CN108473948A - 烟酰胺核糖苷的微生物生产 - Google Patents

烟酰胺核糖苷的微生物生产 Download PDF

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CN108473948A
CN108473948A CN201680078334.8A CN201680078334A CN108473948A CN 108473948 A CN108473948 A CN 108473948A CN 201680078334 A CN201680078334 A CN 201680078334A CN 108473948 A CN108473948 A CN 108473948A
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亚当·G·劳伦斯
席琳·维亚罗格
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Abstract

本公开涉及通过调控导致产生烟酰胺核糖苷的途径的用于生产烟酰胺核糖苷的新方法、表达载体和宿主细胞。

Description

烟酰胺核糖苷的微生物生产
相关申请的交叉引用
本申请要求2015年9月28日递交的美国临时专利申请No.62/233,696和2015年11月13日递交的美国临时专利申请No.62/254,736的申请日的权益，所述临时专利申请的公开内容通过引用整体并入本文。
技术领域
本公开的一些实施方式涉及用于生产烟酰胺核糖苷(nicotinamide riboside)的新方法以及可用于所述方法中的表达载体和宿主细胞。
背景技术
烟酰胺核糖苷(NR)是维生素B3的吡啶-核苷形式，其作为烟酰胺腺嘌呤二核苷酸或NAD+的前体起作用。人们认为：高剂量烟酸可帮助提高高密度脂蛋白胆固醇、降低低密度脂蛋白胆固醇并降低游离脂肪酸，但其机制尚未完全了解。烟酰胺核糖苷在过去已被化学合成。导致烟酰胺核糖苷合成的生物途径是已知的，但通过生物学方法生产烟酰胺核糖苷仍然是一个挑战。因此，期望发现更有效地生产烟酰胺核糖苷的新方法。
细菌中NAD+的生物合成最早在20世纪90年代被阐明，并且显示其依赖于在真核生物中未发现的两种关键酶活性：FAD依赖性L-天冬氨酸氧化酶(NadB，EC1.4.3.16)；和喹啉酸(quinolate)合酶(NadA，EC 2.5.1.72)(Flachmann,1988,European Journal ofBiochemistry,175(2),221-228)。NadB催化L-天冬氨酸氧化为亚氨基琥珀酸，其中利用分子氧作为电子受体并产生过氧化氢，并且有松散结合的黄素腺嘌呤二核苷酸(FAD)辅因子参与(Seifert,1990,Biological chemistry Hoppe-Seyler,371(1),239-248)。已知Esherichia coli中的酶被下游产物NAD+抑制(Nasu S,1982,J Biol Chem,257(2),626-32)，但已经生成了反馈抗性突变体(Hughes,1983,J Bacteriol,154(3),1126-36)。含有铁硫簇的NadA随后进行亚氨基琥珀酸与磷酸二羟丙酮的缩合和环化，从而产生了喹啉酸(Flachmann,1988)。这两种酶的组合活性会从1摩尔天冬氨酸和1摩尔磷酸二羟丙酮产生1摩尔喹啉酸。
NAD+合成的两种典型从头合成途径共有三种另外的酶活性：喹啉酸磷酸核糖基转移酶(NadC，EC 2.4.2.19)；烟酸单核苷酸腺嘌呤基转移酶(NadD，EC 2.7.7.18)；和NAD+合成酶(NadE，EC 6.3.1.5)。NadC将来自磷酸核糖基焦磷酸的磷酸核糖基部分转移到喹啉酸氮并催化随后的中间体脱羧以产生烟酸单核苷酸(NaMN)、焦磷酸和二氧化碳(Begley,2001,Vitamins&Hormones,61,103-119)。NadD利用三磷酸腺苷(ATP)腺苷酸化NaMN，从而产生烟酸二核苷酸(NaAD)和焦磷酸(Begley,2001)。NadD还能够腺苷酸化烟酰胺二核苷酸(NMN)，但与使用NaMN作为底物时相比，具有较低的亲和力(较高的Km)和较低的周转率(turnover)(Vmax)。例如，来自Bacillus subtilis的酶对NaMN的Vmax/Km为其对NMN的Vmax/Km的104倍(Olland,2002,J Biol Chem,277(5),3698-3707)。NAD+生物合成的最后一步由NadE催化，NadE利用氨或谷氨酰胺作为氮供体以将NaAD酰胺化为NAD+，从而将1摩尔ATP水解为AMP和焦磷酸(Begley,2001)。与NadD的底物灵活性类似，该酶可以代替NaAD作用于NaMN以产生NMN，但是底物偏好仍然强烈；在Bacillus anthracis中，Vmax/Km的差异>103倍(Sorci,2009,J Biol Chem,277(5),3698-3707)。
与上述典型途径形成对比，Francisella tularensis中的途径经由NMN作为中间体进行(Sorci,2009)。形成NaMN之后，FtNadE*酶利用NH3以与典型的NadE酶明显类似的机制，即伴随1摩尔ATP的水解(在本文中称为NadE*活性)催化NaMN的酰胺化。FtNadE*酶也酰胺化NaAD，但对NaMN更具特异性，Vmax/Km相对值相差60倍。最后一步由NadM酶催化，NadM酶催化NMN的腺苷酸化。
除了从头合成途径之外，还存在多种补救NMN、NR、烟酰胺(Nam)或烟酸(NA)的途径(Gazzaniga,2009,Microbiol Mol Biol Rev,73(3),529-541)。NMN通过烟酰胺核苷酸酰胺酶(E.coli PncC,B.subtilis CinA,EC 3.5.1.42)的作用被再循环为NaMN；NR通过烟酰胺核糖苷激酶(E.coli NadR,EC 2.7.1.22)被磷酸化成NMN或在可逆反应中通过嘌呤核苷磷酸化酶(E.coli DeoD,B.subtilis DeoD,PupG,Pdp,EC 2.4.2.1)被降解为Nam和磷酸核糖；Nam可以通过DeoD被磷酸核糖基化为NMN或通过烟酰胺酶(PncA，EC 3.5.1.19)被脱酰胺为NA；NA或Nam通过烟酸磷酸核糖基转移酶(E.coli PncB,B.subtilis YueK EC 6.3.4.21)分别被转变为NaMN或NMN。胞外NMN通过周质酸性磷酸酶(E.coli UshA,B.subtilis YfkN,EC3.1.3.5)被去磷酸化为NR，并且胞外NR可以通过NR转运体(E.coli PnuC,B.subtilisNupG)被导入。NAD+自身可被用作嘧啶核苷酸的来源。NAD+通过NAD+二磷酸酶(NudC，EC3.6.1.22)的活性被切割为NMN和腺苷一磷酸。
在细菌中，nad基因的表达通常通过转录阻遏物共同调控。在E.coli中，nadA、nadB和pncB的转录被NadR蛋白抑制，NadR蛋白还具有促成补救途径的催化活性(Raffaelli,1999,J Bacteriol,181(17),5509-5511)。NadR通过在NAD+存在的情况下与保守基序结合来阻断转录。在Bacillus subtilis中，名为YrxA的不同蛋白通过在NA存在的情况下阻断两种分别转录的操纵子nadB-nadA-nadC和nifS-yrxA的转录从而发挥类似作用(Rossolillo,2005,J Bacteriol,187(20),7155-7160)。
发明人现在已经出乎意料地发现了显著提高烟酰胺核糖生产率的新方法，并创建了可用于所述方法中的表达载体和宿主细胞。
发明内容
本发明涉及能够生产烟酰胺核糖苷(NR)的经遗传修饰的细菌，其中所述细菌包含至少一种选自以下的修饰：a)添加异源烟酸酰胺化蛋白(NadE*)的活性；和b)添加或提高烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白的活性，其中与没有任何所述修饰的细菌相比，具有所述至少一种修饰的细菌产生提高量的NR。
在一些实施方式中，所述经遗传修饰的细菌可以进一步包含一种或多种另外的选自以下的修饰：a)阻断或降低以下蛋白的活性，所述蛋白通过抑制nadA基因、nadB基因、nadC基因或其组合的转录起抑制NAD+生物合成的作用；b)阻断或降低作为烟酰胺核糖苷转运蛋白起作用的蛋白质的活性；c)阻断或降低作为烟酸单核苷酸腺嘌呤基转移酶起作用的蛋白质的活性；d)阻断或降低作为烟酰胺单核苷酸酰胺水解酶起作用的蛋白质的活性；e)阻断或降低作为嘌呤核苷磷酸化酶起作用的蛋白质的活性；f)添加或提高作为烟酰胺单核苷酸水解酶起作用的蛋白质的活性；和g)添加或提高编码L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶的基因或其组合的转录。
本发明还涉及生产NR的方法，所述方法包括：在有效产生NR的条件下培养细菌细胞并从培养基中回收NR，从而生产NR，其中宿主微生物包含至少一种选自以下的修饰：a)添加异源烟酸酰胺化蛋白(NadE*)的活性；b)添加或提高烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白的活性；c)阻断或降低作为nadA、nadB、nadC或其组合的转录的负调控因子起作用的蛋白质的活性；d)阻断或降低作为烟酰胺核糖苷转运蛋白起作用的蛋白质的活性；e)阻断或降低作为烟酸单核苷酸腺嘌呤基转移酶起作用的蛋白质的活性；f)阻断或降低作为烟酰胺单核苷酸酰胺水解酶起作用的蛋白质的活性；g)阻断或降低作为嘌呤核苷磷酸化酶起作用的蛋白质的活性；h)添加或提高作为烟酰胺单核苷酸水解酶起作用的蛋白质的活性；和i)添加或提高编码L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶的基因或其组合的转录。
本发明涉及另一种生产NR的方法，所述方法包括：在有效产生NR的条件下培养细菌细胞并从培养基中回收NR，从而生产NR，其中宿主微生物包含至少一种选自以下的修饰：a)添加异源烟酸酰胺化蛋白NadE*的活性；和b)添加或提高烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白的活性。在该方法中，细菌细胞可进一步包含至少一种选自以下的修饰：a)阻断或降低以下蛋白的活性，所述蛋白通过抑制nadA基因、nadB基因、nadC基因或其组合的转录起抑制NAD+生物合成的作用；b)阻断或降低作为烟酰胺核糖苷转运蛋白起作用的蛋白质的活性；c)阻断或降低作为烟酸单核苷酸腺嘌呤基转移酶起作用的蛋白质的活性；d)阻断或降低作为烟酰胺单核苷酸酰胺水解酶起作用的蛋白质的活性；e)阻断或降低作为嘌呤核苷磷酸化酶起作用的蛋白质的活性；f)添加或提高作为烟酰胺单核苷酸水解酶起作用的蛋白质的活性；和g)添加或提高编码L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶的基因或其组合的转录。
在一些实施方式中，NadE*蛋白是包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：1和3至18之任一的同一性为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少97％或至少99％，其中所述多肽具有将烟酸单核苷酸转变为烟酰胺单核苷酸的烟酸酰胺化活性。
在一些实施方式中，当与SEQ ID NO：1的参照氨基酸序列比较时，上述NadE*蛋白还具有一个或多个以下保守氨基酸：a)第287位的酪氨酸，b)第133位的谷氨酰胺，和c)第236位的精氨酸，当与SEQ ID NOS：1和3至18进行比较时，基于ClustalW比对方法，使用缺省参数空位罚分＝10，空位长度罚分＝0.1和Gonnet 250系列蛋白质权重矩阵。
在一些实施方式中，烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白是包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：66至70之任一的同一性为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少97％或至少99％，其中所述多肽具有将NAD+转变为烟酰胺单核苷酸和腺嘌呤的NAD+水解活性。
在一些实施方式中，NAD+生物合成的负调控因子是包含SEQ ID NO：51、52或53的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有抑制NAD+生物合成的活性。
在一些实施方式中，烟酰胺核糖苷转运体是包含SEQ ID NO：54、55、56或71之任一的氨基酸序列的多肽，其中所述多肽具有导入烟酰胺核糖苷的烟酰胺核糖苷转运活性。
在一些实施方式中，核苷水解酶是包含SEQ ID NO：57、58或59之任一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变为烟酰胺核糖苷的核苷水解酶活性。
在一些实施方式中，烟酸单核苷酸腺嘌呤基转移酶蛋白是包含SEQ ID NO：63、64或65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变为烟酸腺嘌呤二核苷酸的烟酸单核苷酸腺嘌呤转移酶活性。
在一些实施方式中，烟酰胺单核苷酸酰胺水解酶蛋白是包含SEQ ID NO：60、61或62之任一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变为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，嘌呤核苷磷酸化酶蛋白是包含SEQ ID NO：72至76之任一的氨基酸序列的多肽，其中所述多肽具有将烟酰胺核糖苷和磷酸转变为烟酰胺和核糖-1-磷酸的嘌呤核苷磷酸化酶活性。在一些实施方式中，喹啉酸合酶是包含SEQ ID NO：77、78或79之任一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亚氨基琥珀酸和磷酸二羟基丙酮转变为喹啉酸和磷酸的活性。
在一些实施方式中，L-天冬氨酸氧化酶是包含SEQ ID NO：80或81之任一的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有在FAD依赖性反应中将天冬氨酸转变为亚氨基琥珀酸的活性。
在一些实施方式中，喹啉酸磷酸核糖基转移酶是包含SEQ ID NO：82、83或84之任一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酸和磷酸核糖基焦磷酸转变为烟酰胺单核苷酸和二氧化碳的活性。
本发明还涉及经遗传修饰的细菌，其特征在于：由于遗传修饰，所述细菌产生NR并且能够在所述细菌生长的发酵液中积累产生的NR至至少100mg/L。
在一些实施方式中，在经遗传修饰的细菌中，遗传修饰选自：a)添加异源烟酸酰胺化蛋白(NadE*)的活性；和b)添加或提高烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白的活性。在一些实施方式中，遗传修饰进一步包含一种或多种另外的选自以下的修饰：a)阻断或降低以下蛋白的活性，所述蛋白通过抑制nadA基因、nadB基因、nadC基因或其组合的转录起抑制NAD+生物合成的作用；b)阻断或降低作为烟酰胺核糖苷转运蛋白起作用的蛋白质的活性；c)阻断或降低作为烟酸单核苷酸腺嘌呤基转移酶起作用的蛋白质的活性；d)阻断或降低作为烟酰胺单核苷酸酰胺水解酶起作用的蛋白质的活性；e)阻断或降低作为嘌呤核苷磷酸化酶起作用的蛋白质的活性；f)添加或提高作为烟酰胺单核苷酸水解酶起作用的蛋白质的活性；和g)添加或提高作为L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶起作用的蛋白质或其组合的活性。
在一些实施方式中，在上述经遗传修饰的细菌中，NadE*蛋白是包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：1和3至18之任一的同一性为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少97％或至少99％，其中所述多肽具有将烟酸单核苷酸转变为烟酰胺单核苷酸的烟酸酰胺化活性。在一个实施方式中，当与SEQ ID NO：1的参照氨基酸序列比较时，NadE*蛋白具有一个或多个以下保守氨基酸：a)第27位的酪氨酸，b)第1343位的谷氨酰胺，和c)第2376位的精氨酸，其中基于ClustalW比对方法与SEQ IDNOS：1和3至18进行比较，使用缺省参数空位罚分＝10，空位长度罚分＝0.1和Gonnet 250系列蛋白质权重矩阵。
在一个实施方式中，烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白是包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：66至70之任一的同一性为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少97％或至少99％，其中所述多肽具有将NAD+转变为烟酰胺单核苷酸和腺嘌呤的NAD+水解活性。
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和R.eutropha。
本发明还涉及从任何上述经遗传修饰的细菌获得的烟酰胺核糖苷化合物。
本发明还涉及组合物，所述组合物包含从上述经遗传修饰的细菌获得的烟酰胺核糖苷化合物。
本发明还涉及食品或饲料，所述食品或饲料包含从上述经遗传修饰的细菌获得的烟酰胺核糖苷化合物。
序列表概述
使用核苷酸碱基的标准字母缩写来示出所附序列表中列出的核酸序列。仅示出了每种核酸序列的一条链，但互补链被理解为通过对所示链的任何提及而包括。在所附序列表中：
SEQ ID NO：1是编码Francisella tularensis NadE*酶(FtNadE*)的氨基酸序列，FtNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：2是编码Francisella tularensis NadE*酶(FtNadE*)的开放阅读框的核苷酸序列。
SEQ ID NO：3是编码Francisella sp.FSC1006NadE*酶(FspFNadE*)的氨基酸序列，FspFNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：4是编码Francisella guangzhouensis NadE*酶(FgNadE*)的氨基酸序列，FgNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：5是编码Francisella sp.TX077308NadE*酶(FspTNadE*)的氨基酸序列，FspTNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：6是编码Francisella philomiragia subsp.philomiragiaATCC25017NadE*酶(FphNadE*)的氨基酸序列，FphNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：7是编码Francisella philomiragia菌株O#319-036[FSC 153]NadE*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。
SEQ ID NO：8是编码Francisella noatunensis supbsp.orientalis str.Toba04NadE*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。
SEQ ID NO：9是编码Francisella philomiragia菌株GA01-2794NadE*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。
SEQ ID NO：10是编码Francisella persica ATCC VR-331NadE*酶(FpeNadE*)的氨基酸序列，FpeNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：11是编码Francisella cf.novicida 3523NadE*酶(FnNadE*)的氨基酸序列，FnNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：12是编码Francisella tularensis subsp.novicida D9876 NadE*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。
SEQ ID NO：13是编码Francisella tularensis subsp.novicida F6168 NadE*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。
SEQ ID NO：14是编码Francisella tularensis subsp.tularensis菌株NIH B-38NadE*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。
SEQ ID NO：15是编码Francisella tularensis subsp.holarctica F92 NadE*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。
SEQ ID NO：16是编码Dichelobacter nodosus VCS1703A NadE*酶(DnNadE*)的氨基酸序列，DnNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：17是编码Mannheimia succinoproducens MBEL55E NadE*酶(MnNadE*)的氨基酸序列，MnNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：18是编码Actinobacillus succinogenes NadE*酶(AsNadE*)的氨基酸序列，AsNadE*是一种烟酸酰胺化蛋白。
SEQ ID NO：19是编码Mannheimia succinoproducens MBEL55E NadE*酶(MnNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：20是编码Dichelobacter nodosus VCS1703A NadE*酶(DnNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：21是编码Actinobacillus succinogenes NadE*酶(AsNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：22是编码Francisella philomiragia subsp.philomiragia ATCC25017NadE*酶(FphNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：23是编码Francisella cf.novicida 3523NadE*酶(FnNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：24是编码Francisella sp.TX077308NadE*酶(FspTNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：25是编码Francisella sp.FSC1006NadE*酶(FspFNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：26是编码Francisella guangzhouensis NadE*酶(FgNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：27是编码Francisella persica ATCC VR-331NadE*酶(FpeNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：28是针对在E.coli中表达优化的编码Mannheimia succinoproducensMBEL55E NadE*酶(MnNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：29是针对在E.coli中表达优化的编码Dichelobacter nodosusVCS1703A NadE*酶(DnNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：30是针对在E.coli中表达优化的编码Actinobacillus succinogenesNadE*酶(AsNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：31是针对在E.coli中表达优化的编码Francisella philomiragiasubsp.philomiragia ATCC 25017NadE*酶(FphNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：32是针对在E.coli中表达优化的编码Francisella cf.novicida3523NadE*酶(FnNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：33是针对在E.coli中表达优化的编码Francisellasp.TX077308NadE*酶(FspTNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：34是针对在E.coli中表达优化的编码Francisella sp.FSC1006NadE*酶(FspFNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：35是针对在E.coli中表达优化的编码Francisella guangzhouensisNadE*酶(FgNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：36是针对在E.coli中表达优化的编码Francisella persica ATCCVR-331NadE*酶(FpeNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：37是针对在E.coli中表达优化的编码Francisella tularensisNadE*酶(FtNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：38是针对在B.subtilis中表达优化的编码Francisella tularensisNadE*酶(FtNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：39是针对在B.subtilis中表达优化的编码Mannheimiasuccinoproducens MBEL55E NadE*酶(MnNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：40是针对在B.subtilis中表达优化的编码Francisella cf.novicida3523NadE*酶(FnNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：41是针对在B.subtilis中表达优化的编码Francisellasp.TX077308NadE*酶(FspTNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：42是针对在E.coli中表达优化的编码Francisella tularensisNadE*酶(FtNadE*)开放阅读框并且编码Y27T、Q133G和R236V突变的核苷酸序列。
SEQ ID NO：43是针对在E.coli中表达优化的编码Mannheimia succinoproducensMBEL55E NadE*酶(MnNadE*)开放阅读框并且编码Y22T、Q128G和R231V突变的核苷酸序列。
SEQ ID NO：44是针对在E.coli中表达优化的编码Francisella cf.novicida3523NadE*酶(FnNadE*)开放阅读框并且编码Y27T、Q133G和R236V突变的核苷酸序列。
SEQ ID NO：45是针对在E.coli中表达优化的编码Francisellasp.TX077308NadE*酶(FspTNadE*)开放阅读框并且编码Y27T、Q133G和R236V突变的核苷酸序列。
SEQ ID NO：46是编码E.coli NadE酶(EcNadE)的核苷酸序列，所述EcNadE编码烟酰胺腺嘌呤二核苷酸酰胺化活性。
SEQ ID NO：47是针对在C.glutamicum中表达优化的编码Francisellacf.novicida 3523NadE*酶(FnNadE*)开放阅读框的核苷酸序列。
SEQ ID NO：48是编码四环素抗性的核苷酸序列。
SEQ ID NO：49是编码新霉素抗性的核苷酸序列。
SEQ ID NO：50是编码壮观霉素抗性的核苷酸序列。
SEQ ID NO：51是编码Escherichia coli NadR酶(NMN合成酶、NR激酶、NAD+生物合成的负调控因子)的氨基酸序列。
SEQ ID NO：52是编码Bacillus subtilis NadR(又名YxrA)酶的氨基酸序列，所述酶是阻遏蛋白。
SEQ ID NO：53是编码Corynebacterium glutamicum NadR(又名CgR_1153)酶的氨基酸序列，所述酶是阻遏蛋白。
SEQ ID NO：54是编码Acinetobacter baylyi PnuC酶的氨基酸序列，所述酶是NR转运蛋白。
SEQ ID NO：55是编码Corynebacterium glutamicum PnuC酶的氨基酸序列，所述酶是NR转运蛋白。
SEQ ID NO：56是编码Escherichia coli PnuC酶的氨基酸序列，所述酶是NR转运蛋白。
SEQ ID NO：57是编码Escherichia coli UshA酶的氨基酸序列，所述酶是烟酰胺单核苷酸水解酶。
SEQ ID NO：58是编码Bacillus subtilis UshA(又名YfkN)酶的氨基酸序列，所述酶是烟酰胺单核苷酸水解酶。
SEQ ID NO：59是编码Corynebacterium glutamicum UshA(又名Cg0397)酶的氨基酸序列，所述酶是烟酰胺单核苷酸水解酶。
SEQ ID NO：60是编码Escherichia coli PncC酶的氨基酸序列，所述酶是烟酰胺单核苷酸酰胺水解酶。
SEQ ID NO：61是编码Bacillus subtilis PncC(又名CinA)酶的氨基酸序列，所述酶是烟酰胺单核苷酸酰胺水解酶。
SEQ ID NO：62是编码Corynebacterium glutamicum PncC(又名Cg2153)酶的氨基酸序列，所述酶是烟酰胺单核苷酸酰胺水解酶。
SEQ ID NO：63是编码Escherichia coli NadD酶的氨基酸序列，所述酶是烟酸单核苷酸腺嘌呤基转移酶。
SEQ ID NO：64是编码Bacillus subtilis NadD酶的氨基酸序列，所述酶是烟酸单核苷酸腺嘌呤基转移酶。
