的一个或多个：i)阻断或减少的用作烟酸核糖苷磷酸化酶的蛋白质；ii)阻断或减少的用作烟酸核糖苷激酶的蛋白质；iii)阻断或减少的用作烟酸核糖转运蛋白的蛋白质；iv)阻断或减少的用作烟酸磷酸核糖基转移酶的蛋白质。
根据本文的实施方式的烟酸核糖苷磷酸化酶可以包括，例如，但不限于，包含SEQID NO:7、SEQ ID NO:8、SEQ ID NO:18或SEQ ID NO:19中任一个的氨基酸序列的多肽或该多肽的变体，其中上述多肽具有用于将烟酸核糖苷转化成烟酸和磷酸核糖的的烟酸核糖苷磷酸化酶的活性。
根据本文的实施方式的烟酸核糖苷激酶可以包括，例如，但不限于，包含SEQ IDNO:1的氨基酸序列的多肽或该多肽的变体，其中上述多肽具有用于将烟酸核糖苷转化成烟酸单核苷酸的烟酸激酶的活性。
根据本文的实施方式的烟酸核糖苷摄取性转运体可以包括，例如，但不限于，包含SEQ ID NO:9、10或11中任一个的氨基酸序列的多肽或该多肽的变体，其中上述多肽具有烟酸核糖苷输入活性。
根据本文的实施方式的烟酸单核苷酸磷酸核糖基转移酶可以包括，例如，但不限于，包含SEQ ID NO:15、SEQ ID NO:16或SEQ ID NO:17的氨基酸序列的多肽或该多肽的变体，其中上述多肽具有用于在可逆反应中将烟酸、5-磷酸-核糖1-二磷酸和三磷酸腺苷转化为烟酸单核苷酸、二磷酸腺苷、二磷酸盐和磷酸盐的烟酸单核苷酸磷酸核糖基转移酶的活性。
在本发明的第四个实施方式中，期望增加例如，由E.coli中的ushA或B.subtilis中的yfkN进行编码的细胞外烟酸单核苷酸水解酶基因的表达水平，且因此使得宿主细胞从NaMN产生过量的细胞外NaR。参见图3。在一个实施方式中，本发明涉及一种细菌菌株，其具有增加的烟酸单核苷酸水解酶活性。根据本文的实施方式的烟酰胺单核苷酸水解酶可以包括，例如但不限于，包含SEQ ID NO:12、13或14的氨基酸序列的多肽或该多肽的变体，其中上述多肽具有用于将烟酸单核苷酸转化成烟酸核糖苷的烟酸单核苷酸水解酶的活性。
除了用作辅因子之外，NAD+还在多种细胞进程中进行消耗，例如作为细菌DNA连接酶的底物，其伴随有NMN的释放。在本发明的第五个实施方式中，NMN向NaMN的快速再循环使***偏向于在NR上产生NaR，且是通过由例如，E.coli中的pncC(SEQ ID NO:20)、B.subtilis中的CinA(SEQ ID NO:21)或C.glutamicum中的cg2153(SEQ ID NO:22)编码的烟酰胺单核苷酸酰胺水解酶，或该烟酰胺单核苷酸酰胺水解酶的变体的过表达来实现的。
在另一些实施方式中，在上述第一个或第二个实施方式中描述的细菌菌株还包括上述第三个实施方式、第四个实施方式或这两者。
例如，在一个实施方式中，本发明涉及一种能够产生烟酸核糖苷的经遗传修饰的细菌，其中该细菌包括下列修饰：i)在带有具有阻断或降低的活性的改变的NAD+生物合成负调节物的宿主中的改变或增加的L-天冬氨酸氧化酶活性，改变或增加的喹啉合酶活性，改变或增加的喹啉磷酸核糖焦磷酸活性或其组合，以及ii)选自以下所组成的组的一种或多种额外的修饰：d)具有阻断或降低的活性的改变的烟酸核糖苷激酶；e)具有阻断或降低的活性的改变的烟酸核糖苷磷酸化酶；f)具有阻断或降低的活性的改变的烟酰胺核糖苷摄取性转运体；g)具有阻断或降低的活性的改变的烟酸磷酸核糖基转移酶；h)具有添加或增加的活性的改变的烟酰胺单核苷酸酰胺水解酶；以及i)具有添加或增加的活性的改变的烟酸单核苷酸水解酶；其中，与没有任何该修饰的细菌相比，具有至少一种该修饰的细菌产生了增加量的NaR。
例如，在一个实施方式中，本发明涉及一种能够产生烟酸核糖苷的经遗传修饰的细菌，其中该细菌包括下列修饰：i)在具有编码这些活性的基因或其组合的增加的转录的宿主中的改变或增加的L-天冬氨酸氧化酶活性，改变或增加的喹啉合酶活性，改变或增加的喹啉磷酸核糖焦磷酸活性或其组合，以及ii)选自以下所组成的组的一种或多种额外的修饰：a)具有阻断或降低的活性的改变的烟酸核糖苷激酶；b)具有阻断或降低的活性的改变的烟酸核糖苷磷酸化酶；c)具有阻断或降低的活性的改变的烟酸核糖苷转运蛋白；d)具有阻断或降低的活性的改变的烟酸磷酸核糖基转移酶；e)具有添加或增加的活性的改变的烟酰胺单核苷酸酰胺水解酶；以及f)具有添加或增加的活性的改变的烟酸单核苷酸水解酶；其中，与没有任何该修饰的细菌相比，具有至少一种该修饰的细菌产生了增加量的NaR。
在另一个实施方式中，本发明涉及一种能够产生烟酸核糖苷的经遗传修饰的细菌，其中该细菌包括下列修饰：i)具有阻断或降低的活性的改变的烟酸单核苷酸腺苷转移酶；以及ii)选自以下所组成的组的一种或多种额外的修饰：a)具有阻断或降低的活性的改变的烟酸核糖苷激酶；b)具有阻断或降低的活性的改变的烟酸核糖苷磷酸化酶；c)具有阻断或降低的活性的改变的烟酸核糖苷转运蛋白；d)具有阻断或降低的活性的改变的烟酸磷酸核糖基转移酶；e)具有添加或增加的活性的改变的烟酰胺单核苷酸酰胺水解酶；以及f)具有添加或增加的活性的改变的烟酸单核苷酸水解酶；其中，与没有任何该修饰的细菌相比，具有至少一种该修饰的细菌产生了增加量的NaR。
在一个实施方式中，具有降低的活性的烟酸单核苷酸腺苷转移酶对于宿主细菌来说是外源的，即，在修饰之前不存在于细胞中，其已使用如本文所述的重组方法引入。
在另一个实施方式中，上述的其他蛋白质对于宿主细菌来说是内源的，即，在修饰之前存在于细胞中，然而可进行交替以增加或降低蛋白质的表达水平。在本发明中表达水平发生改变的内源蛋白质的实例包括，但不限于，烟酸单核苷酸腺苷转移酶、NAD+生物合成的负调节物、烟酸核糖苷激酶、烟酸核糖苷磷酸化酶、烟酸核糖苷转运蛋白、烟酸磷酸核糖基转移酶、烟酰胺单核苷酸酰胺水解酶和烟酸单核苷酸水解酶。
宿主细菌细胞可以通过本领域的技术人员已知适合于该目的的任何方式进行遗传修饰。这包括将感兴趣的基因，诸如编码具有降低的活性的烟酸单核苷酸腺苷化蛋白的基因引入质粒或粘粒或能够在宿主细胞内繁殖的其他表达载体中。替代地，质粒或粘粒DNA或部分质粒或粘粒DNA或线性DNA序列可以整合至宿主基因组中，例如，通过同源重组或随机整合来进行。为了进行遗传修饰，能够通过天然摄取或通过公知的进程，诸如电穿孔来将DNA引入或转化至细胞中。遗传修饰可能涉及在引入的启动子的控制下的基因表达。引入的DNA可以编码一种蛋白质，其可以充当酶或可以调节其他基因的表达。
能够使用经典的菌株开发和/或分子遗传技术来实现微生物的遗传修饰。这种技术在本领域中是已知的且通常被公开用于微生物，例如，在Sambrook等，1989，MolecularCloning:A Laboratory Manual,Cold Spring Harbor Labs Press中所述的。参考文献Sambrook等ibid的全部内容通过引用并入本文。
合适的多核苷酸可以通过随机整合、同源重组被引入细胞中和/或可以形成包括基因组合的表达载体的一部分。这种表达载体形成了本发明的另一个方面。
用于构建这种表达载体的合适载体在本领域中是公知的，且可以被布置成包括可操作性连接至一个或多个表达控制序列的多核苷酸，以用于在宿主细胞中表达所需的酶，例如，如上所述的细菌。例如，包括但不限于，T7启动子、pLac启动子、nudC启动子、ushA启动子的启动子能够与内源基因和/或异源基因结合使用以用于修饰靶向基因的表达模式。类似地，示例性终止子序列包括但不限于使用XPR1、XPR2、CPC1终止子序列。
在一些实施方式中，如本说明书中通篇所述，重组或经遗传修饰的细菌细胞可以是任何***或革兰氏阴性细菌，包括但不限于，Bacillus、Corynebacterium、Escherichia、Acinetobacter、Lactobacillus、Mycobacterium、Pseudomonas和Ralstonia属。在某些实施方式中，示例性细菌种类包括但不限于Bacillus subtilis、Corynebacterium glutamicum、Escherichia coli、Acinetobacter baylyi和Ralstoniaeutropha。
本公开的经遗传修饰的细菌还包含包括如本文定义的多肽的变体的细菌。如本文所使用的，“变体”是指由于在一个或多个(几个)位置上进行了一个或多个(几个)氨基酸残基的替代、***和/或缺失而使氨基酸序列不同于碱基序列的多肽，该氨基酸序列是从碱基序列衍生出来的。替代是指用不同的氨基酸取代占据一个位置的氨基酸；缺失是指去除占据一个位置的氨基酸；且***是指在邻近占据一个位置的氨基酸处添加1-3个氨基酸。
变体是功能变体，这是因为变体序列具有与具有本文指定的天然氨基酸序列的酶相似或相同的功能酶活性特征。
例如，SEQ ID NO:4的功能变体具有与SEQ ID NO:4相似或相同的烟酸单核苷酸腺苷转移酶活性特征。一个实例可以是通过SEQ ID No：4的功能变体进行的烟酸单核苷酸至烟酸腺嘌呤二核苷酸的转化率可以是相同或相似的，然而该功能变体还可以提供其他益处。例如，当使用为SEQ ID NO:4的功能变体的酶时，将实现该速率的至少约80％、90％、95％、96％、97％、98％、99％或100％。
因此，上述SEQ ID No氨基酸序列中的任一个的功能变体或片段是保持在相同酶类别(即具有相同EC编号)内的任何氨基酸序列。确定酶是否落在特定类别内的方法是技术人员所公知的，该技术人员能够在不使用本发明的技术的情况下确定酶的类别。合适的方法可以例如，从国际生物化学与分子生物学联盟获得。
氨基酸替代可以被认为是“保守性的”，其中氨基酸被具有广义上类似的特性的不同氨基酸替代。非保守性替代是氨基酸被不同类型的氨基酸取代的地方。
“保守性替代”是指用同一类别的另一种氨基酸替代氨基酸，其中类别的定义如下：
氨基酸类别实例：
非极性的：A、V、L、I、P、M、F、W
非荷电极性的：G、S、T、C、Y、N、Q
酸性的：D、E
碱性的：K、R、H。
本发明还涉及从上述经遗传修饰的细菌中的任一个获得的烟酸核糖苷化合物。
本发明还涉及一种组合物，其包含从上述经遗传修饰的细菌获得的烟酸核糖苷化合物。
将理解的是，从本发明的经遗传修饰的细菌分离的烟酸核糖化合物能够被重新配制成最终产物。在本公开的一些其他实施方式中，在宿主细胞的背景下，通过如本文所述操纵的宿主细胞产生的烟酸核糖化合物被掺入最终产物(例如，食物或饲料补充剂、药物等)中。例如，宿主细胞可以进行冻干、冷冻干燥、冷冻或其他方式的灭活，且随后全细胞可以被掺入最终产物中或用作最终产物。宿主细胞也可以在掺入产物中之前进行处理，以增加生物利用度(例如，经由裂解进行)。
在本公开的一些实施方式中，将产生的烟酸核糖化合物掺入食物或饲料(例如，食物补充剂)的组分中。根据本发明能够将烟酸核糖化合物掺入至的食品类型没有特别的限制，且包括饮料，诸如牛奶、水、软饮料、能量饮料、茶和果汁；甜点，诸如果冻和饼干；含脂肪的食物和饮料，诸如乳制品；加工食品，诸如大米、面包、早餐麦片等。在一些实施方式中，所产生的烟酸核糖化合物被掺入膳食补充剂，诸如，例如，复合维生素中。
附图说明
图1：使用E.coli命名的用于在NadA和NadB酶的存在下从天冬氨酸和磷酸二羟丙酮合成喹啉的生化途径。
图2：使用E.coli命名的用于合成烟酰胺腺嘌呤二核苷酸的生化途径和酶。
图3：使用E.coli命名的用于从NAD+生物合成的中间物产生烟酸核糖苷的生化途径。
图4：使用E.coli命名的用于产生烟酸核糖苷的具有不良活性的生化途径。ATP：三磷酸腺苷；pRpp：5-磷酸-α-D-核糖1-二磷酸；PPi：二磷酸盐；Pi：磷酸盐。
图5：在菌株ME517的补料分批发酵期间的烟酸核糖苷水平。
序列表的概述
使用核苷酸碱基的标准字母缩写来显示在所附序列表中列出的核酸序列。仅显示了每个核酸序列的一条链，但要理解的是对所显示的链的任何参考均包括互补链。在所附序列表中：
SEQ ID NO:1为编码是抑制蛋白的三功能性的Escherichia coli NadR酶(NMN合成酶、NaR激酶、NAD+生物合成的负调节物)的氨基酸序列。
SEQ ID NO:2为编码是抑制蛋白的Bacillus subtilis YxrA酶的氨基酸序列。
SEQ ID NO:3为编码是抑制蛋白的Corynebacterium glutamicum CgR_1153酶的氨基酸序列。
SEQ ID NO:4为编码是烟酸单核苷酸腺苷转移酶的Escherichia coli NadD酶的氨基酸序列。
SEQ ID NO:5为编码是烟酸单核苷酸腺苷转移酶的Bacillus subtilis NadD酶的氨基酸序列。
SEQ ID NO:6为编码是烟酸单核苷酸腺苷转移酶的Corynebacterium glutamicumNadD Cg2584酶的氨基酸序列。
SEQ ID NO:7为编码是烟酸核糖苷磷酸化酶的Escherichia coli DeoD酶的氨基酸序列。
SEQ ID NO:8为编码是烟酸核糖苷磷酸化酶的Bacillus subtilis DeoD酶的氨基酸序列。
SEQ ID NO:9为编码是NaR转运蛋白的Acinetobacter baylyi PnuC酶的氨基酸序列。
SEQ ID NO:10为编码是NaR转运蛋白的Corynebacterium glutamicum PnuC酶的氨基酸序列。
SEQ ID NO:11为编码是NaR转运蛋白的Escherichia coli PnuC酶的氨基酸序列。
SEQ ID NO:12为编码是烟酸单核苷酸水解酶的Escherichia coli UshA酶的氨基酸序列。
SEQ ID NO:13为编码是烟酸单核苷酸水解酶的Bacillus subtilis YfkN酶的氨基酸序列。
SEQ ID NO:14为编码是烟酸单核苷酸水解酶的Corynebacterium glutamicumCg0397酶的氨基酸序列。
SEQ ID NO:15为编码是烟酸磷酸核糖基转移酶的Escherichia coli PncB酶的氨基酸序列。
SEQ ID NO:16为编码是烟酸磷酸核糖基转移酶的Bacillus subtilis YueK酶的氨基酸序列。
SEQ ID NO:17为编码是烟酸磷酸核糖基转移酶的Corynebacterium glutamicumcg2774酶的氨基酸序列。
SEQ ID NO:18为编码是烟酸核糖苷磷酸化酶的Bacillus subtilis PupG酶的氨基酸序列。
SEQ ID NO:19为编码是烟酸核糖苷磷酸化酶的Bacillus subtilis Pdp酶的氨基酸序列。
SEQ ID NO:20为编码是烟酰胺单核苷酸酰胺水解酶的Escherichia coli PncC酶的氨基酸序列。
SEQ ID NO:21为编码是烟酰胺单核苷酸酰胺水解酶的Bacillus subtilis CinA酶的氨基酸序列。
SEQ ID NO:22为编码是烟酰胺单核苷酸酰胺水解酶的Corynebacteriumglutamicum cg2153酶的氨基酸序列。
SEQ ID NO:23为编码是喹啉合酶的Escherichia coli NadA酶的氨基酸序列。
SEQ ID NO:24为编码是喹啉合酶的Bacillus subtilis NadA酶的氨基酸序列。
SEQ ID NO:25为编码是喹啉合酶的Corynebacterium glutamicum NadA酶的氨基酸序列。
SEQ ID NO:26为编码是L-天冬氨酸氧化酶的Escherichia coli NadB酶的氨基酸序列。
SEQ ID NO:27为编码是L-天冬氨酸氧化酶的Bacillus subtilis NadB酶的氨基酸序列。
SEQ ID NO:28为编码是喹啉磷酸核糖基转移酶的Escherichia coli NadC酶的氨基酸序列。
SEQ ID NO:29为编码是喹啉磷酸核糖基转移酶的Bacillus subtilis NadC酶的氨基酸序列。
SEQ ID NO:30为编码是喹啉磷酸核糖基转移酶的Corynebacterium glutamicumNadC酶的氨基酸序列。
以下实例旨在说明本发明而不以任何方式限制其范围。
实施例
实施例1：破坏NAD+生物合成的负调节物
本文所述的所有基础分子生物学和DNA操纵程序通常是根据Sambrook等或Ausubel等(J.Sambrook、E.F.Fritsch、T.Maniatis(编辑).1989.Molecular Cloning:ALaboratory Manual.Cold Spring Harbor Laboratory Press:New York；以及F.M.Ausubel、R.Brent、R.E.Kingston、D.D.Moore、J.G.Seidman、J.A.Smith、K.Struhl(编辑).1998.Current Protocols in Molecular Biology.Wiley:New York)来执行的。
编码NAD+生物合成的负调节物(nadR)的基因的缺失是由λ红介导的重组工程实现的。抗生素基因，例如具有卡那霉素抗性或氯霉素抗性的，是使用寡核苷酸来进行PCR扩增的，该寡核苷酸具有与在天然nadR开放阅读框的上游和下游的区域同源的为20-50bps的侧翼。替代地，这些侧翼可以在开放阅读框内，导致非功能性蛋白质的翻译。宿主菌株，例如BL21(DE3)，是通过所述的(Datsenko和Wanner，2000)pKD46的转化和诱导来进行制备以用于λ红重组工程的。所制备的细胞通过电穿孔或化学转化进行转化，且通过PCR来筛选转化体以成功破坏靶向基因。
实施例2：增强从L-天冬氨酸、磷酸二羟丙酮和1-α-D-核糖焦磷酸至烟酸单核苷酸的转化
通过在E.coli中由nadB基因编码的L-天冬氨酸氧化酶将天冬氨酸氧化成亚氨基琥珀酸。该实施例描述了E.coli菌株的构建，其改变了天然nadB基因的表达。
将nadB基因置于强组成型启动子的控制下。编码E.coli nadB基团的DNA片断是通过PCR克隆或从头DNA合成获得的。在DNA是通过合成获得的情况下，优化密码子的使用以在E.coli中进行表达。DNA的合成和优化是由GenScript,Inc.进行的。nadB基因在诱导型启动子的控制下在E.coli中进行表达。例如，将开放阅读框克隆至XhoI/NdeI-消化pET24，产生质料pET24-nadB。转化至携带T7聚合酶的菌株，诸如BL21(DE3)中允许对nadB基因进行IPTG诱导，以便促进NaR合成。