SEQ ID NO：65是编码Corynebacterium glutamicum NadD(又名Cg2584)酶的氨基酸序列，所述酶是烟酸单核苷酸腺嘌呤基转移酶。
SEQ ID NO：66是编码Acinetobacter NudC酶的氨基酸序列，所述酶是NAD+二磷酸酶。
SEQ ID NO：67是编码Escherichia coli NudC酶的氨基酸序列，所述酶是NAD+二磷酸酶。
SEQ ID NO：68是编码Corynebacterium glutamicum NudC(又名Cg0888)酶的氨基酸序列，所述酶是NAD+二磷酸酶。
SEQ ID NO：69是编码Burkholderiaceae NudC酶的氨基酸序列，所述酶是NAD+二磷酸酶。
SEQ ID NO：70是编码Haemophilus influenzae NudC酶的氨基酸序列，所述酶是NAD+二磷酸酶。
SEQ ID NO：71是编码B.subtilis NupG蛋白的氨基酸序列，所述蛋白是NR转运蛋白。
SEQ ID NO：72是编码B.subtilis DeoD酶的氨基酸序列，所述酶是核苷磷酸化酶。
SEQ ID NO：73是编码B.subtilis Pdp酶的氨基酸序列，所述酶是核苷磷酸化酶。
SEQ ID NO：74是编码B.subtilis PupG酶的氨基酸序列，所述酶是核苷磷酸化酶。
SEQ ID NO：75是编码E.coli DeoD酶的氨基酸序列，所述酶是核苷磷酸化酶。
SEQ ID NO：76是编码C.glutamicum G18NG酶的氨基酸序列，所述酶是核苷磷酸化酶。
SEQ ID NO：77是编码Escherichia coli NadA酶的氨基酸序列，所述酶是喹啉酸合酶。
SEQ ID NO：78是编码Bacillus subtilis NadA酶的氨基酸序列，所述酶是喹啉酸合酶。
SEQ ID NO：79是编码Corynebacterium glutamicum NadA酶的氨基酸序列，所述酶是喹啉酸合酶。
SEQ ID NO：80是编码Escherichia coli NadB酶的氨基酸序列，所述酶是L-天冬氨酸氧化酶。
SEQ ID NO：81是编码Bacillus subtilis NadB酶的氨基酸序列，所述酶是L-天冬氨酸氧化酶。
SEQ ID NO：82是编码Escherichia coli NadC酶的氨基酸序列，所述酶是喹啉酸磷酸核糖基转移酶。
SEQ ID NO：83是编码Bacillus subtilis NadC酶的氨基酸序列，所述酶是喹啉酸磷酸核糖基转移酶。
SEQ ID NO：84是编码Corynebacterium glutamicum NadC酶的氨基酸序列，所述酶是喹啉酸磷酸核糖基转移酶。
SEQ ID NO：85：是引物10444。
SEQ ID NO：86：是引物10447。
SEQ ID NO：87：是引物11222。
SEQ ID NO：88：是引物11223。
SEQ ID NO：89：是引物11226。
SEQ ID NO：90：是引物11227。
SEQ ID NO：91：是引物11230。
SEQ ID NO：92：是引物11231。
SEQ ID NO：93：是引物11232。
SEQ ID NO：94：是引物11233。
SEQ ID NO：95：是引物11234。
SEQ ID NO：96：是引物11235。
SEQ ID NO：97：是引物11341。
SEQ ID NO：98：是引物11342。
SEQ ID NO：99：是引物11351。
SEQ ID NO：100：是引物11352。
SEQ ID NO：101：是引物11353。
SEQ ID NO：102：是引物11354。
SEQ ID NO：103：是引物11159。
SEQ ID NO：104：是引物11160。
附图简介
现在将仅通过示例的方式参照图1-4示出本发明的一些实施方式，其中：
图1示出了在NadA和NadB酶存在的情况下从天冬氨酸和磷酸二羟丙酮合成喹啉酸的生物化学途径。
图2示出了用于合成烟酰胺腺嘌呤二核苷酸的生物化学途径和酶。
图3示出了可用于从NAD+或NAD+生物合成的中间体生产烟酰胺核糖苷的生物化学途径。
图4示出了对于烟酰胺核糖苷生产而言具有不期望活性的生物化学途径。
图5示出了具有高亮的Y-Q-R基序的NadE*序列的ClustalW比对。
图6示出了具有Y-Q-R基序的独特序列(unique sequences)的邻接共同树。
图7示出了细菌的根源系统发生树(Bern M.和Goldberg,D.,BMCEvol.Bio.2005)，显示了本研究中使用的菌株之间的进化距离。
发明详述
除非本文中另有定义，否则本文中使用的科学和技术术语具有本领域普通技术人员通常理解的含义。
术语“烟酸酰胺化蛋白”表示能够催化烟酸单核苷酸(NaMN)转变为烟酰胺单核苷酸(NMN)的酶。该酶在本文中被称为“NadE*”。烟酸酰胺化蛋白的实例是具有氨基酸序列SEQID NO：1和3至18的多肽。SEQ ID NO：1来源于Francisella tularensis，被称为FtNadE*。FtNadE*蛋白序列在GENBANK登录号YP_170217下提供。SEQ ID NO：3来源于Francisellasp.FSC1006，被称为FspFNadE*；蛋白质登录号可以在登录号WP_040008427.1下获得。SEQID NO：4来源于Francisella guangzhouensis菌株08HL01032，被称为FgNadE*；蛋白质登录号可以在登录号WP_039124332.1下获得。SEQ ID NO：5来源于Francisella sp.TX077308，被称为FspTNadE*；蛋白质登录号可以在登录号WP_013922810.1下获得。SEQ ID NO：6来源于Francisella philomiragia subsp.philomiragia ATCC 25017，被称为FphNadE*；蛋白质登录号可以在登录号WP_004287429.1下获得。SEQ ID NO：7来源于Francisellaphilomiragia菌株O#319-036[FSC 153]，被称为NadE*；蛋白质登录号可以在登录号WP_042517896.1下获得。SEQ ID NO：8来源于Francisella noatunensis subsp.orientalisstr.Toba 04，被称为NadE*；蛋白质登录号可以在登录号WP_014714556.1下获得。SEQ IDNO：9来源于Francisella philomiragia菌株GA01-2794，被称为NadE*；蛋白质登录号可以在登录号WP_044526539.1下获得。SEQ ID NO：10来源于Francisella persica ATCC VR-331，被称为FpeNadE*；蛋白质登录号可以在登录号WP_064461307.1下获得。SEQ ID NO：11来源于Francisella cf.novicida 3523，被称为FnNadE*；蛋白质登录号可以在登录号WP_014548640.1下获得。SEQ ID NO：12来源于Francisella tularensis subsp.novicidaD9876，被称为NadE*；蛋白质登录号可以在登录号WP_003037081.1下获得。SEQ ID NO：13来源于Francisella tularensis subsp.novicida F6168，被称为NadE*；蛋白质登录号可以在登录号WP_003034444.1下获得。SEQ ID NO：14来源于Francisella tularensissubsp.tularensis菌株NIH B-38，被称为NadE*；蛋白质登录号可以在登录号WP_003025712.1下获得。SEQ ID NO：15来源于Francisella tularensis subsp.holarcticaF92，被称为NadE*；蛋白质登录号可以在登录号WP_010032811.1下获得。SEQ ID NO：16来源于Dichelobacter nodosus VCS1703A，被称为DnNadE*；蛋白质登录号可以在登录号WP_011927945.1下获得。SEQ ID NO：17来源于Mannheimia succiniciproducens MBEL55E，被称为MsNadE*；蛋白质登录号可以在登录号WP_011201048.1下获得。SEQ ID NO：18来源于Actinobacillus succinogenes 130Z，被称为AsNadE*；蛋白质登录号可以在登录号WP_012072393.1下获得。
术语“烟酰胺腺嘌呤二核苷酸水解蛋白”或“NAD+二磷酸酶”表示能够催化烟酰胺腺嘌呤二核苷酸(NAD+)转变为烟酰胺单核苷酸(NMN)和腺嘌呤的酶。该酶通常被称为NudC。本发明中使用的烟酰胺腺嘌呤二核苷酸水解蛋白可以来自多种生物体，例如E.coli,C.glutamicum,A.baylyi等。烟酰胺腺嘌呤二核苷酸水解蛋白的例子包括具有氨基酸序列SEQ ID NO：66至70的多肽。编码NAD+二磷酸酶活性的示例性基因在登录号WP_004921449(A.baylyi)、CAF19483(C.glutamicum)、YP_026280(E.coli)和WP_010813670(R.eutropha)下提供。
术语“NAD+生物合成的负调控因子”表示能够通过抑制喹啉酸合酶(NadA)、FAD依赖性L-天冬氨酸氧化酶(NadB)、喹啉酸磷酸核糖基转移酶(NadC)或其任何组合的转录来抑制NAD+生物合成活性的酶。编码NAD+生物合成的负调控因子的示例性基因以登录号WP_004398582.1(B.subtilis)、WP_000093814.1(E.coli)和WP_011014097.1(C.glutamicum)提供。术语“喹啉酸合酶”表示能够将亚氨基琥珀酸和磷酸二羟丙酮转变为喹啉酸和磷酸的酶。本发明中使用的喹啉酸合酶可以来自多种生物体，例如E.coli,B.subtilis,C.glutamicum等。喹啉酸合酶蛋白质的实例包括具有氨基酸序列SEQ ID NO：77、78或79的多肽。编码喹啉酸合成活性的基因在例如登录号ACX40525(E.coli),NP_390663(B.subtilis)和CAF19774(C.glutamicum)下提供。所限定的喹啉酸合酶包括上述喹啉酸合酶的功能性变体。
术语“L-天冬氨酸氧化酶”表示在FAD依赖性反应中能够将天冬氨酸转变为亚氨基琥珀酸的酶。本发明中使用的L-天冬氨酸氧化酶可以来自多种生物体，例如E.coli,B.subtilis,C.glutamicum等。核苷水解酶蛋白质的实例包括具有氨基酸序列SEQ ID NO：80或81的多肽。编码L-天冬氨酸氧化酶活性的基因在例如登录号ACX38768(E.coli)和NP_390665(B.subtilis)下提供。所限定的L-天冬氨酸氧化酶包括上述L-天冬氨酸氧化酶的功能性变体。
术语“喹啉酸磷酸核糖基转移酶”表示能够将喹啉酸和磷酸核糖基焦磷酸转变为烟酰胺单核苷酸和二氧化碳的酶。本发明中使用的喹啉酸磷酸核糖基转移酶可来自多种生物体，例如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：82、83或84的多肽。编码喹啉酸磷酸核糖基转移酶活性的基因在例如登录号ACX41108(E.coli)、NP_390664(B.subtilis)和CAF19773(C.glutamicum)下提供。所限定的喹啉酸磷酸核糖基转移酶包括上述喹啉酸磷酸核糖基转移酶的功能性变体。
术语“烟酰胺核糖苷转运蛋白”表示能够催化烟酰胺核糖苷转运以将烟酰胺核糖苷从周质导入细胞质的酶。该酶通常被称为PnuC。本发明中所述的烟酰胺核糖苷转运蛋白是宿主生物(例如E.coli,B.subtilis,C.glutamicum等)的天然多肽。烟酰胺核糖苷转运蛋白的实例包括具有氨基酸序列SEQ ID NO：54、55、56或71的多肽。编码NR转运活性的基因在例如登录号CAG67923(A.baylyi)、NP_599316(C.glutamicum)、NP_415272(E.coli)和WP_003227216.1(B.subtilis)下提供。
术语“烟酰胺单核苷酸水解酶”表示能够催化烟酰胺单核苷酸水解为烟酰胺核糖苷的酶。该酶通常被称为UshA。本发明中使用的核苷水解酶可以来自多种生物体，例如E.coli,B.subtilis,C,glutamicum等。核苷水解酶蛋白质的实例包括具有氨基酸序列SEQID NO：57、58或59的多肽。编码核苷水解酶活性的基因在例如登录号NP_415013(E.coli)、NP_388665(B.subtilis)和CAF18899(C.glutamicum)下提供。
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转变为烟酸单核苷酸的酶。该酶通常被称为PncC。本发明中所述的烟酰胺单核苷酸酰胺水解酶是宿主生物(例如E.coli,B.subtilis,C.glutamicum等)的天然多肽。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：60、61或62的多肽。编码烟酰胺单核苷酸酰胺水解酶活性的基因在例如登录号NP_417180(E.coli)、AAB00568(B.subtilis)和CAF20304(C.glutamicum)下提供。
术语“烟酸单核苷酸腺嘌呤基转移酶”表示能够催化烟酸单核苷酸转变为烟酸腺嘌呤二核苷酸的酶。该酶通常被称为NadD。本发明中所述的烟酸单核苷酸腺嘌呤基转移酶蛋白是宿主生物(例如E.coli,B.subtilis,C.glutamicum等)的天然多肽。烟酸单核苷酸腺嘌呤基转移酶蛋白的实例包括具有氨基酸序列SEQ ID NO：63、64或65的多肽。编码烟酸单核苷酸腺嘌呤基转移酶活性的基因在例如登录号NP_415172(E.coli)、NP_390442(B.subtilis)和CAF21017(C.glutamicum)下提供。
术语“嘌呤核苷磷酸化酶”表示能够催化烟酰胺核糖苷和磷酸转变为烟酰胺和核糖-1-磷酸的酶。该酶的常用名为DeoD、PupG和Pdp。本发明中所述的嘌呤核苷磷酸化酶是宿主生物(例如E.coli,B.subtilis,C.glutamicum等)的天然多肽。嘌呤核苷磷酸化酶蛋白的实例包括具有氨基酸序列SEQ ID NO：72至75的多肽。编码嘌呤核苷磷酸化酶活性的基因在例如登录号WP_003231176.1(B.subtilis)、WP_003243952.1(B.subtilis)、WP_0032300447.1(B.subtilis)、WP_000224877.1(E.coli)和BAC00196.1(C.glutamicum)下提供。
序列同一性：两个氨基酸序列之间或两个核苷酸序列之间的相关性通过参数“序列同一性”来描述。
为了本公开的目的，使用在EMBOSS包(EMBOSS:The European Molecular BiologyOpen Software Suite,Rice等人,2000,Trends Genet.16:276-277)的Needle程序、优选3.0.0版本或更新版本中执行的Needleman-Wunsch算法(Needleman和Wunsch,1970,J.Mol.Biol.48:443-453)确定两个氨基酸序列之间的序列同一性程度。所使用的任选参数是空位开放罚分为10，空位延伸罚分为0.5，以及EBLOSUM62(BLOSUM62的EMBOSS版本)替代矩阵。标记为“最长同一性”的Needle的输出(使用-nobrief选项获得)被用作同一性百分比，并如下计算：
(相同的残基x 100)/(比对长度-比对中的空位总数)。
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中分离的或被修饰以含有否则不会在自然中存在的形式的核酸的区段或者其是合成的。当核酸构建体含有表达本公开的编码序列所需要的控制序列时，术语核酸构建体与术语“表达盒”的含义相同。
控制序列：术语“控制序列”是指表达编码本公开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而言可以是天然的或外来的，或相对于彼此可以是天然的或外来的。这些控制序列包括但不限于前导序列、聚腺苷酸化序列、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终止信号和翻译终止信号。控制序列可以带有接头，目的是引入特异性限制性位点以促进控制序列与编码多肽的多核苷酸的编码区的连接。
可操作地连接：术语“可操作地连接”意指如下的构造，其中，控制序列相对于多核苷酸的编码序列安置在适当位置处，从而使得该控制序列指导该编码序列的表达。
表达：术语“表达”包括参与产生多肽的任何步骤，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”是指线性或环状的DNA分子，其包含编码多肽的多核苷酸，所述多核苷酸与提供其表达的额外核苷酸可操作地连接。
宿主细胞：术语“宿主细胞”意指任何细菌细胞类型，其对于用包含编码任何一种本公开多肽序列的多核苷酸的核酸构建体或表达载体进行转化、转染、转导等是易感的。术语“宿主细胞”涵盖由于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。
本发明涉及用于生产烟酰胺核糖苷的具有基因工程化特征的细菌菌株。
来自E.coli、B.subtilis和大多数表征的细菌物种以及所有表征的真核生物物种的nadE基因产物利用烟酸腺嘌呤二核苷酸作为酰胺化反应的底物来产生NAD+。凭借该天然途径，通过烟酰胺腺嘌呤二核苷酸(NAD+)的分解获得了烟酰胺核糖苷(NR)，如前述工作中(美国专利No.8,114,626B2)那样。参见图2。
生物体Francisella tularensis经由替代途径合成NAD+，其中NMN是通过F.tularensis NMN合成酶(FtNadE*)的作用从NaMN产生。
出乎意料地，本发明的发明人在细菌中创建了朝向NR的替代途径，其由以下组成：将烟酸单核苷酸酰胺化成NMN，然后去磷酸化成NR。参见图3。例如，本发明的发明人已发现：在E.coli中表达FtNadE*基因或其功能同源物会导致产生过量的NMN。过量的NMN可被输出并通过天然周质酸性磷酸酶被转变为NR。
本发明的发明人进一步发现：FtNadE*不是可用于上述产生NR的替代途径中的唯一蛋白。本发明的发明人从执行相同功能的γ-变形菌的不同菌株中鉴定出了一组NadE*蛋白。例如，编码具有SEQ ID No：3-18的烟酸酰胺化蛋白的NadE*基因或其功能同源物的表达也会导致NR的产生。
因此，在本发明的第一个实施方式中，期望向宿主细胞中引入一种或多种烟酸酰胺化基因。此类基因编码烟酸酰胺化蛋白，其催化NaMN朝向NMN的转变。在一个实施方式中，烟酸酰胺化蛋白是NMN合成酶(NadE*)。在一个具体的实施方式中，烟酸酰胺化蛋白是F.tularensisNMN合成酶(FtNadE*)。根据本文一些实施方式的烟酸酰胺化蛋白可包括，例如但不限于，包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：1和3至18的同一性为至少50％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％，其中上述多肽具有烟酸酰胺化活性或NadE*活性。
本申请的发明人已经确定了SEQ ID NO：1和3至18之中的保守多肽。序列比对的结果示于图5中。另外，以下三个氨基酸被认为对于维持NadE*蛋白活性非常重要：第27位的酪氨酸、第133位的谷氨酰胺和第236位的精氨酸。上述位置基于SEQ ID NO：1编号。参见图5中的SEQ ID No：1和3至18的ClustalW比对。
因此，在一些实施方式中，当与SEQ ID NO：1的参照氨基酸序列比较时，NadE*蛋白可以进一步含有一个或多个以下保守氨基酸：a)第27位的酪氨酸，b)第133位的谷氨酰胺，和c)第236位的精氨酸，其中基于ClustalW比对方法与SEQ ID NOS：1和3至18进行比较，使用缺省参数空位罚分＝10，空位长度罚分＝0.1和Gonnet 250系列蛋白质权重矩阵。SEQ IDNO：1具有第27位的酪氨酸、第133位的谷氨酰胺和第236位的精氨酸。
在E.coli和其他细菌物种中，nudC基因产物催化NADH水解为NMN和单磷酸腺苷(AMP)。在大多数生长条件下，nudC基因以非常低的水平表达。出乎意料地，本发明的发明人通过在含有或不含天然nudC基因的宿主细胞中添加异源nudC基因或通过将天然nudC基因置于强组成型或诱导型启动子的控制之下来创建驱动从NADH产生NMN的替代途径。参见图3。在生产条件下表达nudC导致产生过量的NMN。过量的NMN可被输出并通过天然周质酸性磷酸酶被转变为NR。
因此，在本发明的第二个实施方式中，期望提高nudC基因的表达水平，从而导致宿主细胞产生过量的NMN。在一个实施方式中，本发明涉及具有提高的NAD+二磷酸酶活性的细菌菌株。根据本文的一些实施方式的NAD+二磷酸酶可以包括，例如但不限于，包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：66至70之任一的同一性为至少50％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％，其中上述多肽具有将NAD+转变为NMN的NAD+二磷酸酶活性。
期望修饰用于NR生产的宿主生物体，使得可在细胞内获得更高浓度的NaMN。因此，在本发明的另一个实施方式中，期望引入一种或多种导致宿主细胞内烟酸单核苷酸的生产速率提高的遗传修饰。所述修饰可以包括抑制重新合成NAD+生物合成途径的基因nadA、nadB和/或nadC中的全部或部分的转录的基因的缺失或表达减少。所述修饰可以另外或替代性地包括增加例如由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、喹啉酸合酶基因、喹啉酸磷酸核糖基焦磷酸基因或其组合的表达。所述修饰可以另外或替代性地包括对nadB基因的修饰，这使得该基因对下游代谢物NAD+的抑制具有抗性。
本发明还包括烟酰胺核糖苷导入和补救途径缺陷的基因工程细菌菌株。参见图4。预期细菌中NAD+补救途径的破坏会导致细胞外NR积累，因为这种菌株不能将烟酰胺核糖苷导入细胞质中，并且也不能将烟酰胺核糖苷(NR)磷酸化为烟酰胺单核苷酸(NMN)，或将NMN进一步降解成烟酸单核苷酸(NaMN)。三种酶活性对于细菌NR生产的工程化特别重要。E.coli中的pncC基因产物和B.subtilis中的cinA基因产物是细菌中的补救酶，其将NMN脱酰胺化成NaMN，这与由NadE*催化的反应相反。该基因的缺失阻止NMN向NaMN转化并提高NMN的细胞内浓度。通过核苷磷酸化酶活性将NR降解为Nam和磷酸核糖除去产物，并且编码该活性的基因(例如E.coli中的deoD或B.subtilis中的pdp)的缺失或降低的表达会提高产物形成的速率。在E.coli和许多其他细菌中，pnuC基因产物导入NR，缺失会增加细胞外NR；在B.subtilis中，NR导入是由nupG基因产物完成的，缺失会提高细胞外NR。
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导入和补救途径，因此使宿主细胞保存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一个或多个以下特征：i)烟酰胺摄取转运体的活性被阻断或降低，ii)作为烟酸核苷磷酸化酶起作用的蛋白质被阻断或降低，和iii)烟酰胺单核苷酸酰胺水解酶的活性被阻断或降低，iv)作为NAD+生物合成蛋白(例如L-天冬氨酸氧化酶、喹啉酸合酶和喹啉酸磷酸核糖基转移酶)的负调控因子起作用的蛋白质被阻断或降低，v)作为嘌呤核苷磷酸化酶起作用的蛋白质被阻断或降低；和vi)作为烟酸单核苷酸腺嘌呤基转移酶起作用的蛋白质被阻断或降低。
根据本文一些实施方式的NAD+生物合成的负调控因子可以包括，例如但不限于，包含SEQ ID NO：51、52或53之任一的氨基酸序列的多肽或所述多肽的变体，其中上述多肽具有抑制NAD+生物合成所需基因的活性。
在一些实施方式中，喹啉酸合酶是包含SEQ ID NO：77、78或79的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有从亚氨基琥珀酸和磷酸二羟丙酮形成喹啉酸的活性。
在一些实施方式中，L-天冬氨酸氧化酶是包含SEQ ID NO：80或81的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有从天冬氨酸形成亚氨基琥珀酸的活性。
在一些实施方式中，喹啉酸磷酸核糖基转移酶是包含SEQ ID NO：82、83或84的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有从喹啉酸和磷酸核糖基焦磷酸形成烟酸单核苷酸的活性。
根据本文一些实施方式的烟酰胺摄取转运体(PnuC或NupG)可以包括，例如但不限于，包含SEQ ID NO：54、55、56或71之任一的氨基酸序列的多肽或所述多肽的变体，其中上述多肽具有烟酰胺核糖导入活性。
根据本文一些实施方式的烟酰胺单核苷酸酰胺水解酶(PncC)可包括，例如但不限于，包含SEQ ID NO：15、16或17之任一的氨基酸序列的多肽或所述多肽的变体，其中上述多肽具有烟酰胺单核苷酸酰胺水解酶的活性。
根据本文一些实施方式的烟酸单核苷酸腺嘌呤基转移酶(NadD)可包括，例如但不限于，包含SEQ ID NO：18或SEQ ID NO：19的氨基酸序列的多肽或所述多肽的变体，其中上述多肽具有将烟酸单核苷酸转变为烟酸腺嘌呤二核苷酸的烟酸单核苷酸腺嘌呤基转移酶的活性。
根据本文一些实施方式的嘌呤核苷磷酸化酶(DeoD、PupG、Pdp)可包括，例如但不限于，包含SEQ ID NO：72至76之任一的氨基酸序列的多肽或所述多肽的变体，其中上述多肽具有将烟酰胺核糖苷和磷酸转变为烟酰胺和核糖-1-磷酸的嘌呤核苷磷酸化酶的活性。
在本发明的第四个实施方式中，期望提高ushA基因的表达水平，从而使宿主细胞从NMN产生过量的细胞外NR。在一个实施方式中，本发明涉及具有提高的核苷水解酶活性的细菌菌株。根据本文一些实施方式的烟酰胺单核苷酸水解酶(UshA)可以包括，例如但不限于，包含SEQ ID NO：57、58或59之任一的多肽或所述多肽的变体，其中上述多肽具有将烟酰胺单核苷酸转变为烟酰胺核糖苷的核苷水解酶的活性。
还期望提高NAD+生物合成蛋白(例如L-天冬氨酸氧化酶、喹啉酸合酶和喹啉酸磷酸核糖基转移酶)的表达水平。在一个实施方式中，本发明涉及一种或多种以下蛋白质的活性提高的细菌菌株：L-天冬氨酸氧化酶、喹啉酸合酶和喹啉酸磷酸核糖基转移酶。
在一些实施方式中，所述喹啉酸合酶是包含SEQ ID NO：77、78或79的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有从亚氨基琥珀酸和磷酸二羟丙酮形成喹啉酸的活性。
在一些实施方式中，L-天冬氨酸氧化酶是包含SEQ ID NO：80或81的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有从天冬氨酸形成亚氨基琥珀酸的活性。
在一些实施方式中，喹啉酸磷酸核糖基转移酶是包含SEQ ID NO：82、83或84的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有从喹啉酸和磷酸核糖基焦磷酸形成烟酸单核苷酸的活性。
在另一些实施方式中，以上第一种实施方式或第二种实施方式中所述的细菌菌株进一步包含一种或多种以上第三种实施方式或第四种实施方式中所述的修饰。
例如，在一个实施方式中，本发明涉及能够产生烟酰胺核糖苷的经遗传修饰的细菌，其中所述细菌包含以下修饰：i)添加的异源烟酸酰胺化蛋白NadE*和ii)一种或多种另外的选自以下的修饰：a)具有阻断或降低的活性的改变的NAD+生物合成的负调控因子；b)具有阻断或降低的活性的改变的烟酰胺核糖苷摄取转运体；c)具有阻断或降低的活性的改变的烟酸单核苷酸腺嘌呤基转移酶；d)具有阻断或降低的活性的改变的烟酰胺单核苷酸酰胺水解酶，e)具有阻断或降低的活性的改变的嘌呤核苷磷酸化酶；f)具有添加或提高的活性的改变的烟酰胺单核苷酸水解酶；和g)添加或提高的编码L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶的基因或其组合的转录；其中与没有任何所述修饰的细菌相比，具有所述至少一种修饰的细菌产生提高量的NR。
在另一个实施方式中，本发明涉及能够产生烟酰胺核糖苷的经遗传修饰的细菌，其中所述细菌包含以下修饰：i)具有添加或提高的活性的改变的烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白NudC；和ii)一种或多种另外的选自以下的修饰：a)具有阻断或降低的活性的改变的NAD+生物合成的负调控因子；b)具有阻断或降低的活性的改变的烟酰胺核糖苷摄取转运体；c)具有阻断或降低的活性的改变的烟酸单核苷酸腺嘌呤基转移酶；d)具有阻断或降低的活性的改变的烟酰胺单核苷酸酰胺水解酶，e)具有阻断或降低的活性的改变的嘌呤核苷磷酸化酶；f)具有添加或提高的活性的改变的烟酰胺单核苷酸水解酶；和g)添加或提高的编码L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶的基因或其组合的转录；其中与没有任何所述修饰的细菌相比，具有所述至少一种修饰的细菌产生提高量的NR。
在一个实施方式中，烟酸酰胺化蛋白NadE*对于宿主细菌是外源的，即在已经使用例如本文所述的重组方法引入而修饰之前不存在于该细胞中。
在另一个实施方式中，上述其他蛋白质对于宿主细菌是内源的，即在修饰之前存在于该细胞中，但进行了改变以提高或降低所述蛋白质的表达水平。在本发明中表达水平被改变的内源蛋白质的实例包括但不限于NAD+二磷酸酶、NAD+生物合成的负调控因子、烟酰胺核糖苷摄取转运体、烟酰胺单核苷水解酶、烟酸单核苷酸腺嘌呤基转移酶和烟酰胺单核苷酸酰胺水解酶。
可以通过本领域技术人员已知适合于该目的的任何方式对宿主细菌细胞进行遗传修饰。这包括将感兴趣的基因(例如编码烟酸酰胺化蛋白NadE*的基因)引入能够在宿主细胞内增殖的质粒或粘粒或其他表达载体中。或者，质粒或粘粒DNA或者质粒或粘粒DNA的一部分或线性DNA序列可以整合到宿主基因组中，例如通过同源重组或随机整合。为了进行遗传修饰，可以通过天然摄取或通过公知的方法(例如电穿孔)将DNA引入或转化到细胞中。遗传修饰可涉及在引入的启动子的控制下表达基因。引入的DNA可编码能够充当酶或能够调控其它基因表达的蛋白质。
微生物的遗传修饰可以使用经典菌株开发和/或分子遗传技术完成。此类技术是本领域已知的并且通常关于微生物公开，例如在Sambrook等,1989,Molecular Cloning:ALaboratory Manual,Cold Spring Harbor Labs Press中。上述参考文献Sambrook等通过引用整体并入本文。
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/或可以形成包含基因组合的表达载体的一部分。这种表达载体构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是本领域公知的，并且可以排列成包含与一个或多个表达控制序列可操作地连接的多核苷酸，以便可用于在宿主细胞(例如如上所述的细菌)中表达所需的酶。例如，包括但不限于T7启动子、pLac启动子、nudC启动子、ushA启动子、pVeg启动子的启动子可以与内源基因和/或异源基因组合使用以修饰靶基因的表达模式。类似地，示例性终止子序列包括但不限于XPR1、XPR2、CPC1终止子序列的使用。
在一些实施方式中，本说明书通篇中提及的重组细菌细胞或经遗传修饰的细菌细胞可以是任何革兰氏阳性细菌或革兰氏阴性细菌，包括但不限于Bacillus,Corynebacterium,Escherichia,Acinetobacter,Lactobacillus,Mycobacterium,Pseudomonas和Ralstonia属。在某些实施方式中，示例性细菌物种包括但不限于Bacillussubtilis,Corynebacterium glutamicum,Escherichia coli,Acinetobacter baylyi和Ralstonia eutropha。这些实施方式不限于特定的物种，而是涵盖细菌的所有主要的门(图7)。
本公开的经遗传修饰的细菌还涵盖包含本文所限定的多肽的变体的细菌。在本文中使用时，“变体”意指这样的多肽，其中氨基酸序列与其所来源于的基础序列的不同之处在于：在一个或更多个(几个)位置处进行了一个或更多个(几个)氨基酸残基的替换、插入和/或缺失。替换意指用不同氨基酸置换占据某位置的氨基酸；缺失意指去除占据某位置的氨基酸；插入意指在占据某位置的氨基酸旁添加1-3个氨基酸。
变体是功能性变体，即其中变体序列与具有本文指定的天然氨基酸序列的酶具有相似或相同的功能性酶活性特征。
例如，SEQ ID NO：1和3至18的功能性变体分别与SEQ ID NO：1和3至18具有相似或相同的烟酸酰胺化蛋白FtNadE*活性特征。实例可以是通过SEQ ID NO：1和3至18的功能性变体将烟酸单核苷酸朝向烟酰胺单核苷酸的转变率可以相同或相似，但所述功能性变体还可以提供其他益处。例如，当使用为SEQ ID NO：1和3至18的功能性变体的酶时，将达到至少约80％、90％、95％、96％、97％、98％、99％或100％的转变率。
因此，任何上述SEQ ID NO氨基酸序列的功能性变体或片段是保留在相同酶类别内(即具有相同EC号)的任何氨基酸序列。判定酶是否属于特定类别的方法是本领域技术人员公知的，本领域技术人员可以在不使用创造性技能的情况下判定酶类别。例如，合适的方法可以从国际生物化学和分子生物学联合会(International Union of Biochemistryand Molecular Biology)获得。
当氨基酸被具有广泛相似性质的不同氨基酸置换时，氨基酸替换可被视为“保守的”。非保守替换是氨基酸被不同类型的氨基酸置换的情况。
“保守替换”是指氨基酸被相同类别的另一氨基酸替换，其中所述类别限定如下：
氨基酸类别的例子：
非极性：A、V、L、I、P、M、F、W
不带电极性：G、S、T、C、Y、N、Q
酸性：D、E
碱性：K、R、H。
根据本公开产生的烟酰胺核糖化合物可用于多种应用中的任何，例如利用它们的生物或治疗特性(例如，控制低密度脂蛋白胆固醇，增加高密度脂蛋白胆固醇等)。例如，根据本公开，烟酰胺核糖可用于药物、食品和膳食补充剂等中。
通过本发明公开的方法产生的烟酰胺核糖苷可以在改善血脂谱、预防中风、提供化疗处理的神经保护、治疗真菌感染、预防或减少神经退行、或延长健康和良好状态方面具有治疗价值。因此，本发明进一步涉及从上述经遗传修饰的细菌细胞获得的烟酰胺核糖苷化合物，其用于通过施用有效量的烟酰胺核糖苷组合物来治疗与NAD+生物合成的烟酰胺核糖苷激酶途径相关的疾病或病症。通常具有改变的NAD+或NAD+前体水平或可获益于通过用烟酰胺核糖苷处理而提高的NAD+生物合成的疾病或病症包括但不限于脂质失调(例如血脂异常、高胆固醇血症或高脂血症)、中风、神经退行性疾病(例如阿尔茨海默氏病、帕金森氏症和多发性硬化症)、利用化学疗法时观察到的神经毒性、Candida glabrata感染以及与老化相关的总体健康下降。这种疾病和病症可以通过饮食补充或用烟酰胺核糖苷组合物提供治疗处理方案来预防或治疗。
应该理解的是，从本发明的经遗传修饰的细菌分离的烟酰胺核糖苷化合物可再配制成最终产品。在本公开的一些其他实施方式中，将由本文所述的经操作的宿主细胞产生的烟酰胺核糖苷化合物在宿主细胞的背景下掺入最终产品(例如食物或饲料补充剂、药物等)中。例如，可以将宿主细胞冻干、冷冻干燥、冷冻或以其它方式灭活，然后可以将完整细胞掺入最终产品中或用作最终产品。也可以对宿主细胞进行处理，然后掺入产品中以提高生物利用度(例如，通过裂解)。
在本公开的一些实施方式中，将所产生的烟酰胺核糖苷化合物掺入食物或饲料的组分(例如食物补充剂)中。根据本公开可以掺入烟酰胺核糖苷化合物的食品的类型没有特别限制，包括饮品，诸如乳、水、软饮料、能量饮料、茶和果汁；零食，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，诸如米饭、面包、早餐麦片等。在一些实施方式中，将所产生的烟酰胺核糖苷化合物掺入饮食补充剂(例如综合维生素剂)中。
以下实施例旨在阐释本发明，但不以任何方式限制其范围。
实施例
实施例1
鉴定编码NaMN酰胺化活性(NadE*)的序列
Sorci及其合作者鉴定了由Francisella tularensis的基因组编码的酶FtNadE*(SEQ ID NO：1)，并且证明了其作为烟酰胺单核苷酸(NaMN)酰胺化酶在体内和体外起作用的能力(Sorci L.e.,2009)。此外，他们提出：三个氨基酸残基负责酶对NaMN(相较于NaAD)的底物优先性：Y27；Q133和R236。为了鉴定编码该功能的其他序列，将使用tBlastn的默认参数、利用FtNadE的氨基酸序列(SEQ ID NO：2)、从2016年9月14日的NCBI nr/nt数据库的BLAST搜索得到的50个独特核苷酸序列翻译并使用Geneious比对算法(Biomatters,LLLC.)进行比对。这些序列中有16个具有分别与Y27、Q133和R236对齐的保守的酪氨酸、谷氨酰胺和精氨酸(即含有“Y-Q-R基序”)，并被预测编码NaMN酰胺化酶(SEQ ID NO：3至18和图5)。
实施例2
用于在E.coli中表达NaMN酰胺化活性(NadE*)的基因构建体
基于最大化系统发生距离(图6)，在16个预测的NadE*基因集合中选择10个编码预测的NadE*开放阅读框(SEQ ID NO：2和19至27)的序列，并针对在E.coli中表达进行密码子优化，其中使用Geneious密码子优化算法，利用E.coli K-12密码子使用表，稀有阈值设定为0.4。优化的序列(SEQ ID NO 28至37)由GenScript,Inc.从头合成，并且同样由GenScript克隆到XhoI/NdeI消化的pET24a(+)(Novagen,Inc.)中，从而产生了表1中的质粒。将质粒转化到BL21(DE3)中，这允许IPTG诱导NadE*基因以诱导NR合成并产生了菌株ME407、ME644、ME645、ME646、ME647、ME648、ME649、ME650、ME651、ME652(表2)。
表1：本研究中使用的质粒
表9：图6中序列的查阅表
表2：本研究中使用或描述的菌株
本文所述的所有基本分子生物学和DNA操作程序通常根据Sambrook等或Ausubel等(J.Sambrook,E.F.Fritsch,T.Maniatis(编).1989.Molecular Cloning:A LaboratoryManual.Cold Spring Harbor Laboratory Press:New York；和F.M.Ausubel,R.Brent,R.E.Kingston,D.D.Moore,J.G.Seidman,J.A.Smith,K.Struhl(编).1998.CurrentProtocols in Molecular Biology.Wiley:New York)进行。
实施例3
表达NadE*酶的E.coli菌株的表征
为了检测NadE*表达对NR产生的影响，将E.coli菌株从单菌落接种到LB培养基中并过夜生长(2mL，37℃，15mL试管，250rpm，50ug/mL卡那霉素)。预培养物(200μL)被用于接种2mL含有或不含25μM IPTG的M9nC培养基，并在用AirPore胶带片(Qiagen)密封的24孔深孔板(Whatman Uniplate，10mL，圆底)中生长3天(Infors Multitron摇床，800rpm，80％湿度)。如本文所述通过LC-MS分析样品。没有质粒时，在存在和不存在用25μM IPTG诱导的情况下，NR产生低于定量极限。诱导后，含有用于表达NadE*酶的质粒的菌株产生高达2.7mg/L的NR(表3)。
表3：IPTG诱导后，E.coli摇板培养物中的烟酰胺核糖苷浓度(mg/L)(2个培养物的平均值)
菌株 酶 无IPTG 25uM IPTG
ME407 FtNadE* <LOQ 0.11
ME409 无 <LOQ <LOQ
ME644 DnNadE* <LOQ 0.28
ME645 AsNadE* 0.08 0.31
ME646 FphNadE* 0.02 <LOQ
ME647 FnNadE* <LOQ 1.91
ME648 FspTNadE* 0.03 1.29
ME649 FspFNadE* <LOQ 0.82
ME650 FgNadE* <LOQ 0.64
ME651 FpeNadE* 0.14 1.09
ME652 MnNadE* 0.1 2.73
实施例4
E.coli中增加的NR产生需要具有Y27、Q133和R236的NadE*
为了证明YQR基序对于NR产生的重要性，改变了四个E.coli优化的NadE*序列以除去与Francisella tularensis Y27、Q133、R236残基对齐的残基并用在Bacillusanthracis NadE中编码的氨基酸残基置换(分别为T、G和V；SEQ ID NO：42至45)。由GenScript,Inc进行了相应pET24a(+)质粒的定点诱变，产生了表1中的质粒。将质粒转化到BL21(DE3)中，这允许IPTG诱导nadE-TGV基因并产生了菌株ME708、ME710、ME712和ME714(表2)。具有NadE-TGV的这些菌株没有展示出与具有NadE*的菌株相似的IPTG依赖性的NR产生增加(表4)。
表4：IPTG诱导后，E.coli摇板培养物中的烟酰胺核糖苷浓度(mg/L)
实施例5
E.coli NadE过表达不足以观察到增加的NR产生
为了证明高水平的NaAD酰胺化活性(NadE)不足以产生增加的NR积累，使用分别在起始密码子和终止密码子处添加XhoI/NdeI限制性位点的引物MO11159和MO11160(表5)，通过PCR从BL21(DE3)的基因组扩增野生型nadE开放读码(SEQ ID NO：46)框架。将PCR片段连接到类似地消化的pET24a(+)中，从而产生了质粒pET24b+nadEBL21。将该质粒转化到BL21(DE3)中，这允许IPTG诱导nadE并产生了菌株ME683。当与表达NadE*序列的菌株一起检测NR产生时，额外表达E.coli NadE的该菌株没有展示出IPTG依赖性的NR浓度增加(表4)。
表5：菌株构建中使用的引物
实施例6
构建具有增加的NR累积基础水平的B.subtilis菌株
为了证明NadE*酶在较高产物积累的情况下促进NR积累的功效，将宿主菌株工程化以提高NR积累的基础水平。使E.coli菌株DH5ɑ、Corynebacterium glutamicum菌株ATCC13032和B.subtilis菌株168在丰富培养基(对于E.coli为LB,对于C.glutamiucm和B.subtilis为BHI)中过夜生长，然后以1:10接种到2mL M9nC培养基中。24小时后，对培养物取样用于MS并检查相对NR水平。B.subtilis的NR产生高于E.coli或C.glutamiucm的NR产生，B.subtilis被选为进一步工程的宿主。
通过长侧翼PCR(LF-PCR)构建用于精确缺失nadR、deoD和pupG的盒。通过利用表5中的引物扩增BS168基因组DNA(Roche High Pure PCR模板制备试剂盒)来获得每个基因的侧翼区，所述引物被设计为使得与合适抗生素抗性基因的5'或3'区域同源的序列(分别为壮观霉素、四环素和新霉素，SEQ ID NO：48至50)被并入PCR产物中(Phusion Hot StartFlex DNA聚合酶，每种引物200nM，最初在95℃下变性2分钟，30个循环的在95℃下30秒；在50℃下20秒；在72℃下60秒，最后在72℃下保持7分钟)。利用引物类似地扩增抗生素抗性基因以并入与5'和3'侧翼区同源的序列。将PCR产物凝胶纯化并用于利用合适引物(表5)的LF-PCR(Phusion Hot Start Flex DNA聚合酶，每种引物200nM，每种PCR产物150ng，最初在98℃下变性30秒，35个循环的在98℃下30秒；在55℃下30秒；在72℃下360秒)。将LF-PCR产物纯化并用于转化B.subtilis菌株。
通过自然转化用LF-PCR产物转化BS168(“Molecular Biological Methods forBacillus”.1990.CR Harwood和S.M.Cutting编.John Wiley and Sons)，产生了BS6209(nadR::spe)、ME479(deoD::tet)和ME492(pupG::neo)。使用来自ME492的基因组DNA(如上所述制备)转化BS6209，产生了ME496(nadR::spe pupG::neo)。使用来自ME479的基因组DNA(如上所述制备)转化ME496，产生了ME517(nadR::spe pupG::neo deoD::tet)。
实施例7
表达NadE*的B.subtilis菌株的构建和表征
将4个编码NadE*活性的序列针对在B.subtilis中表达密码子优化(Geneious密码子优化算法，B.subtilis 168密码子使用表，稀有阈值设定为0.4)，优化的序列(SEQ IDNO：38-41)由IDT合成为gBlocks。通过LF-PCR产生用于表达优化的NadE*序列的盒。使用合适的引物(表5)以及pDG1662(Bacillus Genetic Stock Center)和gBlocks作为模板，如上所述扩增含有amyE 5’区域、cat(氯霉素抗性)、pVegI启动子的侧翼区和含有amyE 3’区域的侧翼区。如上所述使用凝胶纯化的侧翼区和gBlocks(上述)进行LF-PCR，并凝胶纯化产物。
如上所述用纯化的DNA转化ME517，并对转化体进行菌落纯化，得到了菌株ME795(MsNadE*)、ME805(FnNadE*)、ME820(FspNadE*)和ME824(FtNadE*)。使用菌株一式两份地接种1mL BHI培养基的培养物，并将ME517一式四份地接种在24孔摇板中并在37℃下孵育过夜(如上所述)。17小时后，离心板，弃上清液并将沉淀重悬于2mL M9nC培养基中。将板放回培养箱中并生长另外24小时。测量NR，含有NadE*过表达构建体的菌株比亲本菌株平均产生多72％-133％的NR(表6)。
表6：B.subtilis摇板培养物中的烟酰胺核糖苷浓度
菌株 酶 NR(mg/L)
ME517 无 53.2
ME517 无 42.2
ME517 无 38.8
ME517 无 37.6
ME795 MsNadE* 72.3
ME795 MsNadE* 75.5
ME814 FnNadE* 102.9
ME814 FnNadE* 98.0
ME820 FspNadE* 99.1
ME820 FspNadE* 94.8
ME824 FtNadE* 76.8
ME824 FtNadE* 79.9
实施例8
表达NadE*的Corynebacterium glutamicum菌株的构建和表征
为了进一步证明这些序列用于在细菌中产生NR的一般用途，针对在C.glutamicum中表达对编码FnNadE*的序列进行密码子优化(Geneious密码子优化算法，C.glutamicum密码子使用表，稀有阈值设定为0.4)，优化的序列(SEQ ID NO：47)由IDT合成为gBlock，所述gBlock含有在编码EcoRI限制位点和Corynebacterium glutamicum共有RBS的开放阅读框上游的附加序列和在编码SmaI限制位点下游的附加序列。用EcoRI/SmaI消化gBlock，产生了760bp的片段，将该片段连接到类似消化的MB4124中，产生了质粒MB4124-FnNadE*。MB4124来源于隐秘(cryptic)C.glutamicum低拷贝pBL1质粒(参见Santamaria等人，J.Gen.Microbiol,130:2237-2246,1984)，这是通过将MB4094(描述于美国专利申请60/692,037中)与来自pTrc99a的IPTG诱导型启动子(Gene.1988年9月30日；69(2):301-15.)组合来实现的。用质粒转化C.glutamicum菌株ATCC13032(Follettie,M.T.等,J.Bacteriol.167:695-702,1993)，以用于FnNadE*的IPTG诱导性表达。
将单菌落接种到2mL VY培养基(视情况而定，+50μg/mL卡那霉素)中并在30℃下生长过夜。将200μL这种培养物用于接种2mL AZ培养基，所述AZ培养基含有2％葡萄糖(视情况而定，+10μg/mL卡那霉素)和不同水平的IPTG。测量NR，含有FnNadE*过表达构建体的菌株展示出IPTG依赖性的NR产生增加(表7)。
表7：IPTG诱导FnNadE*后，Corynebacterium glutamicum摇板培养物中的烟酰胺核糖苷浓度(mg/L)
菌株 0mM IPTG 0.25mM IPTG
ATCC13032 0.02 0.03
ME763 0.03 0.16
ME763 0.01 0.11
实施例9
检测生产培养基中的烟酰胺核糖苷
通过液相色谱/质谱(LCMS)分析NR。培养后，将100μL在96孔深孔板中的900μL MS稀释液(10％水，10mM乙酸铵(pH9.0)，90％乙腈)中稀释。将板离心(10分钟，3000rpm)并将上清液转移到新的板上进行表征。将5μl份的上清液注射到HILIC UPLC柱(Waters BEH酰胺，2.1x75mm P/N 1860005657)上。保留1分钟后，使用从99.9％(pH 9.0的10mM乙酸铵，95％乙腈/5％水)流动相D至70％(pH 9.0的10mM乙酸铵，50/50的乙腈/水)流动相C的线性梯度，以400uL min-1的流速洗脱化合物，持续12分钟，然后在流动相C中保留1分钟，并在流动相D中重新平衡5分钟(表8)。利用三重四极杆质谱仪使用正电喷雾电离检测洗脱的化合物。仪器在MRM模式下运行，使用转变(transition)m/z123>80检测NR。通过与在相同条件下注射的标准品(Chromadex)比较来量化NR。
表8：用于NR的LCMS定量的梯度程序
实施例10
用于细菌生长和生产试验的培养基
1升VY培养基含有25g小牛肉浸液肉汤(Difco)、5g Bacto酵母提取物(Difco)。
1升M9nC培养基含有50g葡萄糖、6g Na2HPO4、3g KH2PO4、0.5g NaCl、1g NH4Cl、2mMMgSO4、15mg Na2EDTA、4.5mg ZnSO4*7H2O、0.3mg CoCl2*6H2O、1mg MnCl2*4H2O、4.5mg CaCl2*2H2O、0.4mg Na2MoO4*2H2O、1mg H3BO3和0.1mg KI。
1升AZ培养基含有20g葡萄糖、2g NaCl、3g柠檬酸钠、0.1g CaCl2*2H2O、4g K2HPO4、2g KH2PO4、7.5g NH4SO4、3.75g尿素、0.5g MgSO4*7H2O、450μg硫胺素、450μg生物素、4mg泛酸盐/酯、15mg Na2EDTA、4.5mg ZnSO4*7H2O、0.3mg CoCl2*6H2O、1mg MnCl2*4H2O、4.5mg CaCl2*2H2O、0.4mg Na2MoO4*2H2O、1mg H3BO3和0.1mg KI。
序列表
<110> 帝斯曼知识产权资产管理有限公司
<120> 烟酰胺核糖苷的微生物生产
<130> 31313-WO-PCT
<140> TBD
<141> 2016-11-14
<150> 62/233696
<151> 2015-09-28
<150> 62/254736
<151> 2015-11-13
<160> 104
<170> PatentIn version 3.5
<210> 1
<211> 249
<212> PRT
<213> Francisella tularensis
<400> 1
Met Lys Ile Val Lys Asp Phe Ser Pro Lys Glu Tyr Ser Gln Lys Leu
1 5 10 15
Val Asn Trp Leu Ser Asp Ser Cys Met Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Leu Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Asp
50 55 60
Asn Asn Gln His Gln Asp Met Gln Asp Ala Leu Glu Leu Ile Glu Met
65 70 75 80
Leu Asn Ile Glu His Tyr Thr Ile Ser Ile Gln Pro Ala Tyr Glu Ala
85 90 95
Phe Leu Ala Ser Thr Gln Ser Phe Thr Asn Leu Gln Asn Asn Arg Gln
100 105 110
Leu Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Tyr Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Asp Val Pro Lys Asn Ile Leu Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asp Phe Leu Asp Gly Lys Gln Val Ser Ala
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Lys Leu Ala Leu Thr Pro Asn Phe
245
<210> 2
<211> 750
<212> DNA
<213> Francisella tularensis
<400> 2
atgaaaatag ttaaagattt tagtcctaaa gaatattcac aaaagttagt taattggcta 60
agtgatagtt gtatgaatta tcctgctgaa ggatttgtga ttggtcttag tggcggtata 120
gattcagcag ttgcggcttc tttagctgtc aaaactggat taccaactac agctttaata 180
ctaccttcag ataataatca acaccaagat atgcaagatg ctctagagct tattgaaatg 240
cttaatattg aacattatac catttcgatt caaccagctt atgaggcttt tcttgcttca 300
acgcaaagct ttacaaatct acaaaacaat agacaacttg tgatcaaggg aaatgctcaa 360
gcacgtttaa ggatgatgta tttgtatgcc tatgcgcaac aatataacag aatagttata 420
ggtactgata atgcttgtga gtggtatatg ggatatttta caaaattcgg tgatggggct 480
gccgatatac ttccactagt taatctcaaa aaatctcaag tttttgaatt aggcaaatac 540
ctagatgtcc ctaaaaacat acttgataaa gctccatctg caggactatg gcaaggacaa 600
actgatgagg atgaaatggg tgtaacttat caagaaattg atgatttctt agatggtaaa 660
caagtttcag caaaagctct agaaagaata aatttctggc ataatcgtag tcaccataag 720
agaaaattag ctttaactcc taatttctag 750
<210> 3
<211> 249
<212> PRT
<213> Francisella sp. FSC1006
<400> 3
Met Ser Val Val Lys Asn Phe Lys Pro Asn Glu Tyr Ala Asn Lys Ile
1 5 10 15
Thr Glu Trp Leu Lys Asp Ser Cys Leu Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Val Gly Ile Ser Gly Gly Ile Asp Ser Ala Val Ala Val Ser Leu
35 40 45
Ala Val Asn Thr Gly Leu Pro Val Thr Gly Leu Ile Met Pro Ser Lys
50 55 60
Asn Asn Asp Asp Lys Asp Thr Leu Asp Ala Ile Glu Leu Ala Lys Lys
65 70 75 80
Leu Asn Ile Glu Tyr His Leu Ile Pro Ile Gln Pro Val Tyr Glu Thr
85 90 95
Phe Leu Asp Ser Ala Glu Asp Ile Lys Asn Ser Ala Asn Asp Arg Gln
100 105 110
His Val Ile Lys Gly Asn Ala Gln Ala Arg Phe Arg Met Ile Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Asn Asn Arg Met Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Ile Lys Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Ser Tyr Leu Asn Val Pro Asn Asn Ile Leu Thr Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Leu Gly Gln Thr Asp Glu Ala Glu Met Gly Val
195 200 205
Ser Tyr Gln Glu Ile Asp Asp Phe Leu Asp Gly Lys His Val Ser Asp
210 215 220
Tyr Ala Leu Asn Gln Ile Lys Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Ile Met Ala Lys Ala Pro Asp Phe
245
<210> 4
<211> 249
<212> PRT
<213> Francisella guangzhouensis strain 08HL01032
<400> 4
Met Asn Val Val Lys Asn Phe Thr Pro Glu Lys Tyr Ser Glu Lys Leu
1 5 10 15
Ile Gln Trp Leu Thr Asn Ser Cys Ile Lys Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Val Ser Gly Gly Ile Asp Ser Ala Val Cys Ala Ser Leu
35 40 45
Leu Ser Lys Thr Asp Leu Pro Thr Thr Ala Phe Ile Leu Pro Ser Lys
50 55 60
Asn Asn Ser Asp Gln Asp Met Ile Asp Ala Leu Glu Leu Ile Asn Lys
65 70 75 80
Leu Asn Ile Pro Tyr His Ile Ile Pro Ile Gln Pro Val Tyr Glu Ser
85 90 95
Phe Leu Lys Ser Thr Gln Leu Phe Thr Asn Pro Gln Asn Asp Arg Gln
100 105 110
Asn Val Ile Lys Gly Asn Ala Gln Ala Arg Phe Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Asn Asn Arg Ile Val Val Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Ile Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Asp Val Pro Arg Asn Ile Leu Thr Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Gly Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asn Phe Leu Asp Gly Lys Glu Val Ser Pro
210 215 220
Ala Thr Phe Glu Lys Ile Ser Tyr Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Lys Met Ala Leu Thr Pro Asp Phe
245
<210> 5
<211> 249
<212> PRT
<213> Francisella sp. TX077308
<400> 5
Met Lys Ile Val Lys Asn Phe Ile Val Glu Gln Tyr Ser Asn Asn Leu
1 5 10 15
Ile Lys Trp Leu Lys Glu Asn Cys Ile Lys Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Ile Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Lys
50 55 60
Asn Asn Gln Asp Gln Asp Met Arg Asp Gly Ile Glu Leu Ile Glu Asn
65 70 75 80
Leu Asn Ile Glu Tyr His Thr Val Ser Ile Gln Pro Ala Tyr Asp Thr
85 90 95
Phe Ile Glu Ser Thr Phe Asn Phe Thr Asn Ser Gln Asn Asp Arg Gln
100 105 110
His Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Asn Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Ile Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Lys Val Pro Lys Asn Ile Ile Gln Lys Asp Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Lys Glu Ile Asp Asp Phe Leu Asp Gly Lys Glu Val Ser Glu
210 215 220
Lys Ala Leu Glu Arg Ile Ser Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Ser Met Ala Phe Thr Pro Asn Phe
245
<210> 6
<211> 249
<212> PRT
<213> Francisella philomiragia subsp. philomiragia ATCC 25017
<400> 6
Met Lys Ile Ile Lys Asn Phe Ile Ala Glu Glu Tyr Ser Lys Lys Leu
1 5 10 15
Ile Glu Trp Leu Lys Lys Ile Cys Ile Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Ile Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Lys