替代地，天然nadB基因的表达还能够通过将nadB基因置于诱导型启动子的控制下来进行改变。编码诱导型或组成型启动子，诸如***糖诱导型pBAD或组成型pLac启动子的DNA片断是通过PCR克隆或从头DNA合成获得的。启动子通过融合PCR在抗生素基因，例如具有卡那霉素抗性或氯霉素抗性的基因的下游融合。该标记-启动子盒将含有为20-50bps的侧翼，其与在天然nadB启动子上游的区域且与nadR开放阅读框的第一核苷酸同源。宿主菌株，例如BL21(DE3)，是通过所述的(Datsenko和Wanner，2000)pKD46的转化和诱导来进行制备以用于λ红重组工程的。所制备的细胞通过电穿孔或化学转化进行转化，且通过PCR来筛选转化体以成功掺入改变的启动子序列。
含有铁-硫簇的喹啉合酶随后进行亚氨基琥珀酸与磷酸二羟丙酮的缩合和环化，产生喹啉，且通过nadA基因在E.coli中进行编码。该实施例描述了E.coli菌株的构建，其改变了天然nadA基因的表达。
将nadA基因置于强组成型启动子的控制下。编码E.coli nadA基团的DNA片断是通过PCR克隆或从头DNA合成获得的。在DNA是通过合成获得的情况下，优化密码子的使用以在E.coli中进行表达。DNA的合成和优化是由GenScript,Inc.进行的。nadA基因在诱导型启动子的控制下在E.coli中进行表达。例如，将开放阅读框克隆至XhoI/NdeI-消化pET24，产生质料pET24-nadA。转化至携带T7聚合酶的菌株，诸如BL21(DE3)中允许对nadA基因进行IPTG诱导，以便促进NaR合成。
替代地，天然nadA基因的表达还能够通过将nadB基因置于诱导型启动子的控制下来进行改变。编码诱导型或组成型启动子，诸如***糖诱导型pBAD或组成型pLac启动子的DNA片断是通过PCR克隆或从头DNA合成获得的。启动子通过融合PCR在抗生素基因，例如具有卡那霉素抗性或氯霉素抗性的基因的下游融合。该标记-启动子盒将含有为20-50bps的侧翼，其与在天然nadB启动子上游的区域且与nadA开放阅读框的第一核苷酸同源。宿主菌株，例如BL21(DE3)，是通过所述的(Datsenko和Wanner，2000,ProC.Natl.Acad.U.S.A.97(12):6640-5.)pKD46的转化和诱导来进行制备以用于λ红重组工程的。所制备的细胞通过电穿孔或化学转化进行转化，且通过PCR来筛选转化体以成功掺入改变的启动子序列。
喹啉磷酸核糖基转移酶将磷酸核糖基部分从磷酸核糖焦磷酸转移至喹啉氮并催化中间物的后续脱羧以产生烟酸单核苷酸且通过nadC基因在E.coli中进行编码。该实施例描述了E.coli菌株的构建，其改变了天然nadC基因的表达。
将nadC基因置于强组成型启动子的控制下。编码E.coli nadC基团的DNA片断是通过PCR克隆或从头DNA合成获得的。在DNA是通过合成获得的情况下，优化密码子的使用以在E.coli中进行表达。DNA的合成和优化是由GenScript,Inc.进行的。nadC基因在诱导型启动子的控制下在E.coli中进行表达。例如，将开放阅读框克隆至XhoI/NdeI-消化pET24，产生质料pET24-nadC。转化至携带T7聚合酶的菌株，诸如BL21(DE3)中允许对nadC基因进行IPTG诱导，以便促进NAR合成。
替代地，天然nadC基因的表达还能够通过将nadC基因置于诱导型启动子的控制下来进行改变。编码诱导型或组成型启动子，诸如***糖诱导型pBAD或组成型pLac启动子的DNA片断是通过PCR克隆或从头DNA合成获得的。启动子通过融合PCR在抗生素基因，例如具有卡那霉素抗性或氯霉素抗性的基因的下游融合。该标记-启动子盒将含有为20-50bps的侧翼，其与在天然nadC启动子上游的区域且与nadC开放阅读框的第一核苷酸同源。宿主菌株，例如BL21(DE3)，是通过所述的(Datsenko和Wanner，2000)pKD46的转化和诱导来进行制备以用于λ红重组工程的。所制备的细胞通过电穿孔或化学转化进行转化，且通过PCR来筛选转化体以成功掺入改变的启动子序列。
替代地，通过在操纵子中的表达来实现nadA、nadB和nadC的表达。编码E.colinadA、nadB和nadC基团的DNA片断是通过PCR克隆或从头DNA合成获得的。在DNA是通过合成获得的情况下，优化密码子的使用以在E.coli中进行表达。DNA合成和优化是由GenScript，Inc进行的。每个基因被连接至5’核糖体结合位点和3’终止子序列。操纵子在诱导型启动子的控制下在E.coli中进行表达。例如，将开放阅读框克隆至XhoI/NdeI-消化pET24，产生质料pET24-nadABC。转化至携带T7聚合酶的菌株，诸如BL21(DE3)中允许对nadABC基因进行IPTG诱导，以便促进NAR合成。
实施例3：阻断或减少从烟酸单核苷酸(NaMN)至烟酸腺嘌呤二核苷酸(NaAD)的转化
在E.coli和B.subtilis中，通过酶NadD使NaMN腺苷酸化。酶活性对于活力来说是必需的，这是因为对NAD+的所有补救和从头途径均需要该腺苷酸化活性，然而，高水平NaMN的累积对于NaR的产生来说是理想的。用诱导型启动子取代nadD基因可阻止这些竞争反应，促进NaMN的过度产生。替代地，已经表征了具有降低的酶活性的nadD基因的等位基因。用具有较低底物亲和力的等位基因取代天然nadD基因将降低NadD酶活性对NaMN水平的影响。
许多诱导型启动子已经在E.coli进行了描述且进行了良好的表征。在这个实施例中，源于E.coli的IPTG诱导型LacI启动子通过融合PCR在抗生素基因，例如具有卡那霉素抗性或氯霉素抗性的基因的下游进行融合。该标记-pLac盒将含有为20-50bps的侧翼，其与天然nadD启动子周围区域同源。宿主菌株，例如BL21(DE3)，是通过所述的(Datsenko和Wanner，2000)pKD46的转化和诱导来进行制备以用于λ红重组工程的。所制备的细胞通过电穿孔或化学转化进行转化，且通过PCR和定序来筛选转化体以成功掺入代替天然nadD启动子的Lac启动子序列。
已经描述了具有较低活性但仍然能够支持在基本培养基上生长的E.coli nadD基因的等位基因，例如N40A或T11A。这些突变用于为NaMN增加NadD的Km，从而增加细胞内的NaMN浓度。经由Stragene位点突变试剂盒将点突变在体外引入nadD基因。突变基因通过融合PCR在抗生素基因，例如具有卡那霉素抗性或氯霉素抗性的基因的下游融合。该标记-nadD*盒将含有为20-50bps的侧翼，其与天然nadD基因周围区域同源。宿主菌株，例如BL21(DE3)，是通过所述的(Datsenko和Wanner，2000)pKD46的转化和诱导来进行制备以用于λ红重组工程的。所制备的细胞通过电穿孔或化学转化进行转化，且通过PCR和定序来筛选转化体以成功掺入改变的nadD序列。
实施例4：增强从烟酸单核苷酸(NaMN)至烟酸核糖苷(NaR)的转化
通过ushA基因经由在E.coli中进行编码的周质酸性磷酸酶将分泌的NaMN去磷酸化成NaR。该实施例描述了E.coli菌株的构建，其改变了天然ushA基因的表达。
为了确保使NMN去磷酸化，将ushA基因置于强组成型启动子的控制下。编码E.coliushA基团的DNA片断是通过PCR克隆或从头DNA合成获得的。在DNA是通过合成获得的情况下，优化密码子的使用以在E.coli中进行表达。DNA的合成和优化是由GenScript,Inc.进行的。ushA基因在诱导型启动子的控制下在E.coli中进行表达。例如，将开放阅读框克隆至XhoI/NdeI-消化pET24，产生质料pET24-ushA。转化至携带T7聚合酶的菌株，诸如BL21(DE3)中允许对ushA基因进行IPTG诱导，以便促进NAR合成。
替代地，天然ushA基因的表达还能够通过将ushA基因置于诱导型启动子的控制下来进行改变。编码诱导型或组成型启动子，诸如***糖诱导型pBAD或组成型pLac启动子的DNA片断是通过PCR克隆或从头DNA合成获得的。启动子通过融合PCR在抗生素基因，例如具有卡那霉素抗性或氯霉素抗性的基因的下游融合。该标记-启动子盒将含有为20-50bps的侧翼，其与在天然ushA启动子上游的区域且与ushA开放阅读框的第一核苷酸同源。宿主菌株，例如BL21(DE3)，是通过所述的(Datsenko和Wanner，2000)pKD46的转化和诱导来进行制备以用于λ红重组工程的。所制备的细胞通过电穿孔或化学转化进行转化，且通过PCR来筛选转化体以成功掺入改变的启动子序列。
实施例5：破坏烟酰胺腺嘌呤二核苷酸(NAD)的补救途径
编码核苷磷酸化酶(deoD)、烟酸/烟酰胺激酶(nadR)的基因和编码烟酰胺核糖苷摄取性转运体(pnuC)的基因的缺失是单独地或组合地由λ红介导的重组工程实现的。抗生素基因，例如具有卡那霉素抗性或氯霉素抗性的，是使用寡核苷酸来进行PCR扩增的，该寡核苷酸具有与在天然deoD、nadR或pnuC开放阅读框的上游和下游的区域同源的为20-50bps的侧翼。替代地，这些侧翼可以在开放阅读框内，导致非功能性蛋白质的翻译。宿主菌株，例如BL21(DE3)，是通过所述的(Datsenko和Wanner，2000)pKD46的转化和诱导来进行制备以用于λ红重组工程的。所制备的细胞通过电穿孔或化学转化进行转化，且通过PCR来筛选转化体以成功破坏靶向基因。这些基因敲除可以通过如上所述用于启动子交换的抗生素基因与用于这些基因的表达盒的装配来与nadR的基因敲除(如在实施例1中公开的)或对NadD活性的修改(如在实施例2中公开的)相结合。
实施例6：细胞生长条件和方案
工程化用于生产NaR的E.coli菌株被接种在含有合适的抗生素的LB培养基中并在37℃下生长过夜。洗涤细胞在具有5％葡萄糖的M9培养基中重悬并在37℃生长3天。在适当的情况下，将IPTG添加至10-50uM的浓度以进行诱导。
实施例7：构建具有升高水平的NaR产生的B.subtilis菌株
通过长侧翼PCR(LF-PCR)构建用于nadR、deoD和pupG精确缺失的盒。用于每个基因的侧翼区是通过扩增具有表5中的引物的BS168基因组DNA(罗氏高纯度PCR模板制备试剂盒)获得的，其被设计成使得与适当的抗生素抗性基因(分别为壮观霉素、四环素和新霉素，SEQ ID No:48至50)的5’或3’区域同源的序列被掺入PCR产物中(Phusion Hot Start FlexDNA聚合酶，每个引物200nM，@95℃下进行2分钟的预变性，进行30个循环：@95℃下进行30秒；@50℃下进行20秒；@72℃下进行60秒，最后在72℃保持7分钟)。类似地，用引物对抗生素抗性基因进行扩增以掺入与5’和3’侧翼区同源的序列。PCR产物进行凝胶纯化并用于以适当的引物(表5)进行LF-PCR(Phusion Hot Start Flex DNA聚合酶，每个引物200nM，每个PCR产物150ng，@98℃下进行30秒的预变性，进行35个循环：@98℃下进行30秒；@55℃下进行30秒；@72℃下进行360秒)。LF-PCR产物被纯化并被用于B.subtilis菌株的转化。
经自然转化用LF-PCR产物转化BS168(“用于Bacillus的分子生物学方法”，1990，由C.R Harwood和S.M.Cutting.John Wiley and Sons编辑)，产生BS6209(nadR::spe)、ME479(deoD::tet)和ME492(pupG::neo)。源于ME492的基因组DNA(如上述方式制备的)被用于转化BS6209，产生ME496(nadR::spe pupG::neo)。源于ME479的基因组DNA(如上述方式制备的)被用于转化ME496，产生ME517(nadR::spe pupG::neo deoD::tet)。
实施例8：产生烟酸核糖苷
使ME517在含有50mL的种子(每升：30g Yeal浸液肉汤、5g细菌培养用酵母提取物、10g山梨糖醇、1滴Basildon 86/013消泡剂)培养基的500mL带挡板烧瓶中在37℃下生长8小时。1mL的预培养物被用于在2L带挡板烧瓶中接种300mL的种子培养基并在37℃下生长16小时。80mL的该种子发酵物被用于接种至产生容器(NBS)，其含有1.2L的分批培养基(1096gH2O、26.4g右旋糖、9.6g KH2PO4、3.6g MgSO4*7H2O、0.24g CaCl2*H2O、2.5g左旋色氨酸、0.036g MnCl2、18g MH4NO3、0.12g柠檬酸钠、0.24mL Clerol消泡剂、12mg Na2EDTA*2H2O、57.5mg ZnSO4*7H2O、3.2mg MnSO4*H2O、3.2mg CuSO4、4.8mg Na2MoO4*2H2O、CoCl2*6H2O、28mg FeSO4*7H2O)。搅拌最初被设定在400rpm，通过添加NH4OH来将pH保持在6.8，且将温度设定在37℃。在消耗分批碳期间，通过根据需要增加搅拌来将dO保持在60％以上，且在消耗分批碳之后，通过葡萄糖进料将dO保持在60％。烟酸核糖苷按下文所述的方式进行定量且在图5中示出其结果。
实施例9：检测在产生培养物中的烟酸核糖苷
产生培养物在含有20％乙腈和0.1％甲酸的水中稀释10倍且进行离心。
通过液相色谱/质谱(LCMS)分析来分析NaR和中间物。在离心(5000xg,10m)之前，用70％乙腈将20μl的肉汤在pH为9.8的水性5mM乙酸铵中按1:50进行稀释。去除上清液并将其按5μl的部分注入HILIC HPLC柱(Waters Atlantis C18,2.1x150mm)。使用从具有70％乙腈pH为9.8的5mM乙酸铵(A)至pH为9.8的5mM乙酸铵(B)线性梯度按50uL/分钟的流速将化合物洗脱20分钟以上，接下来在B中保持5分钟且在A中进行10分钟的再平衡。使用正电喷雾电离来用三重四极杆质谱仪检测洗脱化合物。该仪器以MRM模式操作以检测NaR。NAR是通过与在相同条件下注入的标准物(Sigma Aldrich)比较来进行定量的。
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<213> Escherichia coli
<400> 7
Met Ala Thr Pro His Ile Asn Ala Glu Met Gly Asp Phe Ala Asp Val
1 5 10 15
Val Leu Met Pro Gly Asp Pro Leu Arg Ala Lys Tyr Ile Ala Glu Thr
20 25 30
Phe Leu Glu Asp Ala Arg Glu Val Asn Asn Val Arg Gly Met Leu Gly
35 40 45
Phe Thr Gly Thr Tyr Lys Gly Arg Lys Ile Ser Val Met Gly His Gly
50 55 60
Met Gly Ile Pro Ser Cys Ser Ile Tyr Thr Lys Glu Leu Ile Thr Asp
65 70 75 80
Phe Gly Val Lys Lys Ile Ile Arg Val Gly Ser Cys Gly Ala Val Leu
85 90 95
Pro His Val Lys Leu Arg Asp Val Val Ile Gly Met Gly Ala Cys Thr
100 105 110
Asp Ser Lys Val Asn Arg Ile Arg Phe Lys Asp His Asp Phe Ala Ala
115 120 125
Ile Ala Asp Phe Asp Met Val Arg Asn Ala Val Asp Ala Ala Lys Ala
130 135 140
Leu Gly Ile Asp Ala Arg Val Gly Asn Leu Phe Ser Ala Asp Leu Phe
145 150 155 160
Tyr Ser Pro Asp Gly Glu Met Phe Asp Val Met Glu Lys Tyr Gly Ile
165 170 175
Leu Gly Val Glu Met Glu Ala Ala Gly Ile Tyr Gly Val Ala Ala Glu
180 185 190
Phe Gly Ala Lys Ala Leu Thr Ile Cys Thr Val Ser Asp His Ile Arg
195 200 205
Thr His Glu Gln Thr Thr Ala Ala Glu Arg Gln Thr Thr Phe Asn Asp
210 215 220
Met Ile Lys Ile Ala Leu Glu Ser Val Leu Leu Gly Asp Lys Glu
225 230 235
<210> 8
<211> 233
<212> PRT
<213> Bacillus subtilis
<400> 8
Met Ser Val His Ile Gly Ala Glu Lys Gly Gln Ile Ala Asp Thr Val
1 5 10 15
Leu Leu Pro Gly Asp Pro Leu Arg Ala Lys Phe Ile Ala Glu Thr Tyr
20 25 30
Leu Glu Asn Val Glu Cys Tyr Asn Glu Val Arg Gly Met Tyr Gly Phe
35 40 45
Thr Gly Thr Tyr Lys Gly Lys Lys Ile Ser Val Gln Gly Thr Gly Met
50 55 60
Gly Val Pro Ser Ile Ser Ile Tyr Val Asn Glu Leu Ile Gln Ser Tyr
65 70 75 80
Asp Val Gln Asn Leu Ile Arg Val Gly Ser Cys Gly Ala Ile Arg Lys
85 90 95
Asp Val Lys Val Arg Asp Val Ile Leu Ala Met Thr Ser Ser Thr Asp
100 105 110
Ser Gln Met Asn Arg Val Ala Phe Gly Ser Val Asp Phe Ala Pro Cys
115 120 125
Ala Asp Phe Glu Leu Leu Lys Asn Ala Tyr Asp Ala Ala Lys Asp Lys
130 135 140
Gly Val Pro Val Thr Val Gly Ser Val Phe Thr Ala Asp Gln Phe Tyr
145 150 155 160
Asn Asp Asp Ser Gln Ile Glu Lys Leu Ala Lys Tyr Gly Val Leu Gly
165 170 175
Val Glu Met Glu Thr Thr Ala Leu Tyr Thr Leu Ala Ala Lys His Gly
180 185 190