50 55 60
Asn Asn Gln Asp Gln Asp Met Lys Asp Gly Leu Glu Leu Ile Lys Asn
65 70 75 80
Leu Asp Ile Glu His His Ile Val Pro Ile Gln Pro Ala Tyr Asp Thr
85 90 95
Phe Ile Glu Ser Thr Leu Asn Phe Thr Asn Ser Gln Asn Asp Arg Gln
100 105 110
His Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Asn Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Met Gly Lys Tyr Leu Lys Val Pro Gln Asn Ile Ile Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asn Phe Leu Asp Gly Lys Glu Val Ser Ala
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Ser Met Ala Phe Thr Pro Asn Phe
245
<210> 7
<211> 249
<212> PRT
<213> Francisella philomiragia strain O#319-036 [FSC 153
<400> 7
Met Lys Ile Ile Lys Asn Phe Ile Ala Glu Glu Tyr Ser Lys Lys Leu
1 5 10 15
Ile Glu Trp Leu Lys Lys Ile Cys Ile Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Ile Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Lys
50 55 60
Asn Asn Gln Asp Gln Asp Met Lys Asp Gly Leu Glu Leu Ile Lys Asn
65 70 75 80
Leu Asp Ile Glu His His Ile Val Pro Ile Gln Pro Ala Tyr Asp Thr
85 90 95
Phe Ile Glu Ser Thr Leu Asn Phe Thr Asn Ser Gln Asn Asp Arg Gln
100 105 110
His Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Asn Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Met Gly Glu Tyr Leu Lys Val Pro Gln Asn Ile Ile Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asn Phe Leu Asp Gly Lys Glu Val Ser Ala
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Ser Met Ala Phe Thr Pro Asn Phe
245
<210> 8
<211> 249
<212> PRT
<213> Francisella noatunensis subsp. orientalis str. Toba 04
<400> 8
Met Lys Ile Ile Lys Asn Phe Ile Ala Lys Glu Tyr Ser Lys Lys Leu
1 5 10 15
Ile Glu Trp Leu Lys Lys Ile Cys Ile Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Ile Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Lys
50 55 60
Asn Asn Gln Asp Gln Asp Met Lys Asp Gly Leu Glu Leu Ile Lys Asn
65 70 75 80
Leu Asp Ile Glu His His Ile Val Pro Ile Gln Pro Ala Tyr Asp Thr
85 90 95
Phe Ile Glu Ser Thr Phe Asn Phe Thr Asn Ala Gln Asn Asn Arg Gln
100 105 110
His Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Asn Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Lys Val Pro Gln Asn Ile Ile Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Ser Tyr Lys Glu Ile Asp Asp Phe Leu Asp Gly Lys Glu Val Ser Glu
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Ser Ile Ala Phe Thr Pro Asp Phe
245
<210> 9
<211> 249
<212> PRT
<213> Francisella philomiragia strain GA01-2794
<400> 9
Met Lys Ile Ile Lys Asn Phe Ile Val Glu Lys Tyr Ser Lys Lys Leu
1 5 10 15
Ile Glu Trp Leu Lys Lys Ile Cys Ile Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Ile Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Lys
50 55 60
Asn Asn Gln Asp Gln Asp Met Lys Asp Gly Leu Glu Leu Ile Lys Asn
65 70 75 80
Leu Asp Ile Glu His His Ile Val Pro Ile Gln Pro Ala Tyr Asp Thr
85 90 95
Phe Ile Glu Ser Thr Phe Asn Phe Thr Asn Ala Gln Asn Asn Arg Gln
100 105 110
His Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Asn Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Lys Val Pro Gln Asn Ile Ile Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Ser Tyr Lys Glu Ile Asp Asp Phe Leu Asp Gly Lys Glu Val Ser Glu
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Ser Ile Ala Phe Thr Pro Asp Phe
245
<210> 10
<211> 249
<212> PRT
<213> Francisella persica ATCC VR-331
<400> 10
Met Lys Ile Val Lys Asp Phe Asn Ile Lys Glu Tyr Ser Gln Lys Leu
1 5 10 15
Ile Asp Trp Leu Ser Asp Thr Cys Met Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Leu Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Ser Thr Thr Ala Leu Ile Leu Pro Ser Lys
50 55 60
Asn Asn Gln His Gln Asp Ile Gln Asp Ala Leu Glu Leu Ala Asp Lys
65 70 75 80
Ile Asn Ile Glu His His Thr Ile Thr Ile Gln Thr Val Tyr Glu Thr
85 90 95
Phe Leu Ala Ser Ile Lys Lys Ile Thr Asn Thr Glu Arg Asp Arg Gln
100 105 110
Leu Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Tyr Asn Arg Val Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser His Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Gly Val Pro Lys Asn Ile Leu Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asp Phe Leu Asp Gly Lys Gln Val Ser Ala
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Lys Leu Ala Leu Ile Pro Asn Phe
245
<210> 11
<211> 249
<212> PRT
<213> Francisella cf. novicida 3523
<400> 11
Met Lys Ile Val Lys Asp Phe Ser Pro Lys Glu Tyr Ser Gln Asn Leu
1 5 10 15
Val Asn Trp Leu Ser Asp Thr Cys Ile Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Ile Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Lys
50 55 60
Asn Asn Gln His Gln Asp Ile Gln Asp Ala Leu Glu Leu Val Glu Lys
65 70 75 80
Leu Asn Ile Glu His His Ile Val Thr Ile Gln Pro Ala Tyr Glu Asn
85 90 95
Phe Leu Ala Ser Thr Gln Glu Phe Ile Asn Thr Asp Asn Asn Arg Gln
100 105 110
Leu Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Tyr Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Phe Pro Leu Ile Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Asp Val Pro Lys Asn Ile Ile Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asp Phe Leu Asp Gly Lys Gln Ile Ser Ala
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Lys Leu Ala Leu Thr Pro Asn Phe
245
<210> 12
<211> 249
<212> PRT
<213> Francisella tularensis subsp. novicida D9876
<400> 12
Met Lys Ile Val Lys Asp Phe Ser Pro Lys Glu Tyr Ser Gln Lys Leu
1 5 10 15
Val Asn Trp Leu Ser Asp Ser Cys Met Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Leu Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Asp
50 55 60
Asn Asn Gln His Gln Asp Met Gln Asp Ala Leu Asp Leu Ile Glu Met
65 70 75 80
Leu Asn Ile Glu His Tyr Thr Ile Ser Ile Gln Pro Ala Tyr Glu Ala
85 90 95
Phe Leu Ala Ser Thr Gln Arg Phe Thr Asn Leu Gln Asn Asn Arg Gln
100 105 110
Leu Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Tyr Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Asp Val Pro Lys Asn Ile Leu Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asp Phe Leu Asp Gly Lys Gln Val Ser Ala
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Lys Leu Ala Leu Thr Pro Asn Phe
245
<210> 13
<211> 249
<212> PRT
<213> Francisella tularensis subsp. novicida F6168
<400> 13
Met Lys Ile Val Lys Asp Phe Ser Pro Lys Glu Tyr Ser Gln Lys Leu
1 5 10 15
Val Asn Trp Leu Ser Asp Ser Cys Met Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Leu Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Ile Thr Ala Leu Ile Leu Pro Ser Asp
50 55 60
Asn Asn Gln His Gln Asp Met Gln Asp Ala Leu Asp Leu Ile Glu Met
65 70 75 80
Leu Asn Ile Glu His Tyr Thr Ile Ser Ile Gln Pro Ala Tyr Glu Ala
85 90 95
Phe Leu Ala Ser Thr Gln Ser Phe Thr Asn Leu Gln Asn Asn Arg Gln
100 105 110
Leu Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Tyr Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Asp Val Pro Lys Asn Ile Leu Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asp Phe Leu Asp Gly Lys Gln Val Ser Ala
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Lys Leu Ala Leu Thr Pro Asn Phe
245
<210> 14
<211> 249
<212> PRT
<213> Francisella tularensis subsp. tularensis strain NIH B-38
<400> 14
Met Lys Ile Val Lys Asp Phe Ser Pro Lys Glu Tyr Ser Gln Lys Leu
1 5 10 15
Val Asn Trp Leu Ser Asp Ser Cys Met Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Leu Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Asp
50 55 60
Asn Asn Gln His Gln Asp Met Gln Asp Ala Leu Glu Leu Ile Glu Met
65 70 75 80
Leu Asn Ile Glu His Tyr Thr Ile Ser Ile Gln Leu Ala Tyr Glu Ala
85 90 95
Phe Leu Ala Ser Thr Gln Ser Phe Thr Asn Leu Gln Asn Asn Arg Gln
100 105 110
Leu Val Ile Lys Gly Asn Ala Gln Ala Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Tyr Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Asp Val Pro Lys Asn Ile Leu Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asp Phe Leu Asp Gly Lys Gln Val Ser Ala
210 215 220
Lys Ala Leu Glu Ile Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Lys Leu Ala Leu Thr Pro Asn Phe
245
<210> 15
<211> 249
<212> PRT
<213> Francisella tularensis subsp. holarctica F92
<400> 15
Met Lys Ile Val Lys Asp Phe Ser Pro Lys Glu Tyr Ser Gln Lys Leu
1 5 10 15
Val Asn Trp Leu Ser Asp Ser Cys Met Asn Tyr Pro Ala Glu Gly Phe
20 25 30
Val Ile Gly Leu Ser Gly Gly Ile Asp Ser Ala Val Ala Ala Ser Leu
35 40 45
Ala Val Lys Thr Gly Leu Pro Thr Thr Ala Leu Ile Leu Pro Ser Asp
50 55 60
Asn Asn Gln His Gln Asp Met Gln Asp Ala Leu Glu Leu Ile Glu Met
65 70 75 80
Leu Asn Ile Glu His Tyr Thr Ile Ser Ile Gln Pro Ala Tyr Glu Ala
85 90 95
Phe Leu Ala Ser Thr Gln Ser Phe Thr Asn Leu Gln Asn Asn Arg Gln
100 105 110
Leu Val Ile Lys Gly Asn Ala Gln Thr Arg Leu Arg Met Met Tyr Leu
115 120 125
Tyr Ala Tyr Ala Gln Gln Tyr Asn Arg Ile Val Ile Gly Thr Asp Asn
130 135 140
Ala Cys Glu Trp Tyr Met Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala
145 150 155 160
Ala Asp Ile Leu Pro Leu Val Asn Leu Lys Lys Ser Gln Val Phe Glu
165 170 175
Leu Gly Lys Tyr Leu Asp Val Pro Lys Asn Ile Leu Asp Lys Ala Pro
180 185 190
Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp Glu Asp Glu Met Gly Val
195 200 205
Thr Tyr Gln Glu Ile Asp Asp Phe Leu Asp Gly Lys Gln Val Ser Ala
210 215 220
Lys Ala Leu Glu Arg Ile Asn Phe Trp His Asn Arg Ser His His Lys
225 230 235 240
Arg Lys Leu Ala Leu Thr Pro Asn Phe
245
<210> 16
<211> 239
<212> PRT
<213> Dichelobacter nodosus VCS1703A
<400> 16
Gln Tyr Ile Asp Tyr Leu Leu Val Trp Leu Glu Glu Gln Arg Ala His
1 5 10 15
Leu Tyr Ala Ser Asp Gly Tyr Thr Leu Gly Val Ser Gly Gly Ile Asp
20 25 30
Ser Ala Val Cys Leu His Leu Leu Ala Lys Thr Gly Lys Pro Val Gln
35 40 45
Ala Leu Val Leu Pro Ile Asn Ala Asn Ala Asn Asp Cys Glu Asp Ala
50 55 60
Glu Leu Val Leu Lys Asn Ala Asn Ile Ser Gly Asn Ile Ile Ala Leu
65 70 75 80
Asp Asp Val Tyr Thr Ala Ala Gln Asn Thr Leu Ala Pro Val Leu Asn
85 90 95
Arg Asp Tyr Glu Arg Met Pro Val Leu Asn Gly Asn Leu Met Ala Arg
100 105 110
Leu Arg Met Val Met Leu Tyr Thr Val Ala Gln Ser His Arg Ser Val
115 120 125
Val Val Gly Thr Asp Asn Ala Val Glu Tyr Tyr Leu Gly Tyr Phe Thr
130 135 140
Lys Phe Gly Asp Gly Ala Cys Asp Ile Leu Pro Leu Ala Lys Leu Thr
145 150 155 160
Lys Ser Glu Val Gly Gln Leu Ala Lys Ala Leu Gly Val Pro Lys Lys
165 170 175
Ile Arg Glu Lys Ala Pro Ser Ala Gly Leu Trp Gln Gly Gln Thr Asp
180 185 190
Glu Asn Glu Ile Gly Val Ser Tyr Ala Asp Leu Asp Ala Phe Leu Cys
195 200 205
Gly Lys Thr Val Asp Asp Ala Val Arg Glu Lys Ile Ala Tyr Trp His
210 215 220
Gln Arg Ser His His Lys Arg Met Leu Pro Pro Met Pro Glu Ile
225 230 235
<210> 17
<211> 245
<212> PRT
<213> Mannheimia succiniciproducens MBEL55E
<400> 17
Lys Arg Met Lys Thr Ala Ala Tyr Ala Asp Tyr Leu Ile Gln Trp Leu
1 5 10 15
Glu Asn Gln Arg Thr Glu Leu Tyr Gly Met Asp Gly Tyr Thr Leu Gly
20 25 30
Val Ser Gly Gly Ile Asp Ser Ala Val Cys Ala His Leu Ala Ala Arg
35 40 45
Thr Gly Ala Pro Val Gln Ala Leu Ile Leu Pro Ala Glu Val Thr Ser
50 55 60
Pro Ser Asp Val Ala Asp Ala Gln Ala Thr Leu Glu Ser Ala Gly Ile
65 70 75 80
Asp Gly Gln Ile Ile Ser Ile Ala Pro Trp Tyr Asp Leu Ile Met Gln
85 90 95
Gln Leu Ser Pro Val Leu Asn Ser Glu Pro Glu Arg Val Asn Val Leu
100 105 110
Lys Gly Asn Leu Met Ala Arg Leu Arg Met Ile Ala Leu Phe Thr Thr
115 120 125
Ala Gln Ser His Arg Ser Ile Val Leu Gly Thr Asp Asn Ala Ala Glu
130 135 140
Trp Leu Thr Gly Tyr Phe Thr Lys Phe Gly Asp Gly Ala Ala Asp Val
145 150 155 160
Leu Pro Leu Ala Gly Leu Arg Lys Glu Gln Val Phe Glu Leu Gly Arg
165 170 175
Tyr Leu Gly Val Pro Gln Ser Val Leu Asp Lys Lys Pro Ser Ala Gly
180 185 190
Leu Trp Ala Gly Gln Thr Asp Glu Ala Glu Met Gly Val Thr Tyr Ala
195 200 205
Glu Ile Asp Ala Tyr Leu Arg Gly Glu Thr Val Ser Pro Gln Ala Leu
210 215 220
Gln Gln Ile Arg Phe Trp His Asn Arg Ser His His Lys Arg Met Leu
225 230 235 240
Pro Pro Lys Pro Lys
245
<210> 18
<211> 238
<212> PRT
<213> Actinobacillus succinogenes 130Z
<400> 18
Tyr Val Asp Tyr Leu Val Arg Trp Leu Glu Thr Gln Arg Thr Glu Leu
1 5 10 15
Tyr Gly Met Asp Gly Tyr Thr Leu Gly Val Ser Gly Gly Ile Asp Ser
20 25 30
Ala Val Cys Ala His Leu Ala Ala Arg Thr Gly Ala Pro Val Gln Ala
35 40 45
Leu Ile Leu Pro Ala Glu Val Thr Ser Pro Glu Asp Val Ala Asp Ala
50 55 60
Gln Ile Thr Leu Glu Ser Ala Gly Ile Asp Gly Arg Ile Ile Ser Ile
65 70 75 80
Ala Pro Trp Tyr Asp Leu Ile Met Leu Gln Leu Thr Pro Ala Leu Asn
85 90 95
Ala Glu Ser Glu Arg Ile Asn Val Leu Lys Gly Asn Leu Met Ala Arg
100 105 110
Leu Arg Met Ile Ala Leu Phe Thr Thr Ala Gln Ser His Arg Ser Ile
115 120 125
Val Leu Gly Thr Asp Asn Ala Ala Glu Met Leu Thr Gly Tyr Phe Thr
130 135 140
Lys Phe Gly Asp Gly Ala Ala Asp Val Leu Pro Leu Ala Arg Leu Arg
145 150 155 160
Lys Glu Gln Val Phe Glu Leu Gly Arg Tyr Leu Gly Val Pro Lys Ser
165 170 175
Val Leu Glu Lys Lys Pro Ser Ala Gly Leu Trp Ala Gly Gln Thr Asp
180 185 190
Glu Gly Glu Met Gly Val Ser Tyr Ala Glu Ile Asp Ala Tyr Leu Arg
195 200 205
Gly Glu Thr Val Ser Pro Gln Ala Leu Lys Gln Ile Gln Phe Trp His
210 215 220
Asn Arg Ser His His Lys Arg Met Leu Pro Pro Thr Pro Glu
225 230 235
<210> 19
<211> 753
<212> DNA
<213> Mannheimia succiniciproducens MBEL55E
<400> 19
atgaaaacgg cagcatacgc agattattta attcaatggc tggaaaacca acgaaccgaa 60
ctttacggga tggacggcta tacactgggc gtcagcggcg gtattgacag cgccgtctgc 120
gctcatttgg cagcgcgcac cggcgcaccg gtacaagcct taattttgcc cgccgaagta 180
accagtccgt cagatgtggc ggatgcgcaa gccacactgg aaagcgccgg tattgacgga 240
caaataattt ccattgcacc ttggtacgat ttaattatgc aacaactttc cccggtatta 300
aattccgaac cggagcgcgt taacgtatta aagggtaatt taatggcaag actgcgtatg 360
attgcgctgt ttaccacggc acaaagccat cgttctattg tgttaggcac cgataatgcg 420
gcggaatggc tgacgggtta ttttaccaaa ttcggcgacg gcgcagcgga cgtactgcct 480
ttagcgggat tgcgcaaaga gcaggtattt gaactcggac gttatcttgg cgtaccgcaa 540
agcgtgctgg ataaaaaacc gagcgccggt ttatgggcag gacaaacgga cgaagctgaa 600
atgggtgtta cttatgcgga aatcgacgct tatctgcgcg gcgaaaccgt tagcccgcag 660
gcattgcaac aaatccgttt ctggcacaac cgttctcatc acaaacgtat gttgccacct 720
aaaccgaaat cacccgatga agcggagtgt taa 753
<210> 20
<211> 747
<212> DNA
<213> Dichelobacter nodosus VCS1703A
<400> 20
atgaccgttc atcaatacat cgattattta ctcgtgtggt tagaagagca gcgcgctcat 60
ctttatgcat cagatggtta tacgttgggc gtcagcggcg gcatcgattc cgccgtttgt 120
ctgcatttac tcgccaaaac gggaaaaccc gtgcaagcgt tagttttgcc gatcaatgcg 180