Arg Lys Ala Leu Ser Ile Leu Thr Val Ser Asp His Val Leu Thr Gly
195 200 205
Glu Glu Thr Thr Ala Glu Glu Arg Gln Thr Thr Phe His Asp Met Ile
210 215 220
Glu Val Ala Leu His Ser Val Ser Gln
225 230
<210> 9
<211> 191
<212> PRT
<213> Acinetobacter baylyi
<400> 9
Met Ser Pro Leu Glu Ile Phe Ala Val Ile Ile Ser Val Ile Gly Val
1 5 10 15
Ala Leu Thr Ile Lys Arg Asn Met Trp Cys Trp Gly Phe Asn Phe Leu
20 25 30
Ala Phe Ile Leu Tyr Gly Tyr Leu Phe Phe Ser Phe Lys Leu Tyr Gly
35 40 45
Glu Thr Ile Leu Gln Gly Phe Phe Ile Ile Ile Asn Phe Tyr Gly Phe
50 55 60
Tyr Tyr Trp Leu Lys Gly Lys Gln Thr Glu His Glu Ile Arg Ile Val
65 70 75 80
Ala Ile Pro Ala Lys Thr Val Ile Ile Gln Met Leu Leu Ala Ala Leu
85 90 95
Gly Gly Leu Ile Phe Gly Leu Ser Leu Lys His Phe Thr Asp Ala Ala
100 105 110
Val Pro Met Leu Asp Ser Gln Leu Ala Ala Phe Ser Leu Leu Ala Thr
115 120 125
Tyr Trp Thr Ser Arg Lys His Ile Ala Thr Trp Val Leu Trp Val Phe
130 135 140
Val Asp Ile Val Tyr Val Gly Met Phe Ile Tyr Lys Asp Leu Tyr Leu
145 150 155 160
Thr Ala Gly Leu Tyr Ala Ala Phe Val Val Met Ala Ala Phe Gly Trp
165 170 175
Trp Gln Trp Glu Gln Val Lys Arg Lys Gln Arg Ser Gly Leu Ile
180 185 190
<210> 10
<211> 230
<212> PRT
<213> Corynebacterium glutamicum
<400> 10
Met Asn Pro Ile Thr Glu Leu Leu Asp Ala Thr Leu Trp Ile Gly Gly
1 5 10 15
Val Pro Ile Leu Trp Arg Glu Ile Ile Gly Asn Val Phe Gly Leu Phe
20 25 30
Ser Ala Trp Ala Gly Met Arg Arg Ile Val Trp Ala Trp Pro Ile Gly
35 40 45
Ile Ile Gly Asn Ala Leu Leu Phe Thr Val Phe Met Gly Gly Leu Phe
50 55 60
His Thr Pro Gln Asn Leu Asp Leu Tyr Gly Gln Ala Gly Arg Gln Ile
65 70 75 80
Met Phe Ile Ile Val Ser Gly Tyr Gly Trp Tyr Gln Trp Ser Ala Ala
85 90 95
Lys Arg Arg Ala Leu Thr Pro Glu Asn Ala Val Ala Val Val Pro Arg
100 105 110
Trp Ala Ser Thr Lys Glu Arg Ala Gly Ile Val Ile Ala Ala Val Val
115 120 125
Gly Thr Leu Ser Phe Ala Trp Ile Phe Gln Ala Leu Gly Ser Trp Gly
130 135 140
Pro Trp Ala Asp Ala Trp Ile Phe Val Gly Ser Ile Leu Ala Thr Tyr
145 150 155 160
Gly Met Ala Arg Gly Trp Thr Glu Phe Trp Leu Ile Trp Ile Ala Val
165 170 175
Asp Ile Val Gly Val Pro Leu Leu Leu Thr Ala Gly Tyr Tyr Pro Ser
180 185 190
Ala Val Leu Tyr Leu Val Tyr Gly Ala Phe Val Ser Trp Gly Phe Val
195 200 205
Val Trp Leu Arg Val Gln Lys Ala Asp Lys Ala Arg Ala Leu Glu Ala
210 215 220
Gln Glu Ser Val Thr Val
225 230
<210> 11
<211> 239
<212> PRT
<213> Escherichia coli
<400> 11
Met Asp Phe Phe Ser Val Gln Asn Ile Leu Val His Ile Pro Ile Gly
1 5 10 15
Ala Gly Gly Tyr Asp Leu Ser Trp Ile Glu Ala Val Gly Thr Ile Ala
20 25 30
Gly Leu Leu Cys Ile Gly Leu Ala Ser Leu Glu Lys Ile Ser Asn Tyr
35 40 45
Phe Phe Gly Leu Ile Asn Val Thr Leu Phe Gly Ile Ile Phe Phe Gln
50 55 60
Ile Gln Leu Tyr Ala Ser Leu Leu Leu Gln Val Phe Phe Phe Ala Ala
65 70 75 80
Asn Ile Tyr Gly Trp Tyr Ala Trp Ser Arg Gln Thr Ser Gln Asn Glu
85 90 95
Ala Glu Leu Lys Ile Arg Trp Leu Pro Leu Pro Lys Ala Leu Ser Trp
100 105 110
Leu Ala Val Cys Val Val Ser Ile Gly Leu Met Thr Val Phe Ile Asn
115 120 125
Pro Val Phe Ala Phe Leu Thr Arg Val Ala Val Met Ile Met Gln Ala
130 135 140
Leu Gly Leu Gln Val Val Met Pro Glu Leu Gln Pro Asp Ala Phe Pro
145 150 155 160
Phe Trp Asp Ser Cys Met Met Val Leu Ser Ile Val Ala Met Ile Leu
165 170 175
Met Thr Arg Lys Tyr Val Glu Asn Trp Leu Leu Trp Val Ile Ile Asn
180 185 190
Val Ile Ser Val Val Ile Phe Ala Leu Gln Gly Val Tyr Ala Met Ser
195 200 205
Leu Glu Tyr Ile Ile Leu Thr Phe Ile Ala Leu Asn Gly Ser Arg Met
210 215 220
Trp Ile Asn Ser Ala Arg Glu Arg Gly Ser Arg Ala Leu Ser His
225 230 235
<210> 12
<211> 550
<212> PRT
<213> Escherichia coli
<400> 12
Met Lys Leu Leu Gln Arg Gly Val Ala Leu Ala Leu Leu Thr Thr Phe
1 5 10 15
Thr Leu Ala Ser Glu Thr Ala Leu Ala Tyr Glu Gln Asp Lys Thr Tyr
20 25 30
Lys Ile Thr Val Leu His Thr Asn Asp His His Gly His Phe Trp Arg
35 40 45
Asn Glu Tyr Gly Glu Tyr Gly Leu Ala Ala Gln Lys Thr Leu Val Asp
50 55 60
Gly Ile Arg Lys Glu Val Ala Ala Glu Gly Gly Ser Val Leu Leu Leu
65 70 75 80
Ser Gly Gly Asp Ile Asn Thr Gly Val Pro Glu Ser Asp Leu Gln Asp
85 90 95
Ala Glu Pro Asp Phe Arg Gly Met Asn Leu Val Gly Tyr Asp Ala Met
100 105 110
Ala Ile Gly Asn His Glu Phe Asp Asn Pro Leu Thr Val Leu Arg Gln
115 120 125
Gln Glu Lys Trp Ala Lys Phe Pro Leu Leu Ser Ala Asn Ile Tyr Gln
130 135 140
Lys Ser Thr Gly Glu Arg Leu Phe Lys Pro Trp Ala Leu Phe Lys Arg
145 150 155 160
Gln Asp Leu Lys Ile Ala Val Ile Gly Leu Thr Thr Asp Asp Thr Ala
165 170 175
Lys Ile Gly Asn Pro Glu Tyr Phe Thr Asp Ile Glu Phe Arg Lys Pro
180 185 190
Ala Asp Glu Ala Lys Leu Val Ile Gln Glu Leu Gln Gln Thr Glu Lys
195 200 205
Pro Asp Ile Ile Ile Ala Ala Thr His Met Gly His Tyr Asp Asn Gly
210 215 220
Glu His Gly Ser Asn Ala Pro Gly Asp Val Glu Met Ala Arg Ala Leu
225 230 235 240
Pro Ala Gly Ser Leu Ala Met Ile Val Gly Gly His Ser Gln Asp Pro
245 250 255
Val Cys Met Ala Ala Glu Asn Lys Lys Gln Val Asp Tyr Val Pro Gly
260 265 270
Thr Pro Cys Lys Pro Asp Gln Gln Asn Gly Ile Trp Ile Val Gln Ala
275 280 285
His Glu Trp Gly Lys Tyr Val Gly Arg Ala Asp Phe Glu Phe Arg Asn
290 295 300
Gly Glu Met Lys Met Val Asn Tyr Gln Leu Ile Pro Val Asn Leu Lys
305 310 315 320
Lys Lys Val Thr Trp Glu Asp Gly Lys Ser Glu Arg Val Leu Tyr Thr
325 330 335
Pro Glu Ile Ala Glu Asn Gln Gln Met Ile Ser Leu Leu Ser Pro Phe
340 345 350
Gln Asn Lys Gly Lys Ala Gln Leu Glu Val Lys Ile Gly Glu Thr Asn
355 360 365
Gly Arg Leu Glu Gly Asp Arg Asp Lys Val Arg Phe Val Gln Thr Asn
370 375 380
Met Gly Arg Leu Ile Leu Ala Ala Gln Met Asp Arg Thr Gly Ala Asp
385 390 395 400
Phe Ala Val Met Ser Gly Gly Gly Ile Arg Asp Ser Ile Glu Ala Gly
405 410 415
Asp Ile Ser Tyr Lys Asn Val Leu Lys Val Gln Pro Phe Gly Asn Val
420 425 430
Val Val Tyr Ala Asp Met Thr Gly Lys Glu Val Ile Asp Tyr Leu Thr
435 440 445
Ala Val Ala Gln Met Lys Pro Asp Ser Gly Ala Tyr Pro Gln Phe Ala
450 455 460
Asn Val Ser Phe Val Ala Lys Asp Gly Lys Leu Asn Asp Leu Lys Ile
465 470 475 480
Lys Gly Glu Pro Val Asp Pro Ala Lys Thr Tyr Arg Met Ala Thr Leu
485 490 495
Asn Phe Asn Ala Thr Gly Gly Asp Gly Tyr Pro Arg Leu Asp Asn Lys
500 505 510
Pro Gly Tyr Val Asn Thr Gly Phe Ile Asp Ala Glu Val Leu Lys Ala
515 520 525
Tyr Ile Gln Lys Ser Ser Pro Leu Asp Val Ser Val Tyr Glu Pro Lys
530 535 540
Gly Glu Val Ser Trp Gln
545 550
<210> 13
<211> 1462
<212> PRT
<213> Bacillus subtilis
<400> 13
Met Arg Ile Gln Lys Arg Arg Thr His Val Glu Asn Ile Leu Arg Ile
1 5 10 15
Leu Leu Pro Pro Ile Met Ile Leu Ser Leu Ile Leu Pro Thr Pro Pro
20 25 30
Ile His Ala Glu Glu Ser Ala Ala Pro Gln Val His Leu Ser Ile Leu
35 40 45
Ala Thr Thr Asp Ile His Ala Asn Met Met Asp Tyr Asp Tyr Tyr Ser
50 55 60
Asp Lys Glu Thr Ala Asp Phe Gly Leu Ala Arg Thr Ala Gln Leu Ile
65 70 75 80
Gln Lys His Arg Glu Gln Asn Pro Asn Thr Leu Leu Val Asp Asn Gly
85 90 95
Asp Leu Ile Gln Gly Asn Pro Leu Gly Glu Tyr Ala Val Lys Tyr Gln
100 105 110
Lys Asp Asp Ile Ile Ser Gly Thr Lys Thr His Pro Ile Ile Ser Val
115 120 125
Met Asn Ala Leu Lys Tyr Asp Ala Gly Thr Leu Gly Asn His Glu Phe
130 135 140
Asn Tyr Gly Leu Asp Phe Leu Asp Gly Thr Ile Lys Gly Ala Asp Phe
145 150 155 160
Pro Ile Val Asn Ala Asn Val Lys Thr Thr Ser Gly Glu Asn Arg Tyr
165 170 175
Thr Pro Tyr Val Ile Asn Glu Lys Thr Leu Ile Asp Glu Asn Gly Asn
180 185 190
Glu Gln Lys Val Lys Val Gly Tyr Ile Gly Phe Val Pro Pro Gln Ile
195 200 205
Met Thr Trp Asp Lys Lys Asn Leu Glu Gly Gln Val Gln Val Gln Asp
210 215 220
Ile Val Glu Ser Ala Asn Glu Thr Ile Pro Lys Met Lys Ala Glu Gly
225 230 235 240
Ala Asp Val Ile Ile Ala Leu Ala His Thr Gly Ile Glu Lys Gln Ala
245 250 255
Gln Ser Ser Gly Ala Glu Asn Ala Val Phe Asp Leu Ala Thr Lys Thr
260 265 270
Lys Gly Ile Asp Ala Ile Ile Ser Gly His Gln His Gly Leu Phe Pro
275 280 285
Ser Ala Glu Tyr Ala Gly Val Ala Gln Phe Asn Val Glu Lys Gly Thr
290 295 300
Ile Asn Gly Ile Pro Val Val Met Pro Ser Ser Trp Gly Lys Tyr Leu
305 310 315 320
Gly Val Ile Asp Leu Lys Leu Glu Lys Ala Asp Gly Ser Trp Lys Val
325 330 335
Ala Asp Ser Lys Gly Ser Ile Glu Ser Ile Ala Gly Asn Val Thr Ser
340 345 350
Arg Asn Glu Thr Val Thr Asn Thr Ile Gln Gln Thr His Gln Asn Thr
355 360 365
Leu Glu Tyr Val Arg Lys Pro Val Gly Lys Thr Glu Ala Asp Ile Asn
370 375 380
Ser Phe Phe Ala Gln Val Lys Asp Asp Pro Ser Ile Gln Ile Val Thr
385 390 395 400
Asp Ala Gln Lys Trp Tyr Ala Glu Lys Glu Met Lys Asp Thr Glu Tyr
405 410 415
Lys Asn Leu Pro Ile Leu Ser Ala Gly Ala Pro Phe Lys Ala Gly Gly
420 425 430
Arg Asn Gly Ala Asn Tyr Tyr Thr Asn Ile Pro Ala Gly Asp Leu Ala
435 440 445
Ile Lys Asn Val Gly Asp Leu Tyr Leu Tyr Asp Asn Thr Val Gln Ile
450 455 460
Val Lys Leu Thr Gly Ser Glu Val Lys Asp Trp Leu Glu Met Ser Ala
465 470 475 480
Gly Gln Phe Asn Gln Ile Asp Pro Ala Lys Gly Gly Asp Gln Ala Leu
485 490 495
Leu Asn Glu Asn Phe Arg Ser Tyr Asn Phe Asp Val Ile Asp Gly Val
500 505 510
Thr Tyr Gln Val Asp Val Thr Lys Pro Ala Lys Tyr Asn Glu Asn Gly
515 520 525
Lys Val Ile Asn Ala Asp Ser Ser Arg Ile Ile Asn Leu Ser Tyr Glu
530 535 540
Gly Lys Pro Ile Ser Pro Ser Gln Glu Phe Leu Val Val Thr Asn Asn
545 550 555 560
Tyr Arg Ala Ser Gly Gly Gly Gly Phe Pro His Leu Thr Ser Asp Lys
565 570 575
Ile Val His Gly Ser Ala Val Glu Asn Arg Gln Val Leu Met Asp Tyr
580 585 590
Ile Ile Glu Gln Lys Thr Val Asn Pro Lys Ala Asp Asn Asn Trp Ser
595 600 605
Ile Ala Pro Val Ser Gly Thr Asn Leu Thr Phe Glu Ser Ser Leu Leu
610 615 620
Ala Lys Pro Phe Ala Asp Lys Ala Asp Asp Val Ala Tyr Val Gly Lys
625 630 635 640
Ser Ala Asn Glu Gly Tyr Gly Val Tyr Lys Leu Gln Phe Asp Asp Asp
645 650 655
Ser Asn Pro Asp Pro Pro Lys Asp Gly Leu Trp Asp Leu Thr Val Met
660 665 670
His Thr Asn Asp Thr His Ala His Leu Asp Asp Ala Ala Arg Arg Met
675 680 685
Thr Lys Ile Asn Glu Val Arg Ser Glu Thr Asn His Asn Ile Leu Leu
690 695 700
Asp Ala Gly Asp Val Phe Ser Gly Asp Leu Tyr Phe Thr Lys Trp Asn
705 710 715 720
Gly Leu Ala Asp Leu Lys Met Met Asn Met Met Gly Tyr Asp Ala Met
725 730 735
Thr Phe Gly Asn His Glu Phe Asp Lys Gly Pro Thr Val Leu Ser Asp
740 745 750
Phe Leu Ser Gly Asn Ser Ala Thr Val Asp Pro Ala Asn Arg Tyr His
755 760 765
Phe Glu Ala Pro Glu Phe Pro Ile Val Ser Ala Asn Val Asp Val Ser
770 775 780
Asn Glu Pro Lys Leu Lys Ser Phe Val Lys Lys Pro Gln Thr Phe Thr
785 790 795 800
Ala Gly Glu Lys Lys Glu Ala Gly Ile His Pro Tyr Ile Leu Leu Asp
805 810 815
Val Asp Gly Glu Lys Val Ala Val Phe Gly Leu Thr Thr Glu Asp Thr
820 825 830
Ala Thr Thr Ser Ser Pro Gly Lys Ser Ile Val Phe Asn Asp Ala Phe
835 840 845
Glu Thr Ala Gln Asn Thr Val Lys Ala Ile Gln Glu Glu Glu Lys Val
850 855 860
Asn Lys Ile Ile Ala Leu Thr His Ile Gly His Asn Arg Asp Leu Glu
865 870 875 880
Leu Ala Lys Lys Val Lys Gly Ile Asp Leu Ile Ile Gly Gly His Thr
885 890 895
His Thr Leu Val Asp Lys Met Glu Val Val Asn Asn Glu Glu Pro Thr
900 905 910
Ile Val Ala Gln Ala Lys Glu Tyr Gly Gln Phe Leu Gly Arg Val Asp
915 920 925
Val Ala Phe Asp Glu Lys Gly Val Val Gln Thr Asp Lys Ser Asn Leu
930 935 940
Ser Val Leu Pro Ile Asp Glu His Thr Glu Glu Asn Pro Glu Ala Lys
945 950 955 960
Gln Glu Leu Asp Gln Phe Lys Asn Glu Leu Glu Asp Val Lys Asn Glu
965 970 975
Lys Val Gly Tyr Thr Asp Val Ala Leu Asp Gly Gln Arg Glu His Val
980 985 990
Arg Thr Lys Glu Thr Asn Leu Gly Asn Phe Ile Ala Asp Gly Met Leu
995 1000 1005
Ala Lys Ala Lys Glu Ala Ala Gly Ala Arg Ile Ala Ile Thr Asn
1010 1015 1020
Gly Gly Gly Ile Arg Ala Gly Ile Asp Lys Gly Asp Ile Thr Leu
1025 1030 1035
Gly Glu Val Leu Asn Val Met Pro Phe Gly Asn Thr Leu Tyr Val
1040 1045 1050
Ala Asp Leu Thr Gly Lys Gln Ile Lys Glu Ala Leu Glu Gln Gly
1055 1060 1065
Leu Ser Asn Val Glu Asn Gly Gly Gly Ala Phe Pro Gln Val Ala
1070 1075 1080
Gly Ile Glu Tyr Thr Phe Thr Leu Asn Asn Lys Pro Gly His Arg
1085 1090 1095
Val Leu Glu Val Lys Ile Glu Ser Pro Asn Gly Asp Lys Val Ala
1100 1105 1110
Ile Asn Thr Asp Asp Thr Tyr Arg Val Ala Thr Asn Asn Phe Val
1115 1120 1125
Gly Ala Gly Gly Asp Gly Tyr Ser Val Phe Thr Glu Ala Ser His
1130 1135 1140
Gly Glu Asp Leu Gly Tyr Val Asp Tyr Glu Ile Phe Thr Glu Gln
1145 1150 1155
Leu Lys Lys Leu Gly Asn Lys Val Ser Pro Lys Val Glu Gly Arg
1160 1165 1170
Ile Lys Glu Val Phe Leu Pro Thr Lys Gln Lys Asp Gly Ser Trp
1175 1180 1185
Thr Leu Asp Glu Asp Lys Phe Ala Ile Tyr Ala Lys Asn Ala Asn
1190 1195 1200
Thr Pro Phe Val Tyr Tyr Gly Ile His Glu Gly Ser Gln Glu Lys
1205 1210 1215
Pro Ile Asn Leu Lys Val Lys Lys Asp Gln Val Lys Leu Leu Lys
1220 1225 1230
Glu Arg Glu Ser Asp Pro Ser Leu Thr Met Phe Asn Tyr Trp Tyr
1235 1240 1245
Ser Met Lys Met Pro Met Ala Asn Leu Lys Thr Ala Asp Thr Ala
1250 1255 1260
Ile Gly Ile Lys Ser Thr Gly Glu Leu Asp Val Ser Leu Ser Asp
1265 1270 1275
Val Tyr Asp Phe Thr Val Lys Gln Lys Gly Lys Glu Ile Lys Ser
1280 1285 1290
Phe Lys Glu Pro Val Gln Leu Ser Leu Arg Met Phe Asp Ile Glu
1295 1300 1305
Glu Ala His Asn Pro Ala Ile Tyr His Val Asp Arg Lys Lys Lys
1310 1315 1320
Ala Phe Thr Lys Thr Gly His Gly Ser Val Asp Asp Asp Met Val
1325 1330 1335
Thr Gly Tyr Thr Asn His Phe Ser Glu Tyr Thr Ile Leu Asn Ser
1340 1345 1350
Gly Ser Asn Asn Lys Pro Pro Ala Phe Pro Ser Asp Gln Pro Thr
1355 1360 1365
Gly Gly Asp Asp Gly Asn His Gly Gly Gly Ser Asp Lys Pro Gly
1370 1375 1380
Gly Lys Gln Pro Thr Asp Gly Asn Gly Gly Asn Asp Thr Pro Pro
1385 1390 1395
Gly Thr Gln Pro Thr Asn Gly Ser Gly Gly Asn Gly Ser Gly Gly
1400 1405 1410
Ser Gly Thr Asp Gly Pro Ala Gly Gly Leu Leu Pro Asp Thr Ala
1415 1420 1425
Thr Ser Met Tyr Ser Ile Leu Leu Ala Gly Phe Leu Ile Ser Ala
1430 1435 1440
Leu Gly Thr Ala Met Tyr Leu His Gln Arg Arg Lys Gln Asn Arg
1445 1450 1455
Ala Asn Gln Ala
1460
<210> 14
<211> 694
<212> PRT
<213> Corynebacterium glutamicum
<400> 14
Met Lys Arg Leu Ser Arg Ala Ala Leu Ala Val Val Ala Thr Thr Ala
1 5 10 15
Val Ser Phe Ser Ala Leu Ala Val Pro Ala Phe Ala Asp Glu Ala Ser
20 25 30
Asn Val Glu Leu Asn Ile Leu Gly Val Thr Asp Phe His Gly His Ile
35 40 45
Glu Gln Lys Ala Val Lys Asp Asp Lys Gly Val Ile Thr Gly Tyr Ser
50 55 60
Glu Met Gly Ala Ser Gly Val Ala Cys Tyr Val Asp Ala Glu Arg Ala
65 70 75 80
Asp Asn Pro Asn Thr Arg Phe Ile Thr Val Gly Asp Asn Ile Gly Gly
85 90 95
Ser Pro Phe Val Ser Ser Ile Leu Lys Asp Glu Pro Thr Leu Gln Ala
100 105 110
Leu Ser Ala Ile Gly Val Asp Ala Ser Ala Leu Gly Asn His Glu Phe
115 120 125
Asp Gln Gly Tyr Ser Asp Leu Val Asn Arg Val Ser Leu Asp Gly Ser
130 135 140
Gly Ser Ala Lys Phe Pro Tyr Leu Gly Ala Asn Val Glu Gly Gly Thr
145 150 155 160
Pro Ala Pro Ala Lys Ser Glu Ile Ile Glu Met Asp Gly Val Lys Ile
165 170 175
Ala Tyr Val Gly Ala Val Thr Glu Glu Thr Ala Thr Leu Val Ser Pro
180 185 190
Ala Gly Ile Glu Gly Ile Thr Phe Thr Gly Asp Ile Asp Ala Ile Asn
195 200 205
Ala Glu Ala Asp Arg Val Ile Glu Ala Gly Glu Ala Asp Val Val Ile
210 215 220
Ala Leu Ile His Ala Glu Ala Ala Pro Thr Asp Leu Phe Ser Asn Asn
225 230 235 240
Val Asp Val Val Phe Ser Gly His Thr His Phe Asp Tyr Val Ala Glu
245 250 255
Gly Glu Ala Arg Gly Asp Lys Gln Pro Leu Val Val Ile Gln Gly His
260 265 270
Glu Tyr Gly Lys Val Ile Ser Asp Val Glu Ile Ser Tyr Asp Arg Glu
275 280 285
Ala Gly Lys Ile Thr Asn Ile Glu Ala Lys Asn Val Ser Ala Thr Asp
290 295 300
Val Val Glu Asn Cys Glu Thr Pro Asn Thr Ala Val Asp Ala Ile Val
305 310 315 320
Ala Ala Ala Val Glu Ala Ala Glu Glu Ala Gly Asn Glu Val Val Ala
325 330 335
Thr Ile Asp Asn Gly Phe Tyr Arg Gly Ala Asp Glu Glu Gly Thr Thr
340 345 350
Gly Ser Asn Arg Gly Val Glu Ser Ser Leu Ser Asn Leu Ile Ala Glu
355 360 365
Ala Gly Leu Trp Ala Val Asn Asp Ala Thr Ile Leu Asn Ala Asp Ile
370 375 380
Gly Ile Met Asn Ala Gly Gly Val Arg Ala Asp Leu Glu Ala Gly Glu
385 390 395 400
Val Thr Phe Ala Asp Ala Tyr Ala Thr Gln Asn Phe Ser Asn Thr Tyr
405 410 415
Gly Val Arg Glu Val Ser Gly Ala Gln Phe Lys Glu Ala Leu Glu Gln
420 425 430
Gln Trp Lys Glu Thr Gly Asp Arg Pro Arg Leu Ala Leu Gly Leu Ser
435 440 445
Ser Asn Val Gln Tyr Ser Tyr Asp Glu Thr Arg Glu Tyr Gly Asp Arg
450 455 460
Ile Thr His Ile Thr Phe Asn Gly Glu Pro Met Asp Met Lys Glu Thr
465 470 475 480
Tyr Arg Val Thr Gly Ser Ser Phe Leu Leu Ala Gly Gly Asp Ser Phe
485 490 495
Thr Ala Phe Ala Glu Gly Gly Pro Ile Ala Glu Thr Gly Met Val Asp
500 505 510
Ile Asp Leu Phe Asn Asn Tyr Ile Ala Ala His Pro Asp Ala Pro Ile
515 520 525
Arg Ala Asn Gln Ser Ser Val Gly Ile Ala Leu Ser Gly Pro Ala Val
530 535 540
Ala Glu Asp Gly Thr Leu Val Pro Gly Glu Glu Leu Thr Val Asp Leu
545 550 555 560