aacgcgaacg attgtgaaga tgccgaatta gtgttaaaaa atgctaatat ttccggcaat 240
attatcgcgc tcgatgatgt ttataccgcc gcacaaaaca ccttggcgcc tgttttaaat 300
cgcgattatg aacgtatgcc cgtattaaac ggcaatttaa tggcgcggct gcgtatggtt 360
atgctttata ccgtggcgca aagtcatcgt tcggtggtcg tgggaacgga taacgcggtg 420
gaatattatt taggttactt tacaaaattt ggcgacggcg cctgcgatat tttgccgctg 480
gcaaaactga caaaatcaga agtaggacaa ttggcaaaag cgttaggcgt tccgaaaaaa 540
atccgagaaa aagcgccgag cgcaggcttg tggcaagggc aaaccgatga aaacgaaatc 600
ggcgtatcgt acgcggattt agatgctttt ttgtgcggta aaaccgttga tgatgccgtc 660
agagaaaaaa ttgcttattg gcatcaacgc tcgcatcata aaagaatgtt gccgccgatg 720
ccggaaatcg gattatcttt ggcgtaa 747
<210> 21
<211> 750
<212> DNA
<213> Actinobacillus succinogenes 130Z
<400> 21
atgagaacgg cagcatacgt agattattta gtgcgatggc tggaaaccca gcgtaccgaa 60
ttatacggta tggacggcta cacgctgggg gtcagcggcg gtatcgacag tgccgtttgc 120
gcccatttag cggcacgcac cggcgccccc gtacaggcat tgattttacc cgccgaagtc 180
accagccctg aagatgtggc ggatgctcag attaccttgg aaagtgcagg tattgacggg 240
cggattattt ctatcgctcc ttggtacgat ttaattatgc tacaacttac ccccgcatta 300
aatgcggaat ctgaacgcat taacgtattg aaaggtaact taatggcgcg cttacgtatg 360
atcgcattat ttaccacggc gcaaagccac cgttctatcg tattgggtac ggataacgcc 420
gccgaaatgt taacgggcta tttcaccaaa ttcggcgacg gtgcggcgga cgtattgccg 480
ttagcgaggt tgcgcaaaga acaggtattc gaattagggc gttatcttgg cgtaccgaaa 540
tccgtgctgg agaaaaaacc gagtgcgggc ttatgggcgg ggcaaacgga cgagggggaa 600
atgggtgtca gttatgcgga aatcgacgcc tatctgcgcg gcgaaaccgt cagtccgcag 660
gcgttaaagc agattcaatt ctggcacaac cgttctcatc acaaacgtat gctgccgccg 720
acgccagaac cgccggatga aatcgattaa 750
<210> 22
<211> 750
<212> DNA
<213> Francisella philomiragia subsp. philomiragia
<400> 22
atgaaaataa taaaaaattt tattgcagaa gagtattcta aaaaattaat agaatggtta 60
aaaaaaattt gtataaacta tcctgcagaa ggttttgtta ttggtattag tggtggcata 120
gattcagcag tagcggcatc tttagcggtt aaaactggat tacctacaac agcactaata 180
ctaccatcaa aaaataatca agatcaagat atgaaagatg gactagagct cattaaaaat 240
cttgatatag aacatcatat tgttccaata caacctgctt atgatacatt tatagagtca 300
actcttaact ttacaaactc acaaaatgac cgccaacatg tcatcaaagg taatgctcaa 360
gctcgtctta ggatgatgta tctatatgcc tatgctcaac aaaataacag aattgtaata 420
ggcacagata atgcctgcga atggtatatg ggttatttca caaaatttgg cgatggtgca 480
gcagatatac taccccttgt taatctaaag aaatcacaag tctttgaaat gggcaagtat 540
ctcaaagtgc cgcaaaatat tatagataaa gctccatctg ctggtctatg gcaaggtcaa 600
actgatgaag atgaaatggg tgtcacatat caagaaattg ataacttttt agatggtaaa 660
gaagtctcag ccaaagctct tgagagaata aacttttggc ataatcgtag tcatcacaaa 720
agatctatgg cttttactcc aaacttttaa 750
<210> 23
<211> 750
<212> DNA
<213> Francisella cf. novicida 3523
<400> 23
atgaaaatag ttaaagattt tagtcctaaa gaatattcac aaaatttagt taattggcta 60
agtgatactt gtataaatta tcctgctgaa ggatttgtaa tcggcattag cggtggtata 120
gattcagctg ttgcagcttc tttagctgtc aaaactggat taccaactac agctttaata 180
ctaccttcaa aaaacaatca acaccaagat atccaagatg ctctagaact tgttgagaaa 240
cttaatattg aacatcatat tgttacaatt caaccagcat acgaaaattt tctagcatca 300
acacaggaat ttataaatac agataataat agacaacttg tgatcaaggg aaatgctcaa 360
gcacgtttaa ggatgatgta tttatatgcc tatgcccaac aatataacag aatagttata 420
ggtactgata atgcttgtga gtggtatatg ggatatttta caaaatttgg tgatggcgct 480
gctgatatat ttccgctaat taatcttaaa aaatcacaag tttttgaatt aggtaaatac 540
ttagatgttc cgaaaaatat aattgataaa gctccgtctg ctggactatg gcaaggacaa 600
actgatgagg atgaaatggg cgtaacttat caagaaattg atgatttctt agatggtaaa 660
caaatttcag caaaagccct agaaagaata aacttctggc ataatcgtag tcatcataag 720
agaaaactag ctttaactcc taatttctaa 750
<210> 24
<211> 750
<212> DNA
<213> Francisella sp. TX077308
<400> 24
atgaaaatag taaaaaactt tattgtagaa cagtattcta ataatttaat aaaatggtta 60
aaagagaatt gcataaaata tcctgctgaa ggttttgtga ttggtattag tggtggtatc 120
gattcggcag tagccgcatc tttagcagtc aaaacaggat tacctacaac tgctctaata 180
ttgccatcga agaacaatca agaccaagat atgcgagatg gaatagaact aatcgaaaat 240
cttaatatag agtatcatac tgtttcaata caacctgctt atgacacgtt tatagagtca 300
acatttaact ttacaaactc acaaaatgat cgccaacatg ttatcaaagg aaatgcccaa 360
gcgcgtctta gaatgatgta tttatatgct tatgctcagc aaaataatag aattgttata 420
ggtacagata acgcatgtga atggtacatg ggatatttca ctaaatttgg tgatggtgca 480
gcagatatat taccacttat taatctcaaa aaatctcaag tttttgaact aggtaaatac 540
ttaaaagtgc caaaaaacat tatccaaaaa gatccttctg ccggtctatg gcaaggtcaa 600
actgatgagg atgaaatggg tgtcacatac aaagaaattg atgacttctt agacggtaaa 660
gaagtctcag aaaaagctct cgaaagaata agcttctggc ataatcgtag tcaccataaa 720
agatccatgg cttttacccc taatttttaa 750
<210> 25
<211> 750
<212> DNA
<213> Francisella sp. FSC1006
<400> 25
atgagtgtag taaaaaattt taaacctaat gaatatgcca ataaaattac tgaatggctg 60
aaagactctt gtttaaatta tcccgctgaa ggttttgtgg taggtattag tggaggtata 120
gattcagcag tagcagtctc tttagcagta aatactggac tacctgttac agggctaata 180
atgccatcaa aaaataatga tgataaagat accttagatg ctatagaatt agctaaaaaa 240
ttaaatatag aatatcatct catacccatt caaccagtat atgaaacatt tctagattca 300
gctgaagata tcaaaaacag tgctaatgac cgtcaacatg taatcaaagg aaatgcacaa 360
gctcgtttta gaatgatata cttgtatgct tacgctcagc aaaataatag aatggtaatt 420
ggtacagata atgcttgtga atggtatatg ggctatttta caaaatttgg agatggagcc 480
gctgatatac tgcctcttat aaaattaaaa aaatcacaag tttttgaatt aggtagctat 540
cttaatgtac ccaataacat cctcacaaaa gctccttccg caggactttg gcttggacaa 600
actgatgaag cagagatggg ggtttcatat caagaaatag atgatttcct tgatggtaaa 660
catgtctcag attatgctct taatcaaata aaattctggc ataaccgtag tcatcataaa 720
agaatcatgg ctaaggctcc agatttttaa 750
<210> 26
<211> 753
<212> DNA
<213> Francisella guangzhouensis strain 08HL01032
<400> 26
atgaacgtag taaaaaattt cactcctgaa aaatattcag aaaaacttat acaatggctc 60
actaatagct gtataaaata tcctgcagaa ggtttcgtaa ttggtgtaag tggtggtata 120
gattctgcag tatgtgcatc acttttatcc aaaactgatc ttcctacaac agcttttata 180
ctaccatcaa aaaataactc tgatcaagat atgatcgatg cattagaact tataaataaa 240
ttaaatattc cataccatat aataccaatc cagccagttt atgaaagttt tctaaagtcc 300
acacagctat ttacaaatcc acaaaatgac agacaaaatg tcataaaagg taacgctcaa 360
gctcgtttta gaatgatgta tttatatgct tatgcacaac aaaataatcg tatagtagtt 420
ggaacagata atgcttgtga atggtatatg ggttatttca ccaaatttgg cgatggagct 480
gctgatatac taccattaat aaatcttaaa aagtcccagg tatttgagtt aggtaaatac 540
ttagatgttc caaggaatat cctaactaag gcaccctctg ctggtctttg gcaaggccaa 600
actgatgaag gtgaaatggg agttacttat caggaaatag ataattttct cgacggtaaa 660
gaagtatcgc cagcaacttt tgaaaaaata agctactggc ataatcgctc tcaccacaaa 720
agaaagatgg ctttaacgcc agattttaac taa 753
<210> 27
<211> 750
<212> DNA
<213> Francisella persica ATCC VR-331
<400> 27
atgaaaatag ttaaagattt caacatcaaa gaatattcac aaaagttaat tgattggcta 60
agtgatactt gtatgaatta ccctgctgaa ggatttgtca ttggtcttag cggtggtata 120
gattcggcag ttgcagcttc tttagctgtc aaaactggat tatcaactac agctttaata 180
ttaccatcaa aaaacaatca acaccaagat atacaagatg ctctagaact tgcagataaa 240
attaatattg aacatcatac tattacaatt caaacagtat acgaaacttt tcttgcgtca 300
ataaaaaaaa ttacaaatac cgaacgtgat agacaacttg tcattaaagg aaatgctcaa 360
gctcgtttga ggatgatgta tttatatgcc tatgctcaac aatataatag agtggttatt 420
ggtactgata atgcttgtga atggtatatg ggatatttta caaagtttgg tgatggtgct 480
gctgatattc ttccactagt taatctcaaa aaatctcacg tttttgaatt aggtaaatac 540
ttaggtgttc ctaaaaatat acttgataaa gctccatctg ctgggctatg gcaaggacaa 600
actgatgaag atgaaatggg cgtaacttat caagaaattg atgatttctt agatggtaag 660
caagtttcag cgaaagctct agaaagaata aatttctggc ataatcgtag tcatcataag 720
agaaaactag ctttaattcc taatttctaa 750
<210> 28
<211> 753
<212> DNA
<213> Mannheimia succiniciproducens
<400> 28
atgaaaacgg cagcatacgc agattatctg attcaatggc tggaaaacca acgcaccgaa 60
ctgtacggca tggacggcta taccctgggc gtcagcggcg gtattgacag cgccgtctgc 120
gctcatctgg cagcgcgcac cggcgcgccg gtacaagccc tgattctgcc ggcggaagta 180
accagtccgt cagatgtggc ggatgcgcaa gccaccctgg aaagcgccgg tattgacggc 240
caaattattt ccattgcacc gtggtacgat ctgattatgc aacaactgtc cccggtactg 300
aatagcgaac cggagcgcgt taacgtactg aaaggtaatc tgatggcacg cctgcgtatg 360
attgcgctgt ttaccacggc acaaagccat cgttctattg tgctgggcac cgataatgcg 420
gcggaatggc tgacgggtta ttttaccaaa ttcggcgacg gcgcagcgga cgtactgccg 480
ctggcgggcc tgcgcaaaga gcaggtattt gaactgggcc gttatctggg cgtaccgcaa 540
agcgtgctgg ataaaaaacc gagcgccggt ctgtgggcag gccaaacgga cgaagctgaa 600
atgggtgtta cctatgcgga aatcgacgct tatctgcgcg gcgaaaccgt tagcccgcag 660
gcactgcaac aaatccgttt ctggcacaac cgttctcatc acaaacgtat gctgccgccg 720
aaaccgaaat caccggatga agcggagtgt taa 753
<210> 29
<211> 747
<212> DNA
<213> Dichelobacter nodosus VCS1703A
<400> 29
atgaccgttc atcaatacat tgattatctg ctggtgtggc tggaagagca gcgcgctcat 60
ctgtatgcat cagatggtta tacgctgggc gtcagcggcg gcattgattc cgccgtttgt 120
ctgcatctgc tggccaaaac gggcaaacca gtgcaagcgc tggttctgcc gatcaatgcg 180
aacgcgaacg attgtgaaga tgccgaactg gtgctgaaaa atgctaatat ttccggcaat 240
attatcgcgc tggatgatgt ttataccgcc gcacaaaaca ccctggcgcc ggttctgaat 300
cgcgattatg aacgtatgcc ggtactgaac ggcaatctga tggcgcgcct gcgtatggtt 360
atgctgtata ccgtggcgca aagtcatcgt tcggtggtcg tgggcacgga taacgcggtg 420
gaatattatc tgggttactt taccaaattt ggcgacggcg cctgcgatat tctgccgctg 480
gcaaaactga ccaaatcaga agtaggccaa ctggcaaaag cgctgggcgt tccgaaaaaa 540
atccgcgaaa aagcgccgag cgcgggcctg tggcaaggcc aaaccgatga aaacgaaatc 600
ggcgtatcgt acgcggatct ggatgctttt ctgtgcggta aaaccgttga tgatgccgtc 660
cgcgaaaaaa ttgcttattg gcatcaacgc tcgcatcata aacgtatgct gccgccgatg 720
ccggaaatcg gcctgtctct ggcgtaa 747
<210> 30
<211> 750
<212> DNA
<213> Actinobacillus succinogenes 130Z
<400> 30
atgcgcacgg cagcatacgt agattatctg gtgcgctggc tggaaaccca gcgtaccgaa 60
ctgtacggta tggacggcta cacgctgggc gtcagcggcg gtatcgacag tgccgtttgc 120
gcccatctgg cggcacgcac cggcgccccg gtacaggcac tgattctgcc ggcggaagtc 180
accagcccgg aagatgtggc ggatgctcag attaccctgg aaagtgcagg tattgatggc 240
cgcattattt ctatcgctcc gtggtacgat ctgattatgc tgcaactgac cccggcactg 300
aatgcggaat ctgaacgcat taacgtactg aaaggtaacc tgatggcgcg cctgcgtatg 360
atcgcactgt ttaccacggc gcaaagccac cgttctatcg tactgggtac ggataacgcc 420
gccgaaatgc tgacgggcta tttcaccaaa ttcggcgacg gtgcggcgga cgtactgccg 480
ctggcgcgcc tgcgcaaaga acaggtattc gaactgggcc gttatctggg cgtaccgaaa 540
tccgtgctgg agaaaaaacc gagtgcgggc ctgtgggcgg gccaaacgga cgagggcgaa 600
atgggtgtca gttatgcgga aatcgacgcc tatctgcgcg gcgaaaccgt cagtccgcag 660
gcgctgaaac agattcaatt ctggcacaac cgttctcatc acaaacgtat gctgccgccg 720
acgccggaac cgccggatga aattgattaa 750
<210> 31
<211> 750
<212> DNA
<213> Francisella philomiragia subsp. philomiragia
<400> 31
atgaaaatta ttaaaaattt tattgcagaa gagtattcta aaaaactgat tgaatggctg 60
aaaaaaattt gtattaacta tccggcagaa ggttttgtta ttggtattag tggtggcatt 120
gattcagcag tagcggcatc tctggcggtt aaaaccggcc tgccgaccac cgcactgatt 180
ctgccgtcaa aaaataatca agatcaagat atgaaagatg gcctggagct gattaaaaat 240
ctggatattg aacatcatat tgttccgatt caaccggctt atgatacctt tattgagtca 300
accctgaact ttaccaactc acaaaatgac cgccaacatg tcatcaaagg taatgctcaa 360
gctcgtctgc gcatgatgta tctgtatgcc tatgctcaac aaaataaccg cattgtaatt 420
ggcaccgata atgcctgcga atggtatatg ggttatttca ccaaatttgg cgatggtgca 480
gcagatattc tgccgctggt taatctgaaa aaatcacaag tctttgaaat gggcaaatat 540
ctgaaagtgc cgcaaaatat tattgataaa gctccgtctg ctggtctgtg gcaaggtcaa 600
accgatgaag atgaaatggg tgtcacctat caagaaattg ataactttct ggatggtaaa 660
gaagtctcag ccaaagctct ggagcgcatt aacttttggc ataatcgtag tcatcacaaa 720
cgctctatgg cttttacccc gaacttttaa 750
<210> 32
<211> 750
<212> DNA
<213> Francisella cf. novicida 3523
<400> 32
atgaaaattg ttaaagattt tagtccgaaa gaatattcac aaaatctggt taattggctg 60
agtgatacct gtattaatta tccggctgaa ggctttgtaa tcggcattag cggtggtatt 120
gattcagcgg ttgcagcttc tctggctgtc aaaaccggcc tgccgaccac cgctctgatt 180
ctgccgtcaa aaaacaatca acaccaagat attcaagatg ctctggaact ggttgagaaa 240
ctgaatattg aacatcatat tgttaccatt caaccggcat acgaaaattt tctggcatca 300
acccaggaat ttattaatac cgataataat cgccaactgg tgatcaaagg caatgctcaa 360
gcacgtctgc gcatgatgta tctgtatgcc tatgcccaac aatataaccg cattgttatt 420
ggtaccgata atgcttgtga gtggtatatg ggctatttta ccaaatttgg tgatggcgct 480
gctgatattt ttccgctgat taatctgaaa aaatcacaag tttttgaact gggtaaatac 540
ctggatgttc cgaaaaatat tattgataaa gctccgtctg ctggcctgtg gcaaggccaa 600
accgatgagg atgaaatggg cgtaacctat caagaaattg atgatttcct ggatggtaaa 660
caaatttcag caaaagccct ggaacgcatt aacttctggc ataatcgtag tcatcataaa 720
cgcaaactgg ctctgacccc gaatttctaa 750
<210> 33
<211> 750
<212> DNA
<213> Francisella sp. TX077308
<400> 33
atgaaaattg taaaaaactt tattgtagaa cagtattcta ataatctgat taaatggctg 60
aaagagaatt gcattaaata tccggctgaa ggttttgtga ttggtattag tggtggtatt 120
gattcggcag tagccgcatc tctggcagtc aaaaccggcc tgccgaccac cgctctgatt 180
ctgccgtcga aaaacaatca agaccaagat atgcgcgatg gcattgaact gatcgaaaat 240
ctgaatattg agtatcatac cgtttcaatt caaccggctt atgacacgtt tattgagtca 300
acctttaact ttaccaactc acaaaatgat cgccaacatg ttatcaaagg caatgcccaa 360
gcgcgtctgc gcatgatgta tctgtatgct tatgctcagc aaaataatcg cattgttatt 420
ggtaccgata acgcatgtga atggtacatg ggctatttca ccaaatttgg tgatggtgca 480
gcagatattc tgccgctgat taatctgaaa aaatctcaag tttttgaact gggtaaatac 540
ctgaaagtgc cgaaaaacat tatccaaaaa gatccgtctg ccggtctgtg gcaaggtcaa 600
accgatgagg atgaaatggg tgtcacctac aaagaaattg atgacttcct ggacggtaaa 660
gaagtctcag aaaaagctct ggaacgcatt agcttctggc ataatcgtag tcaccataaa 720
cgcagcatgg cttttacccc gaatttttaa 750
<210> 34
<211> 750
<212> DNA
<213> Francisella sp. FSC
<400> 34
atgagtgtag taaaaaattt taaaccgaat gaatatgcca ataaaattac cgaatggctg 60
aaagactctt gtctgaatta tccggctgaa ggttttgtgg taggtattag tggcggtatt 120
gattcagcag tagcagtctc tctggcagta aataccggcc tgccggttac cggcctgatt 180
atgccgtcaa aaaataatga tgataaagat accctggatg ctattgaact ggctaaaaaa 240
ctgaatattg aatatcatct gattccgatt cagccggtat atgaaacctt tctggattca 300
gcggaagata ttaaaaacag tgctaatgac cgtcaacatg taatcaaagg caatgcacaa 360
gctcgttttc gcatgattta cctgtatgct tacgctcagc aaaataatcg catggtaatt 420
ggtaccgata atgcttgtga atggtatatg ggctatttta ccaaatttgg cgatggcgcc 480
gctgatattc tgccgctgat taaactgaaa aaatcacaag tttttgaact gggtagctat 540
ctgaatgtac cgaataacat cctgaccaaa gctccgagcg cgggcctgtg gctgggccaa 600
accgatgaag cagagatggg cgtttcatat caagaaattg atgatttcct ggatggtaaa 660
catgtctcag attatgctct gaatcaaatt aaattctggc ataaccgtag tcatcataaa 720