Ser Ser Leu Ser Tyr Thr Gly Pro Glu Ala Lys Pro Thr Thr Val Glu
565 570 575
Val Thr Val Gly Thr Glu Lys Lys Thr Ala Asp Val Asp Asn Thr Ile
580 585 590
Val Pro Gln Phe Asp Ser Thr Gly Lys Ala Thr Val Thr Leu Thr Val
595 600 605
Pro Glu Gly Ala Thr Ser Val Lys Ile Ala Thr Asp Asn Gly Thr Thr
610 615 620
Phe Glu Leu Pro Val Thr Val Asn Gly Glu Gly Asn Asn Asp Asp Asp
625 630 635 640
Asp Asp Lys Glu Gln Gln Ser Ser Gly Ser Ser Asp Ala Gly Ser Leu
645 650 655
Val Ala Val Leu Gly Val Leu Gly Ala Leu Gly Gly Leu Val Ala Phe
660 665 670
Phe Leu Asn Ser Ala Gln Gly Ala Pro Phe Leu Ala Gln Leu Gln Ala
675 680 685
Met Phe Ala Gln Phe Met
690
<210> 15
<211> 400
<212> PRT
<213> Escherichia coli
<400> 15
Met Thr Gln Phe Ala Ser Pro Val Leu His Ser Leu Leu Asp Thr Asp
1 5 10 15
Ala Tyr Lys Leu His Met Gln Gln Ala Val Phe His His Tyr Tyr Asp
20 25 30
Val His Val Ala Ala Glu Phe Arg Cys Arg Gly Asp Asp Leu Leu Gly
35 40 45
Ile Tyr Ala Asp Ala Ile Arg Glu Gln Val Gln Ala Met Gln His Leu
50 55 60
Arg Leu Gln Asp Asp Glu Tyr Gln Trp Leu Ser Ala Leu Pro Phe Phe
65 70 75 80
Lys Ala Asp Tyr Leu Asn Trp Leu Arg Glu Phe Arg Phe Asn Pro Glu
85 90 95
Gln Val Thr Val Ser Asn Asp Asn Gly Lys Leu Asp Ile Arg Leu Ser
100 105 110
Gly Pro Trp Arg Glu Val Ile Leu Trp Glu Val Pro Leu Leu Ala Val
115 120 125
Ile Ser Glu Met Val His Arg Tyr Arg Ser Pro Gln Ala Asp Val Ala
130 135 140
Gln Ala Leu Asp Thr Leu Glu Ser Lys Leu Val Asp Phe Ser Ala Leu
145 150 155 160
Thr Ala Gly Leu Asp Met Ser Arg Phe His Leu Met Asp Phe Gly Thr
165 170 175
Arg Arg Arg Phe Ser Arg Glu Val Gln Glu Thr Ile Val Lys Arg Leu
180 185 190
Gln Gln Glu Ser Trp Phe Val Gly Thr Ser Asn Tyr Asp Leu Ala Arg
195 200 205
Arg Leu Ser Leu Thr Pro Met Gly Thr Gln Ala His Glu Trp Phe Gln
210 215 220
Ala His Gln Gln Ile Ser Pro Asp Leu Ala Asn Ser Gln Arg Ala Ala
225 230 235 240
Leu Ala Ala Trp Leu Glu Glu Tyr Pro Asp Gln Leu Gly Ile Ala Leu
245 250 255
Thr Asp Cys Ile Thr Met Asp Ala Phe Leu Arg Asp Phe Gly Val Glu
260 265 270
Phe Ala Ser Arg Tyr Gln Gly Leu Arg His Asp Ser Gly Asp Pro Val
275 280 285
Glu Trp Gly Glu Lys Ala Ile Ala His Tyr Glu Lys Leu Gly Ile Asp
290 295 300
Pro Gln Ser Lys Thr Leu Val Phe Ser Asp Asn Leu Asp Leu Arg Lys
305 310 315 320
Ala Val Glu Leu Tyr Arg His Phe Ser Ser Arg Val Gln Leu Ser Phe
325 330 335
Gly Ile Gly Thr Arg Leu Thr Cys Asp Ile Pro Gln Val Lys Pro Leu
340 345 350
Asn Ile Val Ile Lys Leu Val Glu Cys Asn Gly Lys Pro Val Ala Lys
355 360 365
Leu Ser Asp Ser Pro Gly Lys Thr Ile Cys His Asp Lys Ala Phe Val
370 375 380
Arg Ala Leu Arg Lys Ala Phe Asp Leu Pro His Ile Lys Lys Ala Ser
385 390 395 400
<210> 16
<211> 490
<212> PRT
<213> Bacillus subtilis
<400> 16
Met Leu Glu Tyr Gly Phe Lys Asp Asp Ser Leu Ser Leu His Thr Asp
1 5 10 15
Leu Tyr Gln Ile Asn Met Ala Glu Thr Tyr Trp Arg Asp Gly Ile His
20 25 30
Glu Lys Lys Ala Ile Phe Glu Leu Phe Phe Arg Arg Leu Pro Phe Glu
35 40 45
Asn Gly Tyr Ala Val Phe Ala Gly Leu Glu Lys Ala Ile Glu Tyr Leu
50 55 60
Glu Asn Phe Lys Phe Thr Asp Ser Asp Leu Ser Tyr Leu Gln Asp Glu
65 70 75 80
Leu Gly Tyr His Glu Asp Phe Ile Glu Tyr Leu Arg Gly Leu Ser Phe
85 90 95
Thr Gly Ser Leu Tyr Ser Met Lys Glu Gly Glu Leu Val Phe Asn Asn
100 105 110
Glu Pro Ile Met Arg Val Glu Ala Pro Leu Val Glu Ala Gln Leu Ile
115 120 125
Glu Thr Ala Leu Leu Asn Ile Val Asn Tyr Gln Thr Leu Ile Ala Thr
130 135 140
Lys Ala Ala Arg Ile Lys Gly Val Ile Gly Asp Glu Val Ala Leu Glu
145 150 155 160
Phe Gly Thr Arg Arg Ala His Glu Met Asp Ala Ala Met Trp Gly Ala
165 170 175
Arg Ala Ala Leu Ile Gly Gly Phe Ser Ala Thr Ser Asn Val Arg Ala
180 185 190
Gly Lys Arg Phe Asn Ile Pro Val Ser Gly Thr His Ala His Ala Leu
195 200 205
Val Gln Ala Tyr Arg Asp Glu Tyr Thr Ala Phe Lys Lys Tyr Ala Glu
210 215 220
Thr His Lys Asp Cys Val Phe Leu Val Asp Thr Tyr Asp Thr Leu Arg
225 230 235 240
Ser Gly Met Pro Asn Ala Ile Arg Val Ala Lys Glu Phe Gly Asp Arg
245 250 255
Ile Asn Phe Ile Gly Ile Arg Leu Asp Ser Gly Asp Leu Ala Tyr Leu
260 265 270
Ser Lys Lys Ala Arg Lys Met Leu Asp Glu Ala Gly Phe Thr Asp Ala
275 280 285
Lys Val Ile Ala Ser Ser Asp Leu Asp Glu His Thr Ile Met Asn Leu
290 295 300
Lys Ala Gln Gly Ala Arg Ile Asp Val Trp Gly Val Gly Thr Lys Leu
305 310 315 320
Ile Thr Ala Tyr Asp Gln Pro Ala Leu Gly Ala Val Tyr Lys Leu Val
325 330 335
Ala Ile Glu Glu Asp Gly Lys Met Val Asp Thr Ile Lys Ile Ser Ser
340 345 350
Asn Pro Glu Lys Val Thr Thr Pro Gly Arg Lys Lys Val Tyr Arg Ile
355 360 365
Ile Asn Gln Ser Asn His His Ser Glu Gly Asp Tyr Ile Ala Leu Tyr
370 375 380
Asp Glu Gln Val Asn Asp Gln Lys Arg Leu Arg Met Phe His Pro Val
385 390 395 400
His Thr Phe Ile Ser Lys Phe Val Thr Asn Phe Tyr Ala Lys Asp Leu
405 410 415
His Glu Leu Ile Phe Glu Lys Gly Ile Leu Cys Tyr Gln Asn Pro Glu
420 425 430
Ile Ser Asp Ile Gln Gln Tyr Val Gln Asp Asn Leu Ser Leu Leu Trp
435 440 445
Glu Glu Tyr Lys Arg Ile Ser Lys Pro Glu Glu Tyr Pro Val Asp Leu
450 455 460
Ser Glu Asp Cys Trp Ser Asn Lys Met Gln Arg Ile His Glu Val Lys
465 470 475 480
Ser Arg Ile Glu Glu Glu Leu Glu Glu Glu
485 490
<210> 17
<211> 446
<212> PRT
<213> Corynebacterium glutamicum
<400> 17
Met Asn Thr Asn Pro Ser Glu Phe Ser Ser Asn Arg Ser Thr Ala Leu
1 5 10 15
Leu Thr Asp Lys Tyr Glu Leu Thr Met Leu Gln Ala Ala Leu Ala Asp
20 25 30
Gly Ser Ala Glu Arg Pro Ser Thr Phe Glu Val Phe Ser Arg Arg Leu
35 40 45
Pro Asn Glu Arg Arg Tyr Gly Val Val Ala Gly Thr Ala Arg Val Leu
50 55 60
Lys Ala Ile Arg Asp Phe Val Phe Thr Glu Glu Gln Leu Ala Asp Leu
65 70 75 80
Asp Phe Leu Asp Asp Arg Thr Leu Glu Tyr Leu Arg Asn Tyr Arg Phe
85 90 95
Thr Gly Gln Val Asp Gly Tyr Arg Glu Gly Glu Ile Tyr Phe Pro Gln
100 105 110
Ser Pro Leu Leu Thr Val Arg Gly Thr Phe Ala Glu Cys Val Ile Leu
115 120 125
Glu Thr Val Ile Leu Ser Ile Met Asn Ala Asp Ser Ala Val Ala Ser
130 135 140
Ala Ala Ala Arg Met Val Thr Ala Ala Asp Gly Arg Pro Ile Ile Glu
145 150 155 160
Met Gly Ser Arg Arg Thr His Glu Tyr Ser Ala Val Thr Ala Ser Arg
165 170 175
Ala Ala Tyr Leu Ala Gly Phe Ser Thr Thr Ser Asn Leu Glu Ala Ala
180 185 190
Tyr Arg Tyr Gly Ile Pro Ala Ser Gly Thr Ser Ala His Ala Trp Thr
195 200 205
Leu Leu His Ile Asn Asp Asp Gly Thr Pro Asn Glu Ala Ala Ala Phe
210 215 220
Lys Ala Gln Val Glu Ser Leu Gly Val Asp Thr Thr Leu Leu Val Asp
225 230 235 240
Thr Tyr Asp Ile Thr Gln Gly Val Ala Thr Ala Ile Glu Val Ala Gly
245 250 255
Pro Asp Leu Gly Gly Val Arg Ile Asp Ser Gly Asp Leu Gly Val Leu
260 265 270
Ala Arg Lys Val Arg Lys Gln Leu Asp Asp Leu Asn Ala His Asn Thr
275 280 285
Lys Ile Val Val Ser Ser Asp Leu Asp Glu Phe Ala Ile Ala Gly Leu
290 295 300
Arg Gly Glu Pro Val Asp Val Phe Gly Val Gly Thr Ser Val Val Thr
305 310 315 320
Gly Ser Gly Ala Pro Thr Ala Gly Leu Val Tyr Lys Ile Val Glu Val
325 330 335
Ala Gly His Pro Val Ala Lys Arg Ser Arg Asn Lys Glu Ser Tyr Gly
340 345 350
Gly Gly Lys Lys Ala Val Arg Thr His Arg Lys Ser Gly Thr Ala Ile
355 360 365
Glu Glu Ile Val Tyr Pro Phe Asn Ala Glu Ala Pro Asp Thr Gly Lys
370 375 380
Leu Asp Thr Leu Ser Leu Thr Ile Pro Leu Met Arg Asp Gly Glu Ile
385 390 395 400
Val Pro Gly Leu Pro Thr Leu Glu Asp Ser Arg Ala Tyr Leu Ala Lys
405 410 415
Gln Leu Val Ser Leu Pro Trp Glu Gly Leu Ala Leu Ser Arg Asp Glu
420 425 430
Pro Val Leu His Thr Arg Phe Val Gly Phe Pro Pro Ala Ala
435 440 445
<210> 18
<211> 271
<212> PRT
<213> Bacillus subtilis
<400> 18
Met Lys Asp Arg Ile Glu Arg Ala Ala Ala Phe Ile Lys Gln Asn Leu
1 5 10 15
Pro Glu Ser Pro Lys Ile Gly Leu Ile Leu Gly Ser Gly Leu Gly Ile
20 25 30
Leu Ala Asp Glu Ile Glu Asn Pro Val Lys Leu Lys Tyr Glu Asp Ile
35 40 45
Pro Glu Phe Pro Val Ser Thr Val Glu Gly His Ala Gly Gln Leu Val
50 55 60
Leu Gly Thr Leu Glu Gly Val Ser Val Ile Ala Met Gln Gly Arg Phe
65 70 75 80
His Phe Tyr Glu Gly Tyr Ser Met Glu Lys Val Thr Phe Pro Val Arg
85 90 95
Val Met Lys Ala Leu Gly Val Glu Ala Leu Ile Val Thr Asn Ala Ala
100 105 110
Gly Gly Val Asn Thr Glu Phe Arg Ala Gly Asp Leu Met Ile Ile Thr
115 120 125
Asp His Ile Asn Phe Met Gly Thr Asn Pro Leu Ile Gly Pro Asn Glu
130 135 140
Ala Asp Phe Gly Ala Arg Phe Pro Asp Met Ser Ser Ala Tyr Asp Lys
145 150 155 160
Asp Leu Ser Ser Leu Ala Glu Lys Ile Ala Lys Asp Leu Asn Ile Pro
165 170 175
Ile Gln Lys Gly Val Tyr Thr Ala Val Thr Gly Pro Ser Tyr Glu Thr
180 185 190
Pro Ala Glu Val Arg Phe Leu Arg Thr Met Gly Ser Asp Ala Val Gly
195 200 205
Met Ser Thr Val Pro Glu Val Ile Val Ala Asn His Ala Gly Met Arg
210 215 220
Val Leu Gly Ile Ser Cys Ile Ser Asn Ala Ala Ala Gly Ile Leu Asp
225 230 235 240
Gln Pro Leu Ser His Asp Glu Val Met Glu Val Thr Glu Lys Val Lys
245 250 255
Ala Gly Phe Leu Lys Leu Val Lys Ala Ile Val Ala Gln Tyr Glu
260 265 270
<210> 19
<211> 433
<212> PRT
<213> Bacillus subtilis
<400> 19
Met Arg Met Val Asp Ile Ile Ile Lys Lys Gln Asn Gly Lys Glu Leu
1 5 10 15
Thr Thr Glu Glu Ile Gln Phe Phe Val Asn Gly Tyr Thr Asp Gly Ser
20 25 30
Ile Pro Asp Tyr Gln Ala Ser Ala Leu Ala Met Ala Ile Phe Phe Gln
35 40 45
Asp Met Ser Asp Arg Glu Arg Ala Asp Leu Thr Met Ala Met Val Asn
50 55 60
Ser Gly Glu Thr Ile Asp Leu Ser Ala Ile Glu Gly Ile Lys Val Asp
65 70 75 80
Lys His Ser Thr Gly Gly Val Gly Asp Thr Thr Thr Leu Val Leu Ala
85 90 95
Pro Leu Val Ala Ala Leu Asp Val Pro Val Ala Lys Met Ser Gly Arg
100 105 110
Gly Leu Gly His Thr Gly Gly Thr Ile Asp Lys Leu Glu Ala Ile Met
115 120 125
Gly Phe His Val Glu Leu Thr Lys Asp Glu Phe Ile Lys Leu Val Asn
130 135 140
Arg Asp Lys Val Ala Val Ile Gly Gln Ser Gly Asn Leu Thr Pro Ala
145 150 155 160
Asp Lys Lys Leu Tyr Ala Leu Arg Asp Val Thr Gly Thr Val Asn Ser
165 170 175
Ile Pro Leu Ile Ala Ser Ser Ile Met Ser Lys Lys Ile Ala Ala Gly
180 185 190
Ala Asp Ala Ile Val Leu Asp Val Lys Thr Gly Ala Gly Ala Phe Met
195 200 205
Lys Thr Glu Glu Asp Ala Ala Glu Leu Ala Lys Ala Met Val Arg Ile
210 215 220
Gly Asn Asn Val Gly Arg Gln Thr Met Ala Val Ile Ser Asp Met Ser
225 230 235 240
Gln Pro Leu Gly Phe Ala Ile Gly Asn Ala Leu Glu Val Lys Glu Ala
245 250 255
Ile Asp Thr Leu Lys Gly Glu Gly Pro Glu Asp Leu His Glu Leu Val
260 265 270
Leu Thr Leu Gly Ser Gln Met Val Val Leu Ala Lys Lys Ala Asp Thr
275 280 285
Leu Asp Glu Ala Arg Ala Lys Leu Glu Glu Val Met Lys Asn Gly Lys
290 295 300
Ala Leu Glu Lys Phe Lys Asp Phe Leu Lys Asn Gln Gly Gly Asp Ser
305 310 315 320
Ser Ile Val Asp Asp Pro Ser Lys Leu Pro Gln Ala Ala Tyr Gln Ile
325 330 335
Asp Val Pro Ala Lys Glu Ala Gly Val Val Ser Glu Ile Val Ala Asp
340 345 350
Glu Ile Gly Val Ala Ala Met Leu Leu Gly Ala Gly Arg Ala Thr Lys
355 360 365
Glu Asp Glu Ile Asp Leu Ala Val Gly Ile Met Leu Arg Lys Lys Val
370 375 380
Gly Asp Lys Val Glu Lys Gly Glu Pro Leu Val Thr Leu Tyr Ala Asn
385 390 395 400
Arg Glu Asn Val Asp Glu Val Ile Ala Lys Val Tyr Asp Asn Ile Arg
405 410 415
Ile Ala Ala Glu Ala Lys Ala Pro Lys Leu Ile His Thr Leu Ile Thr
420 425 430
Glu
<210> 20
<211> 165
<212> PRT
<213> Escherichia coli
<400> 20
Met Thr Asp Ser Glu Leu Met Gln Leu Ser Glu Gln Val Gly Gln Ala
1 5 10 15
Leu Lys Ala Arg Gly Ala Thr Val Thr Thr Ala Glu Ser Cys Thr Gly
20 25 30
Gly Trp Val Ala Lys Val Ile Thr Asp Ile Ala Gly Ser Ser Ala Trp
35 40 45
Phe Glu Arg Gly Phe Val Thr Tyr Ser Asn Glu Ala Lys Ala Gln Met
50 55 60
Ile Gly Val Arg Glu Glu Thr Leu Ala Gln His Gly Ala Val Ser Glu
65 70 75 80
Pro Val Val Val Glu Met Ala Ile Gly Ala Leu Lys Ala Ala Arg Ala
85 90 95
Asp Tyr Ala Val Ser Ile Ser Gly Ile Ala Gly Pro Asp Gly Gly Ser
100 105 110
Glu Glu Lys Pro Val Gly Thr Val Trp Phe Ala Phe Ala Thr Ala Arg
115 120 125
Gly Glu Gly Ile Thr Arg Arg Glu Cys Phe Ser Gly Asp Arg Asp Ala
130 135 140
Val Arg Arg Gln Ala Thr Ala Tyr Ala Leu Gln Thr Leu Trp Gln Gln
145 150 155 160
Phe Leu Gln Asn Thr
165
<210> 21
<211> 416
<212> PRT
<213> Bacillus subtilis
<400> 21
Met Glu Phe Pro Lys Lys Ala Glu Ile Ile Ala Val Gly Ser Glu Leu
1 5 10 15
Leu Leu Gly Gln Ile Ala Asn Thr Asn Ala Gln Phe Ile Ser Lys Gln
20 25 30
Leu Ala Glu Ile Gly Val His Val Phe Tyr His Thr Ala Val Gly Asp
35 40 45
Asn Pro Glu Arg Leu Lys Gln Val Ile Arg Ile Ala Glu Glu Arg Ser
50 55 60
Asp Phe Ile Ile Phe Ser Gly Gly Leu Gly Pro Thr Lys Asp Asp Leu
65 70 75 80
Thr Lys Glu Thr Ile Ala Asn Thr Leu Gly Arg Pro Leu Val Leu Asn
85 90 95
Asp Glu Ala Phe Gln Ser Ile Glu Asp Tyr Pro Lys Arg Thr Lys Arg
100 105 110
Thr Met Ser Pro Asn Asn Arg Lys Gln Ala Leu Val Ile Glu Gly Ser
115 120 125
Asp Val Leu Ala Asn His Phe Gly Met Ala Pro Gly Met Leu Thr Glu
130 135 140
His Glu Ser Arg Tyr Tyr Met Leu Leu Pro Gly Pro Pro Ser Glu Leu
145 150 155 160
Arg Pro Met Phe Glu Asn Glu Ala Lys Pro Leu Leu Leu Lys Lys Met
165 170 175
Gly Ser Asn Glu Lys Ile Val Ser Thr Val Leu Arg Phe Phe Gly Ile
180 185 190
Gly Glu Ser Gln Leu Glu Pro Asp Leu Glu Asp Ile Ile Asp Ala Gln
195 200 205
Thr Asn Pro Thr Ile Ala Pro Leu Ala Ala Asp Gly Glu Val Thr Leu
210 215 220
Arg Leu Thr Ala Lys His Ala Asp Glu Lys Glu Thr Glu Arg Leu Leu
225 230 235 240
Lys Glu Thr Glu Ala Val Ile Leu Glu Arg Val Gly Glu Phe Phe Tyr
245 250 255
Gly Tyr Asp Asp Thr Ser Leu Val Lys Glu Leu Ser Ile Ala Cys Lys
260 265 270
Glu Lys Gly Ile Thr Ile Ser Ala Ala Glu Ser Phe Thr Gly Gly Leu
275 280 285
Phe Ser Glu Trp Leu Thr Asp His Ser Gly Ala Ser Lys Leu Phe Ala
290 295 300
Gly Gly Val Val Cys Tyr Thr Asn Asp Val Lys Gln Asn Val Leu Gly
305 310 315 320
Val Lys Lys Glu Thr Leu Asp Arg Phe Gly Ala Val Ser Lys Glu Cys
325 330 335
Ala Ser Glu Leu Ala Lys Gly Val Gln Lys Leu Thr Gly Ser Asp Ile
340 345 350
Gly Ile Ser Phe Thr Gly Val Ala Gly Pro Asp Ala Gln Glu Gly His
355 360 365
Glu Pro Gly His Val Phe Ile Gly Ile Ser Ala Asn Gly Lys Glu Glu
370 375 380
Val His Glu Phe His Phe Ala Gly Ser Arg Thr Gly Ile Arg Lys Arg
385 390 395 400
Gly Ala Lys Tyr Gly Cys His Leu Ile Leu Lys Leu Leu Glu Gln Lys
405 410 415
<210> 22
<211> 172
<212> PRT
<213> Corynebacterium glutamicum
<400> 22
Met Ser Glu Asn Leu Ala Gly Arg Val Val Glu Leu Leu Lys Ser Arg
1 5 10 15
Gly Glu Thr Leu Ala Phe Cys Glu Ser Leu Thr Ala Gly Leu Ala Ser
20 25 30
Ala Thr Ile Ala Glu Ile Pro Gly Ala Ser Val Val Leu Lys Gly Gly
35 40 45
Leu Val Thr Tyr Ala Thr Glu Leu Lys Val Ala Leu Ala Gly Val Pro
50 55 60
Gln Glu Leu Ile Asp Ala His Gly Val Val Ser Pro Gln Cys Ala Arg
65 70 75 80
Ala Met Ala Thr Gly Ala Ala His Arg Cys Gln Ala Asp Trp Ala Val
85 90 95
Ser Leu Thr Gly Val Ala Gly Pro Ser Lys Gln Asp Gly His Pro Val
100 105 110
Gly Glu Val Trp Ile Gly Val Ala Gly Pro Ala His Phe Gly Ala Ser
115 120 125
Gly Thr Ile Asp Ala Tyr Arg Ala Phe Glu Ser Glu Gln Gln Val Ile
130 135 140
Leu Ala Glu Leu Gly Arg His His Ile Arg Glu Ser Ala Val Gln Gln
145 150 155 160
Ser Phe Arg Leu Leu Ile Asp His Ile Glu Ser Gln
165 170
<210> 23
<211> 347
<212> PRT
<213> Escherichia coli
<400> 23
Met Ser Val Met Phe Asp Pro Asp Thr Ala Ile Tyr Pro Phe Pro Pro
1 5 10 15
Lys Pro Thr Pro Leu Ser Ile Asp Glu Lys Ala Tyr Tyr Arg Glu Lys
20 25 30
Ile Lys Arg Leu Leu Lys Glu Arg Asn Ala Val Met Val Ala His Tyr
35 40 45
Tyr Thr Asp Pro Glu Ile Gln Gln Leu Ala Glu Glu Thr Gly Gly Cys
50 55 60
Ile Ser Asp Ser Leu Glu Met Ala Arg Phe Gly Ala Lys His Pro Ala
65 70 75 80
Ser Thr Leu Leu Val Ala Gly Val Arg Phe Met Gly Glu Thr Ala Lys
85 90 95
Ile Leu Ser Pro Glu Lys Thr Ile Leu Met Pro Thr Leu Gln Ala Glu
100 105 110
Cys Ser Leu Asp Leu Gly Cys Pro Val Glu Glu Phe Asn Ala Phe Cys
115 120 125
Asp Ala His Pro Asp Arg Thr Val Val Val Tyr Ala Asn Thr Ser Ala
130 135 140
Ala Val Lys Ala Arg Ala Asp Trp Val Val Thr Ser Ser Ile Ala Val
145 150 155 160
Glu Leu Ile Asp His Leu Asp Ser Leu Gly Glu Lys Ile Ile Trp Ala
165 170 175
Pro Asp Lys His Leu Gly Arg Tyr Val Gln Lys Gln Thr Gly Gly Asp
180 185 190
Ile Leu Cys Trp Gln Gly Ala Cys Ile Val His Asp Glu Phe Lys Thr
195 200 205
Gln Ala Leu Thr Arg Leu Gln Glu Glu Tyr Pro Asp Ala Ala Ile Leu
210 215 220
Val His Pro Glu Ser Pro Gln Ala Ile Val Asp Met Ala Asp Ala Val
225 230 235 240
Gly Ser Thr Ser Gln Leu Ile Ala Ala Ala Lys Thr Leu Pro His Gln
245 250 255
Arg Leu Ile Val Ala Thr Asp Arg Gly Ile Phe Tyr Lys Met Gln Gln
260 265 270
Ala Val Pro Asp Lys Glu Leu Leu Glu Ala Pro Thr Ala Gly Glu Gly
275 280 285
Ala Thr Cys Arg Ser Cys Ala His Cys Pro Trp Met Ala Met Asn Gly
290 295 300
Leu Gln Ala Ile Ala Glu Ala Leu Glu Gln Glu Gly Ser Asn His Glu
305 310 315 320
Val His Val Asp Glu Arg Leu Arg Glu Arg Ala Leu Val Pro Leu Asn
325 330 335