cgcatcatgg ctaaagctcc ggatttttaa 750
<210> 35
<211> 753
<212> DNA
<213> Francisella guangzhouensis strain 08HL01032
<400> 35
atgaacgtag taaaaaattt caccccggaa aaatattcag aaaaactgat tcaatggctg 60
accaatagct gtattaaata tccggcagaa ggtttcgtaa ttggtgtaag tggtggtatt 120
gattctgcgg tatgtgcatc actgctgtcc aaaaccgatc tgccgaccac cgcttttatt 180
ctgccgtcaa aaaataactc tgatcaagat atgattgatg cactggaact gattaataaa 240
ctgaatattc cgtaccatat tattccgatc cagccggttt atgaaagttt tctgaaatcc 300
acccaactgt ttaccaatcc gcaaaatgac cgccaaaatg tcattaaagg taacgctcaa 360
gctcgttttc gcatgatgta tctgtatgct tatgcacaac aaaataatcg tattgtagtt 420
ggcaccgata atgcttgtga atggtatatg ggttatttca ccaaatttgg cgatggcgct 480
gctgatattc tgccgctgat taatctgaaa aaatcccagg tatttgagct gggtaaatac 540
ctggatgttc cgcgcaatat cctgaccaaa gcaccgtctg ctggtctgtg gcaaggccaa 600
accgatgaag gtgaaatggg cgttacctat caggaaattg ataattttct ggacggtaaa 660
gaagtatcgc cggcaacctt tgaaaaaatt agctactggc ataatcgctc tcaccacaaa 720
cgcaaaatgg ctctgacgcc ggattttaac taa 753
<210> 36
<211> 750
<212> DNA
<213> Francisella persica ATCC VR-331
<400> 36
atgaaaattg ttaaagattt caacatcaaa gaatattcac aaaaactgat tgattggctg 60
agtgatacct gtatgaatta cccggctgaa ggctttgtca ttggtctgag cggtggtatt 120
gattcggcag ttgcagcttc tctggctgtc aaaaccggcc tgtcaaccac cgctctgatt 180
ctgccgtcaa aaaacaatca acaccaagat attcaagatg ctctggaact ggcagataaa 240
attaatattg aacatcatac cattaccatt caaaccgtat acgaaacctt tctggcgtca 300
attaaaaaaa ttaccaatac cgaacgtgat cgccaactgg tcattaaagg caatgctcaa 360
gctcgtctgc gcatgatgta tctgtatgcc tatgctcaac aatataatcg cgtggttatt 420
ggtaccgata atgcttgtga atggtatatg ggctatttta ccaaatttgg tgatggtgct 480
gctgatattc tgccgctggt taatctgaaa aaatctcacg tttttgaact gggtaaatac 540
ctgggtgttc cgaaaaatat tctggataaa gctccgtctg ctggcctgtg gcaaggccaa 600
accgatgaag atgaaatggg cgtaacctat caagaaattg atgatttcct ggatggtaaa 660
caagtttcag cgaaagctct ggaacgcatt aatttctggc ataatcgtag tcatcataaa 720
cgcaaactgg ctctgattcc gaatttctaa 750
<210> 37
<211> 750
<212> DNA
<213> FtNadE_star_optimized
<400> 37
atgaaaatcg tcaaagactt ctccccgaaa gaatattccc aaaaactggt gaactggctg 60
agcgactcgt gtatgaacta tccggcagaa ggctttgtca ttggtctgag tggcggtatc 120
gattccgctg tggcggcctc actggccgtt aaaaccggcc tgccgaccac ggcactgatt 180
ctgccgtctg acaacaatca gcatcaagat atgcaggacg cgctggaact gattgaaatg 240
ctgaacatcg aacactacac catttccatc cagccggcgt atgaagcgtt tctggcgagc 300
acccaatctt tcacgaacct gcagaacaat cgtcaactgg tgatcaaagg caatgcgcag 360
gcccgtctgc gcatgatgta tctgtacgcg tatgcgcagc aatacaaccg cattgttatc 420
ggcaccgata atgcctgcga atggtacatg ggttatttta cgaaattcgg cgatggtgca 480
gctgacattc tgccgctggt caacctgaaa aaatcgcagg tgtttgaact gggtaaatac 540
ctggatgttc cgaaaaatat cctggacaaa gcaccgagcg caggtctgtg gcagggtcaa 600
accgatgaag acgaaatggg cgttacgtat caggaaattg atgacttcct ggatggtaaa 660
caagtcagcg cgaaagccct ggaacgtatc aacttctggc acaaccgctc acatcataaa 720
cgcaaactgg cactgacccc gaacttctaa 750
<210> 38
<211> 782
<212> DNA
<213> Optimized_FtNadE_star_Bacillus
<400> 38
gctacctgag aagcttatga aaatcgttaa agacttctct ccgaaagaat attctcaaaa 60
acttgtgaac tggcttagcg actcttgtat gaactatccg gcagaaggct ttgttattgg 120
tctttctggc ggtatcgact ctgctgtggc ggcttcactt gctgttaaaa caggtcttcc 180
gacaacagca cttattcttc cgtctgacaa caatcagcat caagatatgc aggacgcgct 240
tgaacttatt gaaatgctta acatcgaaca ctacacaatt tctatccagc cggcgtatga 300
agcgtttctt gcgagcacac aatctttcac aaaccttcag aacaatcgtc aacttgtgat 360
caaaggcaat gcgcaggctc gtcttcgcat gatgtatctt tacgcgtatg cgcagcaata 420
caaccgcatt gttatcggca cagataatgc ttgcgaatgg tacatgggtt attttacaaa 480
attcggcgat ggtgctgctg acattcttcc gcttgttaac cttaaaaaat ctcaggtgtt 540
tgaacttggt aaataccttg atgttccgaa aaatatcctt gacaaagcac cgagcgcagg 600
tctttggcag ggtcaaacag atgaagacga aatgggcgtt acatatcagg aaattgatga 660
cttccttgat ggtaaacaag ttagcgcgaa agctcttgaa cgtatcaact tctggcacaa 720
ccgctcacat cataaacgca aacttgcact tacaccgaac ttctaagcat gcagtaagta 780
gc 782
<210> 39
<211> 785
<212> DNA
<213> Optimized_AE016827_Bacillus Mannheimia succiniciproducens MBEL55E
<400> 39
gctacctgag aagcttatga aaacagcagc atacgcagat tatcttattc aatggcttga 60
aaaccaacgc acagaacttt acggcatgga cggctataca cttggcgtta gcggcggtat 120
tgacagcgct gtttgcgctc atcttgcagc gcgcacaggc gcgccggtac aagctcttat 180
tcttccggcg gaagtaacat ctccgtcaga tgtggcggat gcgcaagcta cacttgaaag 240
cgctggtatt gacggccaaa ttatttctat tgcaccgtgg tacgatctta ttatgcaaca 300
actttctccg gtacttaata gcgaaccgga acgcgttaac gtacttaaag gtaatcttat 360
ggcacgcctt cgtatgattg cgctttttac aacagcacaa agccatcgtt ctattgtgct 420
tggcacagat aatgcggcgg aatggcttac aggttatttt acaaaattcg gcgacggcgc 480
agcggacgta cttccgcttg cgggccttcg caaagaacag gtatttgaac ttggccgtta 540
tcttggcgta ccgcaaagcg tgcttgataa aaaaccgagc gctggtcttt gggcaggcca 600
aacagacgaa gctgaaatgg gtgttacata cgcggaaatc gacgcttatc ttcgcggcga 660
aacagttagc ccgcaggcac ttcaacaaat ccgtttctgg cacaaccgtt ctcatcacaa 720
acgtatgctt ccgccgaaac cgaaatcacc ggatgaagcg gaatgttaag catgcagtaa 780
gtagc 785
<210> 40
<211> 782
<212> DNA
<213> Optimized_CP002558_Bacillus Francisella cf. novicida 3523
<400> 40
gctacctgag aagcttatga aaattgttaa agatttttct ccgaaagaat attcacaaaa 60
tcttgttaat tggctttctg atacatgtat taattatccg gctgaaggct ttgtaatcgg 120
cattagcggt ggtattgatt cagcggttgc agcttctctt gctgttaaaa caggccttcc 180
gacaacagct cttattcttc cgtcaaaaaa caatcaacac caagatattc aagatgctct 240
tgaacttgtt gaaaaactta atattgaaca tcatattgtt acaattcaac cggcatacga 300
aaattttctt gcatcaacac aggaatttat taatacagat aataatcgcc aacttgtgat 360
caaaggcaat gctcaagcac gtcttcgcat gatgtatctt tatgcttatg ctcaacaata 420
taaccgcatt gttattggta cagataatgc ttgtgaatgg tatatgggct attttacaaa 480
atttggtgat ggcgctgctg atatttttcc gcttattaat cttaaaaaat cacaagtttt 540
tgaacttggt aaataccttg atgttccgaa aaatattatt gataaagctc cgtctgctgg 600
cctttggcaa ggccaaacag atgaagatga aatgggcgta acatatcaag aaattgatga 660
tttccttgat ggtaaacaaa tttcagcaaa agctcttgaa cgcattaact tctggcataa 720
tcgttctcat cataaacgca aacttgctct tacaccgaat ttctaagcat gcagtaagta 780
gc 782
<210> 41
<211> 782
<212> DNA
<213> Optimized_CP002872_Bacillus Francisella sp. TX077308
<400> 41
gctacctgag aagcttatga aaattgtaaa aaactttatt gtagaacagt attctaataa 60
tcttattaaa tggcttaaag aaaattgcat taaatatccg gctgaaggtt ttgtgattgg 120
tatttctggt ggtattgatt ctgcagtagc tgcatctctt gcagttaaaa caggccttcc 180
gacaacagct cttattcttc cgtctaaaaa caatcaagac caagatatgc gcgatggcat 240
tgaacttatc gaaaatctta atattgaata tcatacagtt tcaattcaac cggcttatga 300
cacatttatt gaatcaacat ttaactttac aaactcacaa aatgatcgcc aacatgttat 360
caaaggcaat gctcaagcgc gtcttcgcat gatgtatctt tatgcttatg ctcagcaaaa 420
taatcgcatt gttattggta cagataacgc atgtgaatgg tacatgggct atttcacaaa 480
atttggtgat ggtgcagcag atattcttcc gcttattaat cttaaaaaat ctcaagtttt 540
tgaacttggt aaatacctta aagtgccgaa aaacattatc caaaaagatc cgtctgctgg 600
tctttggcaa ggtcaaacag atgaagatga aatgggtgtt acatacaaag aaattgatga 660
cttccttgac ggtaaagaag tttcagaaaa agctcttgaa cgcattagct tctggcataa 720
tcgttctcac cataaacgca gcatggcttt tacaccgaat ttttaagcat gcagtaagta 780
gc 782
<210> 42
<211> 750
<212> DNA
<213> Reverted FtNadE_star
<400> 42
atgaaaatcg tcaaagactt ctccccgaaa gaatattccc aaaaactggt gaactggctg 60
agcgactcgt gtatgaacac tccggcagaa ggctttgtca ttggtctgag tggcggtatc 120
gattccgctg tggcggcctc actggccgtt aaaaccggcc tgccgaccac ggcactgatt 180
ctgccgtctg acaacaatca gcatcaagat atgcaggacg cgctggaact gattgaaatg 240
ctgaacatcg aacactacac catttccatc cagccggcgt atgaagcgtt tctggcgagc 300
acccaatctt tcacgaacct gcagaacaat cgtcaactgg tgatcaaagg caatgcgcag 360
gcccgtctgc gcatgatgta tctgtacgcg tatgcgggtc aatacaaccg cattgttatc 420
ggcaccgata atgcctgcga atggtacatg ggttatttta cgaaattcgg cgatggtgca 480
gctgacattc tgccgctggt caacctgaaa aaatcgcagg tgtttgaact gggtaaatac 540
ctggatgttc cgaaaaatat cctggacaaa gcaccgagcg caggtctgtg gcagggtcaa 600
accgatgaag acgaaatggg cgttacgtat caggaaattg atgacttcct ggatggtaaa 660
caagtcagcg cgaaagccct ggaacgtatc aacttctggc acaacgtctc acatcataaa 720
cgcaaactgg cactgacccc gaacttctaa 750
<210> 43
<211> 753
<212> DNA
<213> Optimized_AE016827_Reverted Mannheimia succiniciproducens MBEL55E
<400> 43
atgaaaacgg cagcatacgc agattatctg attcaatggc tggaaaacca acgcaccgaa 60
ctgaccggca tggacggcta taccctgggc gtcagcggcg gtattgacag cgccgtctgc 120
gctcatctgg cagcgcgcac cggcgcgccg gtacaagccc tgattctgcc ggcggaagta 180
accagtccgt cagatgtggc ggatgcgcaa gccaccctgg aaagcgccgg tattgacggc 240
caaattattt ccattgcacc gtggtacgat ctgattatgc aacaactgtc cccggtactg 300
aatagcgaac cggagcgcgt taacgtactg aaaggtaatc tgatggcacg cctgcgtatg 360
attgcgctgt ttaccacggc aggcagccat cgttctattg tgctgggcac cgataatgcg 420
gcggaatggc tgacgggtta ttttaccaaa ttcggcgacg gcgcagcgga cgtactgccg 480
ctggcgggcc tgcgcaaaga gcaggtattt gaactgggcc gttatctggg cgtaccgcaa 540
agcgtgctgg ataaaaaacc gagcgccggt ctgtgggcag gccaaacgga cgaagctgaa 600
atgggtgtta cctatgcgga aatcgacgct tatctgcgcg gcgaaaccgt tagcccgcag 660
gcactgcaac aaatccgttt ctggcacaac gtttctcatc acaaacgtat gctgccgccg 720
aaaccgaaat caccggatga agcggagtgt taa 753
<210> 44
<211> 750
<212> DNA
<213> Optimized_CP002558_Reverted Francisella cf. novicida 3523
<400> 44
atgaaaattg ttaaagattt tagtccgaaa gaatattcac aaaatctggt taattggctg 60
agtgatacct gtattaatac tccggctgaa ggctttgtaa tcggcattag cggtggtatt 120
gattcagcgg ttgcagcttc tctggctgtc aaaaccggcc tgccgaccac cgctctgatt 180
ctgccgtcaa aaaacaatca acaccaagat attcaagatg ctctggaact ggttgagaaa 240
ctgaatattg aacatcatat tgttaccatt caaccggcat acgaaaattt tctggcatca 300
acccaggaat ttattaatac cgataataat cgccaactgg tgatcaaagg caatgctcaa 360
gcacgtctgc gcatgatgta tctgtatgcc tatgccggcc aatataaccg cattgttatt 420
ggtaccgata atgcttgtga gtggtatatg ggctatttta ccaaatttgg tgatggcgct 480
gctgatattt ttccgctgat taatctgaaa aaatcacaag tttttgaact gggtaaatac 540
ctggatgttc cgaaaaatat tattgataaa gctccgtctg ctggcctgtg gcaaggccaa 600
accgatgagg atgaaatggg cgtaacctat caagaaattg atgatttcct ggatggtaaa 660
caaatttcag caaaagccct ggaacgcatt aacttctggc ataatgttag tcatcataaa 720
cgcaaactgg ctctgacccc gaatttctaa 750
<210> 45
<211> 750
<212> DNA
<213> Optimized_CP002872_Reverted Francisella sp. TX077308
<400> 45
atgaaaattg taaaaaactt tattgtagaa cagtattcta ataatctgat taaatggctg 60
aaagagaatt gcattaaaac tccggctgaa ggttttgtga ttggtattag tggtggtatt 120
gattcggcag tagccgcatc tctggcagtc aaaaccggcc tgccgaccac cgctctgatt 180
ctgccgtcga aaaacaatca agaccaagat atgcgcgatg gcattgaact gatcgaaaat 240
ctgaatattg agtatcatac cgtttcaatt caaccggctt atgacacgtt tattgagtca 300
acctttaact ttaccaactc acaaaatgat cgccaacatg ttatcaaagg caatgcccaa 360
gcgcgtctgc gcatgatgta tctgtatgct tatgctgggc aaaataatcg cattgttatt 420
ggtaccgata acgcatgtga atggtacatg ggctatttca ccaaatttgg tgatggtgca 480
gcagatattc tgccgctgat taatctgaaa aaatctcaag tttttgaact gggtaaatac 540
ctgaaagtgc cgaaaaacat tatccaaaaa gatccgtctg ccggtctgtg gcaaggtcaa 600
accgatgagg atgaaatggg tgtcacctac aaagaaattg atgacttcct ggacggtaaa 660
gaagtctcag aaaaagctct ggaacgcatt agcttctggc ataatgttag tcaccataaa 720
cgcagcatgg cttttacccc gaatttttaa 750
<210> 46
<211> 828
<212> DNA
<213> Escherichia coli
<400> 46
atgacattgc aacaacaaat aataaaggcg ctgggcgcaa aaccgcagat taatgccgaa 60
gaggaaattc gtcgtagtgt cgattttctg aaaagctacc tgcgaactta tccgttcatt 120
aaatcactgg tgctcgggat cagcggcggt caggactcca cgcttgccgg aaagctgtgc 180
cagatggcga ttaatgagct gcgccaggaa accggcaacg aatcactgca atttattgcc 240
gtacgcctgc cctatggtgt tcaggccgac gaacaagatt gccaggatgc cattgccttt 300
attcaaccgg atcgcgtatt aaccgttaat atcaagggcg cggtattggc tagcgagcag 360
gcattgcggg aagcaggcat tgaactgagc gattttgtcc gtggcaatga aaaagcgcgt 420
gagcggatga aagcacaata tagcattgcg ggtatgacca gcggtgtcgt ggtgggcacc 480
gatcatgcag cagaagccat taccggattc ttcactaaat atggtgacgg cggtacggat 540
attaatccgc tgtatcgtct caacaaacgt cagggtaaac agttactggc ggcattaggt 600
tgcccggaac acctttataa gaaagcgcca acggccgatc tggaagatga tcgcccttct 660
ctgccagatg aagtggcact cggcgtgacc tatgacaata tcgacgacta tctggaaggg 720
aaaaacgtac ctcaacaggt cgccagaaca atagagaact ggtatctgaa aaccgaacat 780
aaacgccgtc cgccaattac cgttttcgat gatttctgga aaaagtaa 828
<210> 47
<211> 789
<212> DNA
<213> Optimized_CP002558_Corynebacterium Francisella cf. novicida 3523
<400> 47
cagagaattc aaaggaggac aaccatggat gaaaattgtg aaagattttt ccccaaaaga 60
atattcccaa aatctggtga attggctgtc cgatacctgt attaattatc cagcagaagg 120
ctttgtgatc ggcatttccg gtggtattga ttccgcagtg gcagcatccc tggcagtgaa 180
aaccggcctg ccaaccaccg cactgattct gccatccaaa aacaatcaac accaagatat 240
tcaagatgca ctggaactgg tggaaaaact gaatattgaa catcatattg tgaccattca 300
accagcatac gaaaattttc tggcatccac ccaggaattt attaataccg ataataatcg 360
ccaactggtg atcaaaggca atgcacaagc acgcctgcgc atgatgtatc tgtatgcata 420
tgcacaacaa tataaccgca ttgtgattgg caccgataat gcatgtgaat ggtatatggg 480
ctattttacc aaatttggtg atggcgcagc agatattttt ccactgatta atctgaaaaa 540
atcccaagtg tttgaactgg gtaaatacct ggatgtgcca aaaaatatta ttgataaagc 600
accatccgca ggcctgtggc aaggccaaac cgatgaagat gaaatgggcg tgacctatca 660
agaaattgat gatttcctgg atggtaaaca aatttccgca aaagcactgg aacgcattaa 720
cttctggcat aatcgctccc atcataaacg caaactggca ctgaccccaa atttctaagg 780
tacccgggg 789
<210> 48
<211> 1611
<212> DNA
<213> B. subtilis Tetracyclin cassette 1611bp
<400> 48
aaagagtagt tcaacaaacg ggccatattg ttgtataagt gatgaaatac tgaatttaaa 60
acttagttta tatgtggtaa aatgttttaa tcaagtttag gaggaattaa ttatgaagtg 120
taatgaatgt aacagggttc aattaaaaga gggaagcgta tcattaaccc tataaactac 180
gtctgccctc attattggag ggtgaaatgt gaatacatcc tattcacaat cgaatttacg 240
acacaaccaa attttaattt ggctttgcat tttatctttt tttagcgtat taaatgaaat 300
ggttttgaac gtctcattac ctgatattgc aaatgatttt aataaaccac ctgcgagtac 360
aaactgggtg aacacagcct ttatgttaac cttttccatt ggaacagctg tatatggaaa 420
gctatctgat caattaggca tcaaaaggtt actcctattt ggaattataa taaattgttt 480
cgggtcggta attgggtttg ttggccattc tttcttttcc ttacttatta tggctcgttt 540
tattcaaggg gctggtgcag ctgcatttcc agcactcgta atggttgtag ttgcgcgcta 600
tattccaaag gaaaataggg gtaaagcatt tggtcttatt ggatcgatag tagccatggg 660
agaaggagtc ggtccagcga ttggtggaat gatagcccat tatattcatt ggtcctatct 720
tctactcatt cctatgataa caattatcac tgttccgttt cttatgaaat tattaaagaa 780
agaagtaagg ataaaaggtc attttgatat caaaggaatt atactaatgt ctgtaggcat 840
tgtatttttt atgttgttta caacatcata tagcatttct tttcttatcg ttagcgtgct 900