Arg Met Leu Asp Phe Ala Ala Thr Leu Arg Gly
340 345
<210> 24
<211> 368
<212> PRT
<213> Bacillus subtilis
<400> 24
Met Ser Ile Leu Asp Val Ile Lys Gln Ser Asn Asp Met Met Pro Glu
1 5 10 15
Ser Tyr Lys Glu Leu Ser Arg Lys Asp Met Glu Thr Arg Val Ala Ala
20 25 30
Ile Lys Lys Lys Phe Gly Ser Arg Leu Phe Ile Pro Gly His His Tyr
35 40 45
Gln Lys Asp Glu Val Ile Gln Phe Ala Asp Gln Thr Gly Asp Ser Leu
50 55 60
Gln Leu Ala Gln Val Ala Glu Lys Asn Lys Glu Ala Asp Tyr Ile Val
65 70 75 80
Phe Cys Gly Val His Phe Met Ala Glu Thr Ala Asp Met Leu Thr Ser
85 90 95
Glu Gln Gln Thr Val Val Leu Pro Asp Met Arg Ala Gly Cys Ser Met
100 105 110
Ala Asp Met Ala Asp Met Gln Gln Thr Asn Arg Ala Trp Lys Lys Leu
115 120 125
Gln His Ile Phe Gly Asp Thr Ile Ile Pro Leu Thr Tyr Val Asn Ser
130 135 140
Thr Ala Glu Ile Lys Ala Phe Val Gly Lys His Gly Gly Ala Thr Val
145 150 155 160
Thr Ser Ser Asn Ala Lys Lys Val Leu Glu Trp Ala Phe Thr Gln Lys
165 170 175
Lys Arg Ile Leu Phe Leu Pro Asp Gln His Leu Gly Arg Asn Thr Ala
180 185 190
Tyr Asp Leu Gly Ile Ala Leu Glu Asp Met Ala Val Trp Asp Pro Met
195 200 205
Lys Asp Glu Leu Val Ala Glu Ser Gly His Thr Asn Val Lys Val Ile
210 215 220
Leu Trp Lys Gly His Cys Ser Val His Glu Lys Phe Thr Thr Lys Asn
225 230 235 240
Ile His Asp Met Arg Glu Arg Asp Pro Asp Ile Gln Ile Ile Val His
245 250 255
Pro Glu Cys Ser His Glu Val Val Thr Leu Ser Asp Asp Asn Gly Ser
260 265 270
Thr Lys Tyr Ile Ile Asp Thr Ile Asn Gln Ala Pro Ala Gly Ser Lys
275 280 285
Trp Ala Ile Gly Thr Glu Met Asn Leu Val Gln Arg Ile Ile His Glu
290 295 300
His Pro Asp Lys Gln Ile Glu Ser Leu Asn Pro Asp Met Cys Pro Cys
305 310 315 320
Leu Thr Met Asn Arg Ile Asp Leu Pro His Leu Leu Trp Ser Leu Glu
325 330 335
Gln Ile Glu Lys Gly Glu Pro Ser Gly Val Ile Lys Val Pro Lys Ala
340 345 350
Ile Gln Glu Asp Ala Leu Leu Ala Leu Asn Arg Met Leu Ser Ile Thr
355 360 365
<210> 25
<211> 428
<212> PRT
<213> Corynebacterium glutamicum
<400> 25
Met Thr Thr Ser Ile Thr Pro Ser Val Asn Leu Ala Leu Lys Asn Ala
1 5 10 15
Asn Ser Cys Asn Ser Glu Leu Lys Asp Gly Pro Trp Phe Leu Asp Gln
20 25 30
Pro Gly Met Pro Asp Val Tyr Gly Pro Gly Ala Ser Gln Asn Asp Pro
35 40 45
Ile Pro Ala His Ala Pro Arg Gln Gln Val Leu Pro Glu Glu Tyr Gln
50 55 60
Arg Ala Ser Asp Asp Glu Leu His Arg Arg Ile Arg Glu Ala Lys Asp
65 70 75 80
Thr Leu Gly Asp Lys Val Val Ile Leu Gly His Phe Tyr Gln Arg Asp
85 90 95
Glu Val Ile Gln His Ala Asp Phe Val Gly Asp Ser Phe Gln Leu Ala
100 105 110
Arg Ala Ala Lys Thr Arg Pro Glu Ala Glu Ala Ile Val Phe Cys Gly
115 120 125
Val His Phe Met Ala Glu Thr Ala Asp Leu Leu Ser Thr Asp Glu Gln
130 135 140
Ser Val Ile Leu Pro Asn Leu Ala Ala Gly Cys Ser Met Ala Asp Met
145 150 155 160
Ala Asp Leu Asp Ser Val Glu Asp Cys Trp Glu Gln Leu Thr Ser Ile
165 170 175
Tyr Gly Asp Asp Thr Leu Ile Pro Val Thr Tyr Met Asn Ser Ser Ala
180 185 190
Ala Leu Lys Gly Phe Val Gly Glu His Gly Gly Ile Val Cys Thr Ser
195 200 205
Ser Asn Ala Arg Ser Val Leu Glu Trp Ala Phe Glu Arg Gly Gln Arg
210 215 220
Val Leu Phe Phe Pro Asp Gln His Leu Gly Arg Asn Thr Ala Lys Ala
225 230 235 240
Met Gly Ile Gly Ile Asp Gln Met Pro Leu Trp Asn Pro Asn Lys Pro
245 250 255
Leu Gly Gly Asn Thr Val Ser Glu Leu Glu Asn Ala Lys Val Leu Leu
260 265 270
Trp His Gly Phe Cys Ser Val His Lys Arg Phe Thr Val Glu Gln Ile
275 280 285
Asn Lys Ala Arg Ala Glu Tyr Pro Asp Val His Val Ile Val His Pro
290 295 300
Glu Ser Pro Met Pro Val Val Asp Ala Ala Asp Ser Ser Gly Ser Thr
305 310 315 320
Asp Phe Ile Val Lys Ala Ile Gln Ala Ala Pro Ala Gly Ser Thr Phe
325 330 335
Ala Ile Gly Thr Glu Ile Asn Leu Val Gln Arg Leu Ala Ala Gln Tyr
340 345 350
Pro Gln His Thr Ile Phe Cys Leu Asp Pro Val Ile Cys Pro Cys Ser
355 360 365
Thr Met Tyr Arg Ile His Pro Gly Tyr Leu Ala Trp Ala Leu Glu Glu
370 375 380
Leu Val Ala Gly Asn Val Ile Asn Gln Ile Ser Val Ser Glu Ser Val
385 390 395 400
Ala Ala Pro Ala Arg Val Ala Leu Glu Arg Met Leu Ser Val Val Pro
405 410 415
Ala Ala Pro Val Thr Pro Ser Ser Ser Lys Asp Ala
420 425
<210> 26
<211> 540
<212> PRT
<213> Escherichia coli
<400> 26
Met Asn Thr Leu Pro Glu His Ser Cys Asp Val Leu Ile Ile Gly Ser
1 5 10 15
Gly Ala Ala Gly Leu Ser Leu Ala Leu Arg Leu Ala Asp Gln His Gln
20 25 30
Val Ile Val Leu Ser Lys Gly Pro Val Thr Glu Gly Ser Thr Phe Tyr
35 40 45
Ala Gln Gly Gly Ile Ala Ala Val Phe Asp Glu Thr Asp Ser Ile Asp
50 55 60
Ser His Val Glu Asp Thr Leu Ile Ala Gly Ala Gly Ile Cys Asp Arg
65 70 75 80
His Ala Val Glu Phe Val Ala Ser Asn Ala Arg Ser Cys Val Gln Trp
85 90 95
Leu Ile Asp Gln Gly Val Leu Phe Asp Thr His Ile Gln Pro Asn Gly
100 105 110
Glu Glu Ser Tyr His Leu Thr Arg Glu Gly Gly His Ser His Arg Arg
115 120 125
Ile Leu His Ala Ala Asp Ala Thr Gly Arg Glu Val Glu Thr Thr Leu
130 135 140
Val Ser Lys Ala Leu Asn His Pro Asn Ile Arg Val Leu Glu Arg Ser
145 150 155 160
Asn Ala Val Asp Leu Ile Val Ser Asp Lys Ile Gly Leu Pro Gly Thr
165 170 175
Arg Arg Val Val Gly Ala Trp Val Trp Asn Arg Asn Lys Glu Thr Val
180 185 190
Glu Thr Cys His Ala Lys Ala Val Val Leu Ala Thr Gly Gly Ala Ser
195 200 205
Lys Val Tyr Gln Tyr Thr Thr Asn Pro Asp Ile Ser Ser Gly Asp Gly
210 215 220
Ile Ala Met Ala Trp Arg Ala Gly Cys Arg Val Ala Asn Leu Glu Phe
225 230 235 240
Asn Gln Phe His Pro Thr Ala Leu Tyr His Pro Gln Ala Arg Asn Phe
245 250 255
Leu Leu Thr Glu Ala Leu Arg Gly Glu Gly Ala Tyr Leu Lys Arg Pro
260 265 270
Asp Gly Thr Arg Phe Met Pro Asp Phe Asp Glu Arg Gly Glu Leu Ala
275 280 285
Pro Arg Asp Ile Val Ala Arg Ala Ile Asp His Glu Met Lys Arg Leu
290 295 300
Gly Ala Asp Cys Met Phe Leu Asp Ile Ser His Lys Pro Ala Asp Phe
305 310 315 320
Ile Arg Gln His Phe Pro Met Ile Tyr Glu Lys Leu Leu Gly Leu Gly
325 330 335
Ile Asp Leu Thr Gln Glu Pro Val Pro Ile Val Pro Ala Ala His Tyr
340 345 350
Thr Cys Gly Gly Val Met Val Asp Asp His Gly Arg Thr Asp Val Glu
355 360 365
Gly Leu Tyr Ala Ile Gly Glu Val Ser Tyr Thr Gly Leu His Gly Ala
370 375 380
Asn Arg Met Ala Ser Asn Ser Leu Leu Glu Cys Leu Val Tyr Gly Trp
385 390 395 400
Ser Ala Ala Glu Asp Ile Thr Arg Arg Met Pro Tyr Ala His Asp Ile
405 410 415
Ser Thr Leu Pro Pro Trp Asp Glu Ser Arg Val Glu Asn Pro Asp Glu
420 425 430
Arg Val Val Ile Gln His Asn Trp His Glu Leu Arg Leu Phe Met Trp
435 440 445
Asp Tyr Val Gly Ile Val Arg Thr Thr Lys Arg Leu Glu Arg Ala Leu
450 455 460
Arg Arg Ile Thr Met Leu Gln Gln Glu Ile Asp Glu Tyr Tyr Ala His
465 470 475 480
Phe Arg Val Ser Asn Asn Leu Leu Glu Leu Arg Asn Leu Val Gln Val
485 490 495
Ala Glu Leu Ile Val Arg Cys Ala Met Met Arg Lys Glu Ser Arg Gly
500 505 510
Leu His Phe Thr Leu Asp Tyr Pro Glu Leu Leu Thr His Ser Gly Pro
515 520 525
Ser Ile Leu Ser Pro Gly Asn His Tyr Ile Asn Arg
530 535 540
<210> 27
<211> 531
<212> PRT
<213> Bacillus subtilis
<400> 27
Met Ser Lys Lys Thr Ile Ala Val Ile Gly Ser Gly Ala Ala Ala Leu
1 5 10 15
Ser Leu Ala Ala Ala Phe Pro Pro Ser Tyr Glu Val Thr Val Ile Thr
20 25 30
Lys Lys Ser Val Lys Asn Ser Asn Ser Val Tyr Ala Gln Gly Gly Ile
35 40 45
Ala Ala Ala Tyr Ala Lys Asp Asp Ser Ile Glu Ala His Leu Glu Asp
50 55 60
Thr Leu Tyr Ala Gly Cys Gly His Asn Asn Leu Ala Ile Val Ala Asp
65 70 75 80
Val Leu His Asp Gly Lys Met Met Val Gln Ser Leu Leu Glu Arg Gly
85 90 95
Phe Pro Phe Asp Arg Asn Glu Arg Gly Gly Val Cys Leu Gly Arg Glu
100 105 110
Gly Ala His Ser Tyr Asn Arg Ile Phe His Ala Gly Gly Asp Ala Thr
115 120 125
Gly Arg Leu Leu Ile Asp Tyr Leu Leu Lys Arg Ile Asn Ser Lys Ile
130 135 140
Lys Leu Ile Glu Asn Glu Thr Ala Ala Asp Leu Leu Ile Glu Asp Gly
145 150 155 160
Arg Cys Ile Gly Val Met Thr Lys Asp Ser Lys Gly Arg Leu Lys Val
165 170 175
Arg His Ala Asp Glu Val Val Leu Ala Ala Gly Gly Cys Gly Asn Leu
180 185 190
Phe Leu His His Thr Asn Asp Leu Thr Val Thr Gly Asp Gly Leu Ser
195 200 205
Leu Ala Tyr Arg Ala Gly Ala Glu Leu Thr Asp Leu Glu Phe Thr Gln
210 215 220
Phe His Pro Thr Leu Leu Val Lys Asn Gly Val Ser Tyr Gly Leu Val
225 230 235 240
Ser Glu Ala Val Arg Gly Glu Gly Gly Cys Leu Val Asp Glu Asn Gly
245 250 255
Arg Arg Ile Met Ala Glu Arg His Pro Leu Gly Asp Leu Ala Pro Arg
260 265 270
Asp Ile Val Ser Arg Val Ile His Glu Glu Met Ala Lys Gly Asn Arg
275 280 285