gtcattcctg atatttgtaa aacatatcag gaaagtaaca gatccttttg ttgatcccgg 960
attagggaaa aatatacctt ttatgattgg agttctttgt gggggaatta tatttggaac 1020
agtagcaggg tttgtctcta tggttcctta tatgatgaaa gatgttcacc agctaagtac 1080
tgccgaaatc ggaagtgtaa ttattttccc tggaacaatg agtgtcatta ttttcggcta 1140
cattggtggg atacttgttg atagaagagg tcctttatac gtgttaaaca tcggagttac 1200
atttctttct gttagctttt taactgcttc ctttctttta gaaacaacat catggttcat 1260
gacaattata atcgtatttg ttttaggtgg gctttcgttc accaaaacag ttatatcaac 1320
aattgtttca agtagcttga aacagcagga agctggtgct ggaatgagtt tgcttaactt 1380
taccagcttt ttatcagagg gaacaggtat tgcaattgta ggtggtttat tatccatacc 1440
cttacttgat caaaggttgt tacctatgga agttgatcag tcaacttatc tgtatagtaa 1500
tttgttatta cttttttcag gaatcattgt cattagttgg ctggttacct tgaatgtata 1560
taaacattct caaagggatt tctaaatcgt taagggatca actttgggag a 1611
<210> 49
<211> 1430
<212> DNA
<213> B. subtilis Neomycin cassette 1430bp
<400> 49
gcttgggctg caggtcgaga tcagggaatg agtttataaa ataaaaaaag cacctgaaaa 60
ggtgtctttt tttgatggtt ttgaacttgt tctttcttat cttgatacat atagaaataa 120
cgtcattttt attttagttg ctgaaaggtg cgttgaagtg ttggtatgta tgtgttttaa 180
agtattgaaa acccttaaaa ttggttgcac agaaaaaccc catctgttaa agttataagt 240
gactaaacaa ataactaaat agatgggggt ttcttttaat attatgtgtc ctaatagtag 300
catttattca gatgaaaaat caagggtttt agtggacaag acaaaaagtg gaaaagtgag 360
accatgtgct taggaagacg agttattaat agctgaataa gaacggtgct ctccaaatat 420
tcttatttag aaaagcaaat ctaaaattat ctgaaaaggg aatgagaata gtgaatggac 480
caataataat gactagagaa gaaagaatga agattgttca tgaaattaag gaacgaatat 540
tggataaata tggggatgat gttaaggcta ttggtgttta tggctctctt ggtcgtcaga 600
ctgatgggcc ctattcggat attgagatga tgtgtgtcat gtcaacagag gaagcagagt 660
tcagccatga atggacaacc ggtgagtgga aggtggaagt gaattttgat agcgaagaga 720
ttctactaga ttatgcatct caggtggaat cagattggcc gcttacacat ggtcaatttt 780
tctctatttt gccgatttat gattcaggtg gatacttaga gaaagtgtat caaactgcta 840
aatcggtaga agcccaaaag ttccacgatg cgatttgtgc ccttatcgta gaagagctgt 900
ttgaatatgc aggcaaatgg cgtaatattc gtgtgcaagg accgacaaca tttctaccat 960
ccttgactgt acaggtagca atggcaggtg ccatgttgat tggtctgcat catcgcatct 1020
gttatacgac gagcgcttcg gtcttaactg aagcagttaa gcaatcagat cttccttcag 1080
gttatgacca tctgtgccag ttcgtaatgt ctggtcaact ttccgactct gagaaacttc 1140
tggaatcgct agagaatttc tggaatggga ttcaggagtg gacagaacga cacggatata 1200
tagtggatgt gtcaaaacgc ataccatttt gaacgatgac ctctaataat tgttaatcat 1260
gttggttacg tatttattaa cttctcctag tattagtaat tatcatggct gtcatggcgc 1320
attaacggaa taaagggtgt gcttaaatcg ggccattttg cgtaataaga aaaaggatta 1380
attatgagcg aattgaatta ataataaggt aatagattta cattagaaaa 1430
<210> 50
<211> 1158
<212> DNA
<213> B. subtilis Spectinomycin cassette 1158 bp
<400> 50
catatgcaag ggtttattgt tttctaaaat ctgattacca attagaatga atatttccca 60
aatattaaat aataaaacaa aaaaattgaa aaaagtgttt ccaccatttt ttcaattttt 120
ttataatttt tttaatctgt tatttaaata gtttatagtt aaatttacat tttcattagt 180
ccattcaata ttctctccaa gataactacg aactgctaac aaaattctct ccctatgttc 240
taatggagaa gattcagcca ctgcatttcc cgcaatatct tttggtatga ttttacccgt 300
gtccatagtt aaaatcatac ggcataaagt taatatagag ttggtttcat catcctgata 360
attatctatt aattcctctg acgaatccat aatggctctt ctcacatcag aaaatggaat 420
atcaggtagt aattcctcta agtcataatt tccgtatatt cttttatttt ttcgttttgc 480
ttggtaaagc attatggtta aatctgaatt taattccttc tgaggaatgt atccttgttc 540
ataaagctct tgtaaccatt ctccataaat aaattcttgt ttgggaggat gattccacgg 600
taccatttct tgctgaataa taattgttaa ttcaatatat cgtaagttgc ttttatctcc 660
tatttttttt gaaataggtc taattttttg tataagtatt tctttacttt gatctgtcaa 720
tggttcagat acgacgacta aaaagtcaag atcactattt ggttttagtc cactctcaac 780
tcctgatcca aacatgtaag taccaataag gttatttttt aaatgtttcc gaagtatttt 840
tttcacttta ttaatttgtt cgtatgtatt caaatatatc ctcctcacta ttttgattag 900
tacctatttt atatccatag ttgttaatta aataaactta atttagttta tttatagatt 960
tcattggctt ctaaattttt tatctagata ataattattt tagttaattt tattctagat 1020
tatatatgat atgatctttc atttccataa aactaaagta agtgtaaacc tattcattgt 1080
tttaaaaata tctcttgcca gtcacgttac gttattagtt atagttatta taacatgtat 1140
tcacgaacga aaatcgat 1158
<210> 51
<211> 410
<212> PRT
<213> Escherichia coli NadR
<400> 51
Met Ser Ser Phe Asp Tyr Leu Lys Thr Ala Ile Lys Gln Gln Gly Cys
1 5 10 15
Thr Leu Gln Gln Val Ala Asp Ala Ser Gly Met Thr Lys Gly Tyr Leu
20 25 30
Ser Gln Leu Leu Asn Ala Lys Ile Lys Ser Pro Ser Ala Gln Lys Leu
35 40 45
Glu Ala Leu His Arg Phe Leu Gly Leu Glu Phe Pro Arg Gln Lys Lys
50 55 60
Thr Ile Gly Val Val Phe Gly Lys Phe Tyr Pro Leu His Thr Gly His
65 70 75 80
Ile Tyr Leu Ile Gln Arg Ala Cys Ser Gln Val Asp Glu Leu His Ile
85 90 95
Ile Met Gly Phe Asp Asp Thr Arg Asp Arg Ala Leu Phe Glu Asp Ser
100 105 110
Ala Met Ser Gln Gln Pro Thr Val Pro Asp Arg Leu Arg Trp Leu Leu
115 120 125
Gln Thr Phe Lys Tyr Gln Lys Asn Ile Arg Ile His Ala Phe Asn Glu
130 135 140
Glu Gly Met Glu Pro Tyr Pro His Gly Trp Asp Val Trp Ser Asn Gly
145 150 155 160
Ile Lys Lys Phe Met Ala Glu Lys Gly Ile Gln Pro Asp Leu Ile Tyr
165 170 175
Thr Ser Glu Glu Ala Asp Ala Pro Gln Tyr Met Glu His Leu Gly Ile
180 185 190
Glu Thr Val Leu Val Asp Pro Lys Arg Thr Phe Met Ser Ile Ser Gly
195 200 205
Ala Gln Ile Arg Glu Asn Pro Phe Arg Tyr Trp Glu Tyr Ile Pro Thr
210 215 220
Glu Val Lys Pro Phe Phe Val Arg Thr Val Ala Ile Leu Gly Gly Glu
225 230 235 240
Ser Ser Gly Lys Ser Thr Leu Val Asn Lys Leu Ala Asn Ile Phe Asn
245 250 255
Thr Thr Ser Ala Trp Glu Tyr Gly Arg Asp Tyr Val Phe Ser His Leu
260 265 270
Gly Gly Asp Glu Ile Ala Leu Gln Tyr Ser Asp Tyr Asp Lys Ile Ala
275 280 285
Leu Gly His Ala Gln Tyr Ile Asp Phe Ala Val Lys Tyr Ala Asn Lys
290 295 300
Val Ala Phe Ile Asp Thr Asp Phe Val Thr Thr Gln Ala Phe Cys Lys
305 310 315 320
Lys Tyr Glu Gly Arg Glu His Pro Phe Val Gln Ala Leu Ile Asp Glu
325 330 335
Tyr Arg Phe Asp Leu Val Ile Leu Leu Glu Asn Asn Thr Pro Trp Val
340 345 350
Ala Asp Gly Leu Arg Ser Leu Gly Ser Ser Val Asp Arg Lys Glu Phe
355 360 365
Gln Asn Leu Leu Val Glu Met Leu Glu Glu Asn Asn Ile Glu Phe Val
370 375 380
Arg Val Glu Glu Glu Asp Tyr Asp Ser Arg Phe Leu Arg Cys Val Glu
385 390 395 400
Leu Val Arg Glu Met Met Gly Glu Gln Arg
405 410
<210> 52
<211> 180
<212> PRT
<213> Bacillus subtilis YxrA (repressor protein
<400> 52
Met Thr Glu Glu Leu Lys Leu Met Gly Ala Asn Arg Arg Asp Gln Leu
1 5 10 15
Leu Leu Trp Leu Lys Glu Ser Lys Ser Pro Leu Thr Gly Gly Glu Leu
20 25 30
Ala Lys Lys Ala Asn Val Ser Arg Gln Val Ile Val Gln Asp Ile Ser
35 40 45
Leu Leu Lys Ala Lys Asn Val Pro Ile Ile Ala Thr Ser Gln Gly Tyr
50 55 60
Val Tyr Met Asp Ala Ala Ala Gln Gln His Gln Gln Ala Glu Arg Ile
65 70 75 80
Ile Ala Cys Leu His Gly Pro Glu Arg Thr Glu Glu Glu Leu Gln Leu
85 90 95
Ile Val Asp Glu Gly Val Thr Val Lys Asp Val Lys Ile Glu His Pro
100 105 110
Val Tyr Gly Asp Leu Thr Ala Ala Ile Gln Val Gly Thr Arg Lys Glu
115 120 125
Val Ser His Phe Ile Lys Lys Ile Asn Ser Thr Asn Ala Ala Tyr Leu
130 135 140
Ser Gln Leu Thr Asp Gly Val His Leu His Thr Leu Thr Ala Pro Asp
145 150 155 160
Glu His Arg Ile Asp Gln Ala Cys Gln Ala Leu Glu Glu Ala Gly Ile
165 170 175
Leu Ile Lys Asp
180
<210> 53
<211> 214
<212> PRT
<213> Corynebacterium glutamicum CgR_1153 (repressor protein)
<400> 53
Met Pro Ala Ser Pro Glu Ile Gln Met Ala Val Ser Thr Ile Ile Phe
1 5 10 15
Ala Leu Arg Pro Gly Pro Gln Asp Leu Pro Ser Leu Trp Ala Pro Phe
20 25 30
Val Pro Arg Thr Arg Glu Pro His Leu Asn Lys Trp Ala Leu Pro Gly
35 40 45
Gly Trp Leu Pro Pro His Glu Glu Leu Glu Asp Ala Ala Ala Arg Thr
50 55 60
Leu Ala Glu Thr Thr Gly Leu His Pro Ser Tyr Leu Glu Gln Leu Tyr
65 70 75 80
Thr Phe Gly Lys Val Asp Arg Ser Pro Thr Gly Arg Val Ile Ser Val
85 90 95
Val Tyr Trp Ala Leu Val Arg Ala Asp Glu Ala Leu Lys Ala Ile Pro
100 105 110
Gly Glu Asn Val Gln Trp Phe Pro Ala Asp His Leu Pro Glu Leu Ala
115 120 125
Phe Asp His Asn Asp Ile Val Lys Tyr Ala Leu Glu Arg Leu Arg Thr
130 135 140
Lys Val Glu Tyr Ser Glu Ile Ala His Ser Phe Leu Gly Glu Thr Phe
145 150 155 160
Thr Ile Ala Gln Leu Arg Ser Val His Glu Ala Val Leu Gly His Lys
165 170 175
Leu Asp Ala Ala Asn Phe Arg Arg Ser Val Ala Thr Ser Pro Asp Leu
180 185 190
Ile Asp Thr Gly Glu Val Leu Ala Gly Thr Pro His Arg Pro Pro Lys
195 200 205
Leu Phe Arg Phe Gln Arg
210
<210> 54
<211> 191
<212> PRT
<213> Acinetobacter baylyi PnuC (NR transporter protein)
<400> 54
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> 55
<211> 230
<212> PRT
<213> Corynebacterium glutamicum NR transporter protein
<400> 55
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> 56
<211> 239
<212> PRT
<213> Escherichia coli PnuC (NR transporter protein)
<400> 56
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> 57
<211> 550
<212> PRT
<213> Escherichia coli UshA (nucleotide hydrolase)
<400> 57
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> 58
<211> 1462
<212> PRT
<213> Bacillus subtilis YfkN (nucleotide hydrolase)
<400> 58
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> 59
<211> 693
<212> PRT
<213> Corynebacterium glutamicum Cg0397 (nucleotide hydrolase)
<400> 59
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 Val Glu Ile Ser Tyr Asp Arg Glu Ala
275 280 285
Gly Lys Ile Thr Asn Ile Glu Ala Lys Asn Val Ser Ala Thr Asp Val
290 295 300
Val Glu Asn Cys Glu Thr Pro Asn Thr Ala Val Asp Ala Ile Val Ala
305 310 315 320
Ala Ala Val Glu Ala Ala Glu Glu Ala Gly Asn Glu Val Val Ala Thr
325 330 335
Ile Asp Asn Gly Phe Tyr Arg Gly Ala Asp Glu Glu Gly Thr Thr Gly
340 345 350
Ser Asn Arg Gly Val Glu Ser Ser Leu Ser Asn Leu Ile Ala Glu Ala
355 360 365
Gly Leu Trp Ala Val Asn Asp Ala Thr Ile Leu Asn Ala Asp Ile Gly
370 375 380
Ile Met Asn Ala Gly Gly Val Arg Ala Asp Leu Glu Ala Gly Glu Val
385 390 395 400
Thr Phe Ala Asp Ala Tyr Ala Thr Gln Asn Phe Ser Asn Thr Tyr Gly
405 410 415
Val Arg Glu Val Ser Gly Ala Gln Phe Lys Glu Ala Leu Glu Gln Gln
420 425 430
Trp Lys Glu Thr Gly Asp Arg Pro Arg Leu Ala Leu Gly Leu Ser Ser
435 440 445
Asn Val Gln Tyr Ser Tyr Asp Glu Thr Arg Glu Tyr Gly Asp Arg Ile
450 455 460
Thr His Ile Thr Phe Asn Gly Glu Pro Met Asp Met Lys Glu Thr Tyr
465 470 475 480
Arg Val Thr Gly Ser Ser Phe Leu Leu Ala Gly Gly Asp Ser Phe Thr
485 490 495
Ala Phe Ala Glu Gly Gly Pro Ile Ala Glu Thr Gly Met Val Asp Ile
500 505 510
Asp Leu Phe Asn Asn Tyr Ile Ala Ala His Pro Asp Ala Pro Ile Arg
515 520 525
Ala Asn Gln Ser Ser Val Gly Ile Ala Leu Ser Gly Pro Ala Val Ala
530 535 540
Glu Asp Gly Thr Leu Val Pro Gly Glu Glu Leu Thr Val Asp Leu Ser
545 550 555 560
Ser Leu Ser Tyr Thr Gly Pro Glu Ala Lys Pro Thr Thr Val Glu Val
565 570 575
Thr Val Gly Thr Glu Lys Lys Thr Ala Asp Val Asp Asn Thr Ile Val
580 585 590
Pro Gln Phe Asp Ser Thr Gly Lys Ala Thr Val Thr Leu Thr Val Pro
595 600 605
Glu Gly Ala Thr Ser Val Lys Ile Ala Thr Asp Asn Gly Thr Thr Phe
610 615 620
Glu Leu Pro Val Thr Val Asn Gly Glu Gly Asn Asn Asp Asp Asp Asp
625 630 635 640
Asp Lys Glu Gln Gln Ser Ser Gly Ser Ser Asp Ala Gly Ser Leu Val
645 650 655
Ala Val Leu Gly Val Leu Gly Ala Leu Gly Gly Leu Val Ala Phe Phe
660 665 670
Leu Asn Ser Ala Gln Gly Ala Pro Phe Leu Ala Gln Leu Gln Ala Met
675 680 685
Phe Ala Gln Phe Met
690
<210> 60
<211> 165
<212> PRT
<213> Escherichia coli PncC (nicotinamide mononucleotide amidohydrolase)
<400> 60
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> 61
<211> 415
<212> PRT
<213> Bacillus subtilis CinA (nicotinamide mononucleotide amidohydrolase)
<400> 61
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 Gly Gly Leu Gly Pro Thr Lys Asp Asp Leu Thr
65 70 75 80
Lys Glu Thr Ile Ala Asn Thr Leu Gly Arg Pro Leu Val Leu Asn Asp
85 90 95
Glu Ala Phe Gln Ser Ile Glu Asp Tyr Pro Lys Arg Thr Lys Arg Thr
100 105 110
Met Ser Pro Asn Asn Arg Lys Gln Ala Leu Val Ile Glu Gly Ser Asp
115 120 125
Val Leu Ala Asn His Phe Gly Met Ala Pro Gly Met Leu Thr Glu His
130 135 140
Glu Ser Arg Tyr Tyr Met Leu Leu Pro Gly Pro Pro Ser Glu Leu Arg
145 150 155 160
Pro Met Phe Glu Asn Glu Ala Lys Pro Leu Leu Leu Lys Lys Met Gly
165 170 175
Ser Asn Glu Lys Ile Val Ser Thr Val Leu Arg Phe Phe Gly Ile Gly
180 185 190
Glu Ser Gln Leu Glu Pro Asp Leu Glu Asp Ile Ile Asp Ala Gln Thr
195 200 205
Asn Pro Thr Ile Ala Pro Leu Ala Ala Asp Gly Glu Val Thr Leu Arg
210 215 220
Leu Thr Ala Lys His Ala Asp Glu Lys Glu Thr Glu Arg Leu Leu Lys
225 230 235 240
Glu Thr Glu Ala Val Ile Leu Glu Arg Val Gly Glu Phe Phe Tyr Gly
245 250 255
Tyr Asp Asp Thr Ser Leu Val Lys Glu Leu Ser Ile Ala Cys Lys Glu
260 265 270
Lys Gly Ile Thr Ile Ser Ala Ala Glu Ser Phe Thr Gly Gly Leu Phe
275 280 285
Ser Glu Trp Leu Thr Asp His Ser Gly Ala Ser Lys Leu Phe Ala Gly
290 295 300
Gly Val Val Cys Tyr Thr Asn Asp Val Lys Gln Asn Val Leu Gly Val
305 310 315 320
Lys Lys Glu Thr Leu Asp Arg Phe Gly Ala Val Ser Lys Glu Cys Ala
325 330 335
Ser Glu Leu Ala Lys Gly Val Gln Lys Leu Thr Gly Ser Asp Ile Gly
340 345 350
Ile Ser Phe Thr Gly Val Ala Gly Pro Asp Ala Gln Glu Gly His Glu
355 360 365
Pro Gly His Val Phe Ile Gly Ile Ser Ala Asn Gly Lys Glu Glu Val
370 375 380
His Glu Phe His Phe Ala Gly Ser Arg Thr Gly Ile Arg Lys Arg Gly
385 390 395 400
Ala Lys Tyr Gly Cys His Leu Ile Leu Lys Leu Leu Glu Gln Lys
405 410 415
<210> 62
<211> 172
<212> PRT
<213> Corynebacterium glutamicum Cg2153 (nicotinamide mononucleotideamidohydrolase)
<400> 62
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> 63
<211> 213
<212> PRT
<213> Escherichia coli NadD (nicotinic acid mononucleotideadenyltransferase)
<400> 63
Met Lys Ser Leu Gln Ala Leu Phe Gly Gly Thr Phe Asp Pro Val His
1 5 10 15
Tyr Gly His Leu Lys Pro Val Glu Thr Leu Ala Asn Leu Ile Gly Leu
20 25 30
Thr Arg Val Thr Ile Ile Pro Asn Asn Val Pro Pro His Arg Pro Gln
35 40 45
Pro Glu Ala Asn Ser Val Gln Arg Lys His Met Leu Glu Leu Ala Ile
50 55 60
Ala Asp Lys Pro Leu Phe Thr Leu Asp Glu Arg Glu Leu Lys Arg Asn
65 70 75 80
Ala Pro Ser Tyr Thr Ala Gln Thr Leu Lys Glu Trp Arg Gln Glu Gln
85 90 95
Gly Pro Asp Val Pro Leu Ala Phe Ile Ile Gly Gln Asp Ser Leu Leu
100 105 110
Thr Phe Pro Thr Trp Tyr Glu Tyr Glu Thr Ile Leu Asp Asn Ala His
115 120 125
Leu Ile Val Cys Arg Arg Pro Gly Tyr Pro Leu Glu Met Ala Gln Pro
130 135 140
Gln Tyr Gln Gln Trp Leu Glu Asp His Leu Thr His Asn Pro Glu Asp
145 150 155 160