Val Tyr Ile Asp Phe Ser Ala Ile Ser Asp Phe Glu Thr Arg Phe Pro
290 295 300
Thr Ile Thr Ala Ile Cys Glu Lys Ala Gly Ile Asp Ile His Ser Gly
305 310 315 320
Lys Ile Pro Val Ala Pro Gly Met His Phe Leu Met Gly Gly Val Ser
325 330 335
Val Asn Arg Trp Gly Glu Thr Thr Val Pro Gly Leu Tyr Ala Ile Gly
340 345 350
Glu Thr Ala Cys Ser Gly Leu His Gly Ala Asn Arg Leu Ala Ser Asn
355 360 365
Ser Leu Leu Glu Ala Leu Val Phe Gly Lys Arg Ala Ala Glu His Ile
370 375 380
Ile Gln Lys Pro Val Tyr Asn Arg Gln Tyr Gln Ser Gly Leu Glu Thr
385 390 395 400
Ser Val Phe Tyr Glu Val Pro Asp Ile Glu Gly His Glu Leu Gln Ser
405 410 415
Lys Met Thr Ser His Met Ser Ile Leu Arg Glu Gln Ser Ser Leu Ile
420 425 430
Glu Leu Ser Ile Trp Leu His Thr Leu Pro Phe Gln Glu Val Asn Val
435 440 445
Lys Asp Ile Thr Ile Arg Gln Met Glu Leu Ser His Leu Trp Gln Thr
450 455 460
Ala Lys Leu Met Thr Phe Ser Ala Leu Leu Arg Glu Glu Ser Arg Gly
465 470 475 480
Ala His Phe Arg Thr Asp Phe Pro His Ala Glu Val Ser Trp Gln Gly
485 490 495
Arg Gln Ile Val His Thr Lys Lys Gly Thr Lys Ile Arg Lys Asn Glu
500 505 510
Gly Ile Trp Asn Asn Glu Ser Phe Thr Ala Glu Lys Ile Thr Glu Ser
515 520 525
Leu Phe Ser
530
<210> 28
<211> 297
<212> PRT
<213> Escherichia coli
<400> 28
Met Pro Pro Arg Arg Tyr Asn Pro Asp Thr Arg Arg Asp Glu Leu Leu
1 5 10 15
Glu Arg Ile Asn Leu Asp Ile Pro Gly Ala Val Ala Gln Ala Leu Arg
20 25 30
Glu Asp Leu Gly Gly Thr Val Asp Ala Asn Asn Asp Ile Thr Ala Lys
35 40 45
Leu Leu Pro Glu Asn Ser Arg Ser His Ala Thr Val Ile Thr Arg Glu
50 55 60
Asn Gly Val Phe Cys Gly Lys Arg Trp Val Glu Glu Val Phe Ile Gln
65 70 75 80
Leu Ala Gly Asp Asp Val Thr Ile Ile Trp His Val Asp Asp Gly Asp
85 90 95
Val Ile Asn Ala Asn Gln Ser Leu Phe Glu Leu Glu Gly Pro Ser Arg
100 105 110
Val Leu Leu Thr Gly Glu Arg Thr Ala Leu Asn Phe Val Gln Thr Leu
115 120 125
Ser Gly Val Ala Ser Lys Val Arg His Tyr Val Glu Leu Leu Glu Gly
130 135 140
Thr Asn Thr Gln Leu Leu Asp Thr Arg Lys Thr Leu Pro Gly Leu Arg
145 150 155 160
Ser Ala Leu Lys Tyr Ala Val Leu Cys Gly Gly Gly Ala Asn His Arg
165 170 175
Leu Gly Leu Ser Asp Ala Phe Leu Ile Lys Glu Asn His Ile Ile Ala
180 185 190
Ser Gly Ser Val Arg Gln Ala Val Glu Lys Ala Ser Trp Leu His Pro
195 200 205
Asp Ala Pro Val Glu Val Glu Val Glu Asn Leu Glu Glu Leu Asp Glu
210 215 220
Ala Leu Lys Ala Gly Ala Asp Ile Ile Met Leu Asp Asn Phe Glu Thr
225 230 235 240
Glu Gln Met Arg Glu Ala Val Lys Arg Thr Asn Gly Lys Ala Leu Leu
245 250 255
Glu Val Ser Gly Asn Val Thr Asp Lys Thr Leu Arg Glu Phe Ala Glu
260 265 270
Thr Gly Val Asp Phe Ile Ser Val Gly Ala Leu Thr Lys His Val Gln
275 280 285
Ala Leu Asp Leu Ser Met Arg Phe Arg
290 295
<210> 29
<211> 289
<212> PRT
<213> Bacillus subtilis
<400> 29
Met Asn His Leu Gln Leu Lys Lys Leu Leu Asn His Phe Phe Leu Glu
1 5 10 15
Asp Ile Gly Thr Gly Asp Leu Thr Ser Gln Ser Ile Phe Gly Glu Gln
20 25 30
Ser Cys Glu Ala Glu Ile Val Ala Lys Ser Glu Gly Ile Phe Ala Gly
35 40 45
Ala Ala Ile Ile Lys Glu Gly Phe Ser Leu Leu Asp Glu Asn Val Gln
50 55 60
Ser Ile Leu His Lys Lys Asp Gly Asp Met Leu His Lys Gly Glu Val
65 70 75 80
Ile Ala Glu Leu His Gly Pro Ala Ala Ala Leu Leu Ser Gly Glu Arg
85 90 95
Val Val Leu Asn Leu Ile Gln Arg Leu Ser Gly Ile Ala Thr Met Thr
100 105 110
Arg Glu Ala Val Arg Cys Leu Asp Asp Glu Gln Ile Lys Ile Cys Asp
115 120 125
Thr Arg Lys Thr Thr Pro Gly Leu Arg Met Leu Glu Lys Tyr Ala Val
130 135 140
Arg Ala Gly Gly Gly Tyr Asn His Arg Phe Gly Leu Tyr Asp Gly Ile
145 150 155 160
Met Ile Lys Asp Asn His Ile Ala Ala Cys Gly Ser Ile Leu Glu Ala
165 170 175
Cys Lys Lys Ala Arg Gln Ala Ala Gly His Met Val Asn Ile Glu Val
180 185 190
Glu Ile Glu Thr Glu Glu Gln Leu Arg Glu Ala Ile Ala Ala Gly Ala
195 200 205
Asp Val Ile Met Phe Asp Asn Cys Pro Pro Asp Thr Val Arg His Phe
210 215 220
Ala Lys Leu Thr Pro Ala Asn Ile Lys Thr Glu Ala Ser Gly Gly Ile
225 230 235 240
Thr Leu Glu Ser Leu Pro Ala Phe Lys Gly Thr Gly Val Asn Tyr Ile
245 250 255
Ser Leu Gly Phe Leu Thr His Ser Val Lys Ser Leu Asp Ile Ser Met
260 265 270
Asp Val Thr Leu Ser Asn Glu Ser Val Glu Glu Cys Cys Tyr Val Asn
275 280 285
Ser
<210> 30
<211> 279
<212> PRT
<213> Corynebacterium glutamicum
<400> 30
Met Thr Thr His Ile Asp Arg Ile Val Gly Ala Ala Leu Ser Glu Asp
1 5 10 15
Ala Pro Trp Gly Asp Ile Thr Ser Asp Thr Phe Ile Pro Gly Ser Ala
20 25 30
Gln Leu Ser Ala Lys Val Val Ala Arg Glu Pro Gly Val Phe Ser Gly
35 40 45
Gln Ala Leu Phe Asp Ala Ser Phe Arg Leu Val Asp Pro Arg Ile Asn
50 55 60
Ala Ser Leu Lys Val Ala Asp Gly Asp Ser Phe Glu Thr Gly Asp Ile
65 70 75 80
Leu Gly Thr Ile Thr Gly Ser Ala Arg Ser Ile Leu Arg Ser Glu Arg
85 90 95
Ile Ala Leu Asn Phe Ile Gln Arg Thr Ser Gly Ile Ala Thr Leu Thr
100 105 110
Ser Cys Tyr Val Ala Glu Val Lys Gly Thr Lys Ala Arg Ile Val Asp
115 120 125
Thr Arg Lys Thr Thr Pro Gly Leu Arg Ile Ile Glu Arg Gln Ala Val
130 135 140
Arg Asp Gly Gly Gly Phe Asn His Arg Ala Thr Leu Ser Asp Ala Val
145 150 155 160
Met Val Lys Asp Asn His Leu Ala Ala Ile Ala Ser Gln Gly Leu Ser
165 170 175
Ile Thr Glu Ala Leu Ser Asn Met Lys Ala Lys Leu Pro His Thr Thr
180 185 190
His Val Glu Val Glu Val Asp His Ile Glu Gln Ile Glu Pro Val Leu
195 200 205
Ala Ala Gly Val Asp Thr Ile Met Leu Asp Asn Phe Thr Ile Asp Gln
210 215 220
Leu Ile Glu Gly Val Asp Leu Ile Gly Gly Arg Ala Leu Val Glu Ala
225 230 235 240
Ser Gly Gly Val Asn Leu Asn Thr Ala Gly Lys Ile Ala Ser Thr Gly
245 250 255
Val Asp Val Ile Ser Val Gly Ala Leu Thr His Ser Val His Ala Leu
260 265 270
Asp Leu Gly Leu Asp Ile Phe
275

Claims (19)

1.一种能够产生烟酸核糖苷(NaR)的经遗传修饰的细菌，其中所述细菌包括选自以下所组成的组的至少一种修饰：
a)通过抑制nadA、nadB、nadC基因或其组合的转录来阻断或降低具有抑制NAD+生物合成作用的蛋白质的活性；
b)添加或增加编码L-天冬氨酸氧化酶、喹啉合酶、喹啉磷酸核糖基转移酶的基因或其组合的转录；以及
c)阻断或降低用作烟酸单核苷酸腺苷转移酶的蛋白质的活性；
其中，与没有任何所述修饰的所述细菌相比，具有所述至少一种修饰的所述细菌产生增加量的NaR。
2.根据权利要求1所述的细菌，其中所述细菌还包括选自以下所组成的组的一种或多种额外的修饰：
d)阻断或降低用作烟酸核糖苷磷酸化酶的蛋白质的活性；
e)阻断或降低用作烟酸核糖苷激酶的蛋白质的活性；
f)阻断或降低用作烟酸核糖苷转运蛋白的蛋白质的活性；
g)阻断或降低用作烟酸磷酸核糖基转移酶的蛋白质的活性；
h)添加或增加用作烟酰胺单核苷酸酰胺水解酶的蛋白质的活性；以及
i)添加或增加用作烟酸单核苷酸水解酶的蛋白质的活性。
3.根据权利要求1所述的细菌，其中具有抑制NAD+生物合成作用的所述蛋白质是包含SEQ ID NO：1、2或3中的任一个的氨基酸序列的多肽或其变体，其中所述多肽具有抑制NAD+生物合成的活性。
4.根据权利要求1所述的细菌，其中所述L-天冬氨酸氧化酶是包含SEQ ID NO：26或27中的任一个的氨基酸序列的多肽或其变体，其中所述多肽具有在FAD依赖型反应中将天冬氨酸转化成亚氨基琥珀酸的活性。
5.根据权利要求1所述的细菌，其中所述喹啉合酶是包含SEQ ID NO：23、24或25中的任一个的氨基酸序列的多肽或其变体，其中所述多肽具有将亚氨基琥珀酸和磷酸二羟丙酮转化成喹啉和磷酸的活性。
6.根据权利要求1所述的细菌，其中所述喹啉磷酸核糖基转移酶是包含SEQ ID NO：28、29或30中的任一个的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有将喹啉和磷酸核糖焦磷酸转化成烟酸单核苷酸和二氧化碳的活性。
7.根据权利要求1所述的细菌，其中所述烟酸单核苷酸腺苷转移酶蛋白是包含SEQ IDNO：4、5或6中的任一个的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有将烟酸单核苷酸转化成烟酸腺嘌呤二核苷酸的烟酸单核苷酸腺苷转移酶活性。
8.根据权利要求2所述的细菌，其中所述烟酸核糖苷磷酸化酶是包含SEQ ID NO：7、8、18或19中的任一个的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有将烟酸核糖苷转化成烟酸和磷酸核糖的核苷裂解活性。
9.根据权利要求2所述的细菌，其中所述烟酸核糖苷激酶是包含SEQ ID NO：1的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有将烟酸核糖苷转化成烟酸单核苷酸的活性。
10.根据权利要求2所述的细菌，其中所述烟酸核糖苷转运蛋白是包含SEQ ID NO：9、10或11中的任一个的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有输入烟酸核糖苷的烟酸核糖苷转运活性。
11.根据权利要求2所述的细菌，其中所述烟酸磷酸核糖基转移酶是包含SEQ ID NO：15、16或17中的任一个的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有将烟酸、5-磷酸-核糖1-二磷酸和三磷酸腺苷转化为烟酸单核苷酸、二磷酸腺苷、二磷酸盐和磷酸盐的烟酸磷酸核糖基转移酶活性。
12.根据权利要求2所述的细菌，其中所述烟酰胺单核苷酸酰胺水解酶是包含SEQ IDNO：20、21或22中的任一个的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有将烟酰胺单核苷酸转化成烟酸单核苷酸的烟酰胺单核苷酸酰胺水解酶活性。
13.根据权利要求2所述的细菌，其中所述烟酸单核苷酸水解酶是包含SEQ ID NO：12、13或14中的任一个的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有将烟酸单核苷酸转化成烟酸核糖苷的烟酸单核苷酸水解酶活性。
14.根据权利要求1至13中任一项所述的细菌，其中所述细菌选自E.coli、B.subtilis、C.glutamicum、A.baylyi和R.eutropha所组成的组。
15.一种产生NaR的方法，其包括：在有效产生NaR的条件下培养细菌细胞并从培养基回收NaR，从而产生NaR，其中宿主微生物包括选自以下所组成的组的至少一种修饰：
a)通过抑制nadA、nadB、nadC基因或其组合的转录来阻断或降低具有抑制NAD+生物合成作用的蛋白质的活性；
b)添加或增加编码L-天冬氨酸氧化酶、喹啉合酶、喹啉磷酸核糖基转移酶的基因或其组合的转录；以及
c)阻断或降低用作烟酸单核苷酸腺苷转移酶的蛋白质的活性。
16.根据权利要求15所述的方法，其中所述细菌细胞还包括选自以下所组成的组的至少一种修饰：
d)阻断或降低用作烟酸核糖苷磷酸化酶的蛋白质的活性；
e)阻断或降低用作烟酸核糖苷激酶的蛋白质的活性；
f)阻断或降低用作烟酸核糖苷转运蛋白的蛋白质的活性；
g)阻断或降低用作烟酸磷酸核糖基转移酶的蛋白质的活性；
h)添加或增加用作烟酰胺单核苷酸酰胺水解酶的蛋白质的活性；以及
i)添加或增加用作烟酸单核苷酸水解酶的蛋白质的活性。
17.从根据权利要求1至14中任一项所述的经遗传修饰的细菌获得的烟酸核糖苷化合物。
18.一种组合物，其包含从根据权利要求1至14中任一项所述的经遗传修饰的细菌获得的烟酸核糖苷化合物。
19.一种食品或饲料，其包含从根据权利要求1至14中任一项所述的经遗传修饰的细菌获得的烟酸核糖苷化合物。
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