Leu His Leu Gln Pro Ala Gly Lys Ile Tyr Leu Ala Glu Thr Pro Trp
165 170 175
Phe Asn Ile Ser Ala Thr Ile Ile Arg Glu Arg Leu Gln Asn Gly Glu
180 185 190
Ser Cys Glu Asp Leu Leu Pro Glu Pro Val Leu Thr Tyr Ile Asn Gln
195 200 205
Gln Gly Leu Tyr Arg
210
<210> 64
<211> 189
<212> PRT
<213> Bacillus subtilis NadD (nicotinic acid mononucleotideadenyltransferase)
<400> 64
Met Lys Lys Ile Gly Ile Phe Gly Gly Thr Phe Asp Pro Pro His Asn
1 5 10 15
Gly His Leu Leu Met Ala Asn Glu Val Leu Tyr Gln Ala Gly Leu Asp
20 25 30
Glu Ile Trp Phe Met Pro Asn Gln Ile Pro Pro His Lys Gln Asn Glu
35 40 45
Asp Tyr Thr Asp Ser Phe His Arg Val Glu Met Leu Lys Leu Ala Ile
50 55 60
Gln Ser Asn Pro Ser Phe Lys Leu Glu Leu Val Glu Met Glu Arg Glu
65 70 75 80
Gly Pro Ser Tyr Thr Phe Asp Thr Val Ser Leu Leu Lys Gln Arg Tyr
85 90 95
Pro Asn Asp Gln Leu Phe Phe Ile Ile Gly Ala Asp Met Ile Glu Tyr
100 105 110
Leu Pro Lys Trp Tyr Lys Leu Asp Glu Leu Leu Asn Leu Ile Gln Phe
115 120 125
Ile Gly Val Lys Arg Pro Gly Phe His Val Glu Thr Pro Tyr Pro Leu
130 135 140
Leu Phe Ala Asp Val Pro Glu Phe Glu Val Ser Ser Thr Met Ile Arg
145 150 155 160
Glu Arg Phe Lys Ser Lys Lys Pro Thr Asp Tyr Leu Ile Pro Asp Lys
165 170 175
Val Lys Lys Tyr Val Glu Glu Asn Gly Leu Tyr Glu Ser
180 185
<210> 65
<211> 226
<212> PRT
<213> Corynebacterium glutamicum Cg2584 (nicotinic acid mononucleotideadenyltransferase)
<400> 65
Met Arg Thr Leu Tyr Cys Pro Leu Met Thr Thr Thr Val Lys Arg Arg
1 5 10 15
Ala Arg Ile Gly Ile Met Gly Gly Thr Phe Asp Pro Ile His Asn Gly
20 25 30
His Leu Val Ala Gly Ser Glu Val Ala Asp Arg Phe Asp Leu Asp Leu
35 40 45
Val Val Tyr Val Pro Thr Gly Gln Pro Trp Gln Lys Ala Asn Lys Lys
50 55 60
Val Ser Pro Ala Glu Asp Arg Tyr Leu Met Thr Val Ile Ala Thr Ala
65 70 75 80
Ser Asn Pro Arg Phe Met Val Ser Arg Val Asp Ile Asp Arg Gly Gly
85 90 95
Asp Thr Tyr Thr Ile Asp Thr Leu Gln Asp Leu Ser Lys Gln Tyr Pro
100 105 110
Asp Ala Gln Leu Tyr Phe Ile Thr Gly Ala Asp Ala Leu Ala Gln Ile
115 120 125
Val Thr Trp Arg Asp Trp Glu Lys Thr Phe Glu Leu Ala His Phe Val
130 135 140
Gly Val Thr Arg Pro Gly Tyr Glu Leu Asp Gly Asn Ile Ile Pro Glu
145 150 155 160
Met His Gln Asp Arg Val Ser Leu Val Asp Ile Pro Ala Met Ala Ile
165 170 175
Ser Ser Thr Asp Cys Arg Glu Arg Ser Ser Glu Glu Arg Pro Val Trp
180 185 190
Tyr Leu Val Pro Asp Gly Val Val Gln Tyr Ile Ala Lys Arg Gln Leu
195 200 205
Tyr Arg Pro Glu Gly Ser Asp Lys Asp Met Asp Pro Lys Gly Gln Asn
210 215 220
Gln Ala
225
<210> 66
<211> 249
<212> PRT
<213> Acinetobacter NudC enzyme, (NAD+ diphosphatase)
<400> 66
Met Ser Glu Leu Ser Leu Ala Tyr Ile Phe His Asn Gln Gln Leu Leu
1 5 10 15
Val Asn Glu Lys Leu Glu Leu Pro Lys Val Glu Thr Leu Ala Ser Asp
20 25 30
Leu Gln Phe Gly Ser Gly Asp Asn Val Ile Ala Arg Asp Leu Gly Ala
35 40 45
Asp Glu Thr Ile Pro Glu Gly Trp His Leu Val Ser Ile Arg Gln Leu
50 55 60
Ile Ser Ser Trp Ser Thr Glu Glu Phe Met Arg Ala Ser Arg Ala Val
65 70 75 80
Gln Leu Leu Glu Trp Arg Arg Asn His Lys Phe Cys Ser His Cys Gly
85 90 95
His Glu Thr Glu Ile His Pro Thr Glu Tyr Ala Met Val Cys Pro Ala
100 105 110
Cys Gln Tyr Arg Gln Tyr Pro Arg Val Gln Pro Cys Val Ile Thr Val
115 120 125
Ile Thr Arg Gly Asp Asn Glu Ile Leu Leu Ala Lys Asn Ala Asn Asn
130 135 140
Lys Ser Asn Met Tyr Gly Leu Ile Ala Gly Phe Val Glu Val Ala Glu
145 150 155 160
Thr Leu Glu Glu Ala Val Gln Arg Glu Thr Leu Glu Glu Val Gly Leu
165 170 175
Lys Leu Lys Asn Ile Arg Tyr Leu Ala Ser Gln Pro Trp Pro Phe Pro
180 185 190
Ser Asn Leu Met Leu Ala Phe His Ala Glu Tyr Glu Ser Gly Asp Ile
195 200 205
Lys Leu Gln Glu Glu Glu Ile Ser Asp Ala Gln Phe Phe Lys Phe Asp
210 215 220
Gln Leu Pro Glu Ile Pro Phe Lys Gly Ser Ile Ala His Ala Met Ile
225 230 235 240
Met His Val Ile Gln Lys Gln Pro Ile
245
<210> 67
<211> 257
<212> PRT
<213> Escherichia coli NudC enzyme, (NAD+ diphosphatase)
<400> 67
Met Asp Arg Ile Ile Glu Lys Leu Asp His Gly Trp Trp Val Val Ser
1 5 10 15
His Glu Gln Lys Leu Trp Leu Pro Lys Gly Glu Leu Pro Tyr Gly Glu
20 25 30
Ala Ala Asn Phe Asp Leu Val Gly Gln Arg Ala Leu Gln Ile Gly Glu
35 40 45
Trp Gln Gly Glu Pro Val Trp Leu Val Gln Gln Gln Arg Arg His Asp
50 55 60
Met Gly Ser Val Arg Gln Val Ile Asp Leu Asp Val Gly Leu Phe Gln
65 70 75 80
Leu Ala Gly Arg Gly Val Gln Leu Ala Glu Phe Tyr Arg Ser His Lys
85 90 95
Tyr Cys Gly Tyr Cys Gly His Glu Met Tyr Pro Ser Lys Thr Glu Trp
100 105 110
Ala Met Leu Cys Ser His Cys Arg Glu Arg Tyr Tyr Pro Gln Ile Ala
115 120 125
Pro Cys Ile Ile Val Ala Ile Arg Arg Asp Asp Ser Ile Leu Leu Ala
130 135 140
Gln His Thr Arg His Arg Asn Gly Val His Thr Val Leu Ala Gly Phe
145 150 155 160
Val Glu Val Gly Glu Thr Leu Glu Gln Ala Val Ala Arg Glu Val Met
165 170 175
Glu Glu Ser Gly Ile Lys Val Lys Asn Leu Arg Tyr Val Thr Ser Gln
180 185 190
Pro Trp Pro Phe Pro Gln Ser Leu Met Thr Ala Phe Met Ala Glu Tyr
195 200 205
Asp Ser Gly Asp Ile Val Ile Asp Pro Lys Glu Leu Leu Glu Ala Asn
210 215 220
Trp Tyr Arg Tyr Asp Asp Leu Pro Leu Leu Pro Pro Pro Gly Thr Val
225 230 235 240
Ala Arg Arg Leu Ile Glu Asp Thr Val Ala Met Cys Arg Ala Glu Tyr
245 250 255
Glu
<210> 68
<211> 238
<212> PRT
<213> Corynebacterium glutamicum NudC (a.k.a. Cg0888) enzyme, (NAD+diphosphatase)
<400> 68
Met Arg Ile Leu Pro Ile Gly Pro His Asp Glu Ile Ala Val Asn Gly
1 5 10 15
Ser Ile Val Leu Leu Ser Glu His Asp Gly Asp Ile Val Ser Val Gly
20 25 30
Pro Asp Leu Gly Thr Val Arg Val Thr Leu Glu Glu Ile Glu Ser Leu
35 40 45
Gly Thr Pro Thr Ala Pro Arg Asp Leu Gly Ser Arg Glu Val Asp Ala
50 55 60
Cys Val Ser Leu Leu Arg Asn Arg Glu Leu Val Arg Phe Asp Pro His
65 70 75 80
Asp Gly Ser Glu Leu Thr Tyr Arg Glu His Ser Val Ala Tyr Gly Ala
85 90 95
Ser Gly Lys Pro Leu Phe Pro Arg Leu Asp Pro Ala Val Ile Gly Ile
100 105 110
Val Glu Leu Arg Gly Glu Asp Arg Leu Leu Leu Gly Met Asn Ala Gln
115 120 125
Lys Arg Gln Arg Tyr Ser Leu Ile Ala Gly Tyr Val Ser His Gly Glu
130 135 140
Ser Leu Glu Asp Ala Phe Thr Arg Glu Val Phe Glu Glu Ala Ala Arg
145 150 155 160
Arg Val Ser Glu Ile Ser Tyr Val Ser Ser Gln Pro Trp Pro Ile Ser
165 170 175
Gly Ser Leu Met Leu Gly Met Lys Gly Phe Thr Glu Asp Glu Leu Pro
180 185 190
Gln Gly Glu Thr Asp Gly Glu Leu Ala Glu Thr Ile Trp Ala Ser Pro
195 200 205
Leu Asp Ile Ile Asp Arg Lys Ile Pro Ile Ala Pro Pro Gly Ser Ile
210 215 220
Ala Tyr Asp Met Ile Asn Ala Trp Ala Arg Asp Lys Gln Asn
225 230 235
<210> 69
<211> 194
<212> PRT
<213> Burkholderiaceae NudC enzyme, (NAD+ diphosphatase)
<400> 69
Met Gln Tyr Arg Phe Cys Pro Gln Cys Gly Ala Pro Leu Glu Val Leu
1 5 10 15
Pro Leu Ser Gly Arg Glu Arg His Ala Cys Val Gln Gln Glu Cys Gly
20 25 30
Phe Val His Trp Asn Asn Pro Leu Pro Val Leu Ala Ala Val Val Glu
35 40 45
Tyr Gln Asp Lys Leu Leu Leu Ala Arg Asn Ala Ala Trp Pro Glu Thr
50 55 60
Met Phe Ala Leu Val Thr Gly Phe Leu Glu Arg Asp Glu Pro Pro Glu
65 70 75 80
Leu Gly Val Ala Arg Glu Leu Lys Glu Glu Thr Asn Leu Asp Thr Glu
85 90 95
Ser Val Ser Leu Ile Gly Val Tyr Glu Phe Met Arg Lys Asn Glu Leu
100 105 110
Ile Ile Ala Tyr His Val Lys Ala Thr Gly Thr Val Ala Leu Ser Glu
115 120 125
Glu Leu Ala Asp Tyr Lys Leu Val Ala Pro Glu Asp Val Arg Val Trp
130 135 140
Ser Ala Gly Thr Gly Phe Ala Val Ala Asp Trp Leu Ala Ala Arg Gly
145 150 155 160
Tyr Pro Val Arg Phe Phe Asp Arg Gln Thr Gly Ala Asp Ile Pro Asp
165 170 175
Pro Arg Arg Pro His Asp Phe Ser Leu Val Gly Gly Ser Leu Gln Tyr
180 185 190
Arg Trp
<210> 70
<211> 603
<212> PRT
<213> Haemophilus influenzae NadN (NAD+ diphosphatase)
<400> 70
Met Leu Leu Ser Lys Lys Ser Ala Thr Phe Ala Leu Ser Ala Phe Ala
1 5 10 15
Met Leu Phe Thr Ser Ala Ala Leu Ala Lys Glu Ala Pro Gln Ala His
20 25 30
Lys Ala Val Glu Leu Ser Ile Leu His Ile Asn Asp His His Ser Tyr
35 40 45
Leu Glu Pro His Glu Thr Arg Ile Asn Leu Asn Gly Gln Gln Thr Lys
50 55 60
Val Asp Ile Gly Gly Phe Ser Ala Val Asn Ala Lys Leu Asn Glu Leu
65 70 75 80
Arg Lys Lys Tyr Lys Asn Pro Leu Val Leu His Ala Gly Asp Ala Ile
85 90 95
Thr Gly Thr Leu Tyr Phe Thr Leu Phe Gly Gly Ser Ala Asp Ala Ala
100 105 110
Val Met Asn Ala Gly Asn Phe His Tyr Phe Thr Leu Gly Asn His Glu
115 120 125
Phe Asp Ala Gly Asn Glu Gly Leu Leu Lys Leu Leu Glu Pro Leu Lys
130 135 140
Ile Pro Val Leu Ser Ala Asn Val Ile Pro Asp Lys Ser Ser Ile Leu
145 150 155 160
Tyr Asn Lys Trp Lys Pro Tyr Asp Ile Phe Thr Val Asp Gly Glu Lys
165 170 175
Ile Ala Ile Ile Gly Leu Asp Thr Val Asn Lys Thr Val Asn Ser Ser
180 185 190
Ser Pro Gly Lys Asp Val Lys Phe Tyr Asp Glu Ile Ala Thr Ala Gln
195 200 205
Ile Met Ala Asn Ala Leu Lys Gln Gln Gly Ile Asn Lys Ile Ile Leu
210 215 220
Leu Ser His Ala Gly Ser Glu Lys Asn Ile Glu Ile Ala Gln Lys Val
225 230 235 240
Asn Asp Ile Asp Val Ile Val Thr Gly Asp Ser His Tyr Leu Tyr Gly
245 250 255
Asn Asp Glu Leu Arg Gly Leu Lys Leu Pro Val Ile Tyr Glu Tyr Pro
260 265 270
Leu Glu Phe Lys Asn Pro Asn Gly Glu Pro Val Phe Val Met Glu Gly
275 280 285
Trp Ala Tyr Ser Ala Val Val Gly Asp Leu Gly Val Lys Phe Ser Pro
290 295 300
Gln Gly Ile Ala Ser Ile Thr Arg Lys Ile Pro His Val Leu Met Ser
305 310 315 320
Ser His Lys Leu Gln Val Lys Asn Ser Glu Gly Lys Trp Ala Glu Leu
325 330 335
Ala Gly Asp Glu Arg Lys Lys Ala Leu Asp Thr Leu Lys Ser Met Lys
340 345 350
Ser Ile Ser Leu Asp Asp His Asp Ala Lys Thr Asp Lys Leu Ile Ala
355 360 365
Lys Tyr Lys Ser Glu Lys Asp His Leu Ala Gln Glu Ile Val Gly Val
370 375 380
Ile Thr Gly Ser Ala Met Pro Gly Gly Ser Ala Asn Arg Ile Pro Asn
385 390 395 400
Lys Ala Gly Ser Asn Pro Glu Gly Ser Ile Ala Thr Arg Phe Ile Ala
405 410 415
Glu Thr Met Tyr Asn Glu Leu Lys Thr Val Asp Leu Thr Ile Gln Asn
420 425 430
Ala Gly Gly Val Arg Ala Asp Ile Leu Pro Gly Asn Val Thr Phe Asn
435 440 445
Asp Ala Tyr Thr Phe Leu Pro Phe Gly Asn Thr Leu Tyr Thr Tyr Lys
450 455 460
Met Glu Gly Ser Leu Val Lys Gln Val Leu Glu Asp Ala Met Gln Phe
465 470 475 480
Ala Leu Val Asp Gly Ser Thr Gly Ala Phe Pro Tyr Gly Ala Gly Ile
485 490 495
Arg Tyr Glu Ala Asn Glu Thr Pro Asn Ala Glu Gly Lys Arg Leu Val
500 505 510
Ser Val Glu Val Leu Asn Lys Gln Thr Gln Gln Trp Glu Pro Ile Asp
515 520 525
Asp Asn Lys Arg Tyr Leu Val Gly Thr Asn Ala Tyr Val Ala Gly Gly
530 535 540
Lys Asp Gly Tyr Lys Thr Phe Gly Lys Leu Phe Asn Asp Pro Lys Tyr
545 550 555 560
Glu Gly Val Asp Thr Tyr Leu Pro Asp Ala Glu Ser Phe Ile Lys Phe
565 570 575
Met Lys Lys His Pro His Phe Glu Ala Tyr Thr Ser Ser Asn Val Lys
580 585 590
Phe Asn Ala Ser Thr Asp Ala Leu Pro Lys Lys
595 600
<210> 71
<211> 397
<212> PRT
<213> Bs168|nupG_purine_nucleoside_transport_protein
<400> 71
Met Tyr Phe Leu Leu Asn Leu Val Gly Leu Ile Val Ile Met Ala Val
1 5 10 15
Val Phe Leu Cys Ser Pro Gln Lys Lys Lys Ile Lys Trp Arg Pro Ile
20 25 30
Ile Thr Leu Ile Val Leu Glu Leu Leu Ile Thr Trp Phe Met Leu Gly
35 40 45
Thr Lys Val Gly Ser Trp Ala Ile Gly Lys Ile Gly Asp Phe Phe Thr
50 55 60
Trp Leu Ile Ala Cys Ala Ser Asp Gly Ile Ala Phe Ala Phe Pro Ser
65 70 75 80
Val Met Ala Asn Glu Thr Val Asp Phe Phe Phe Ser Ala Leu Leu Pro
85 90 95
Ile Ile Phe Ile Val Thr Phe Phe Asp Ile Leu Thr Tyr Phe Gly Ile
100 105 110
Leu Pro Trp Leu Ile Asp Lys Ile Gly Trp Val Ile Ser Lys Ala Ser
115 120 125
Arg Leu Pro Lys Leu Glu Ser Phe Phe Ser Ile Gln Met Met Phe Leu
130 135 140
Gly Asn Thr Glu Ala Leu Ala Val Ile Arg Gln Gln Leu Thr Val Leu
145 150 155 160
Ser Asn Asn Arg Leu Leu Thr Phe Gly Leu Met Ser Met Ser Ser Ile
165 170 175
Ser Gly Ser Ile Ile Gly Ser Tyr Leu Ser Met Val Pro Ala Thr Tyr
180 185 190
Val Phe Thr Ala Ile Pro Leu Asn Cys Leu Asn Ala Leu Ile Ile Ala
195 200 205
Asn Leu Leu Asn Pro Val His Val Pro Glu Asp Glu Asp Ile Ile Tyr
210 215 220
Thr Pro Pro Lys Glu Glu Lys Lys Asp Phe Phe Ser Thr Ile Ser Asn
225 230 235 240
Ser Met Leu Val Gly Met Asn Met Val Ile Val Ile Leu Ala Met Val
245 250 255
Ile Gly Tyr Val Ala Leu Thr Ser Ala Val Asn Gly Ile Leu Gly Val
260 265 270
Phe Val His Gly Leu Thr Ile Gln Thr Ile Phe Ala Tyr Leu Phe Ser
275 280 285
Pro Phe Ala Phe Leu Leu Gly Leu Pro Val His Asp Ala Met Tyr Val
290 295 300
Ala Gln Leu Met Gly Met Lys Leu Ala Thr Asn Glu Phe Val Ala Met
305 310 315 320
Leu Asp Leu Lys Asn Asn Leu Lys Ser Leu Pro Pro His Thr Val Ala
325 330 335
Val Ala Thr Thr Phe Leu Thr Ser Phe Ala Asn Phe Ser Thr Val Gly
340 345 350
Met Ile Tyr Gly Thr Tyr Asn Ser Ile Leu Asp Gly Glu Lys Ser Thr
355 360 365
Val Ile Gly Arg Asn Val Trp Lys Leu Leu Val Ser Gly Ile Ala Val
370 375 380
Ser Leu Leu Ser Ala Ala Ile Val Gly Leu Phe Val Trp
385 390 395
<210> 72
<211> 233
<212> PRT
<213> Bs168|deoD_-_purine_nucleoside_phosphorylase
<400> 72
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> 73
<211> 433
<212> PRT
<213> Bs168|pdp_-_pyrimidine-nucleoside_phosphorylase
<400> 73
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> 74
<211> 271
<212> PRT
<213> Bs168|pupG_-_purine_nucleoside_phosphorylase
<400> 74
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> 75
<211> 239
<212> PRT
<213> gnl|ECOLI|DEOD-MONOMER purine nucleoside phosphorylase
<400> 75
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> 76
<211> 211
<212> PRT
<213> gnl|CORYNE|G18NG-MONOMER nucleoside phosphorylase
<400> 76
Met Lys Leu Phe Gln Ser Cys Phe Leu Leu Asn Asn Ser Ala Cys Asn
1 5 10 15
Leu Leu Val Val Leu Cys Leu Val Thr Met Thr Glu Thr Leu Phe Val
20 25 30
Ser Ala Thr Thr Glu Glu Ala Val Tyr Leu Pro Asp Gly Ile Asp Leu
35 40 45
Leu Val Thr Gly Ile Gly Thr Thr Ala Ala Thr Met Ile Leu Thr Lys
50 55 60
Glu Leu Ala Thr Arg Glu Val Leu Pro Ala Arg Ile Val Asn Ile Gly
65 70 75 80
Thr Ala Gly Ala Leu Val Asp Gly Leu Ala Gly Val Tyr Glu Ile Glu
85 90 95
Tyr Val Leu Gln His Asp Phe Ser Ser Glu Leu Ile Ala Glu Met Thr
100 105 110
Gly Lys Pro Cys Ser Asn Gly Ser Thr Leu Ala Thr Ser Gly His Phe
115 120 125
Pro Val Ala Ser Leu Ala Thr Gly Asn Ser Phe Ile Ala Asp Ser Glu
130 135 140
Thr Arg Asn His Leu Ala Thr Arg Ala Ser Leu Cys Asp Met Glu Gly
145 150 155 160
Ala Ala Leu Val Gly Val Ala Lys His Phe Gly Val Pro Ile Thr Leu
165 170 175
Leu Lys Gln Val Ser Asp Ser Ala Asp Glu Glu Ala Ser Gly Ser Trp
180 185 190
Phe Asp Ala Val Asp Ala Gly Ala Arg Gln Leu Ala Glu Ala Val Lys
195 200 205
Glu Phe Lys
210
<210> 77
<211> 346
<212> PRT
<213> Escherichia coli NadA (quinolate synthase)
<400> 77
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 Leu Thr Arg Leu Gln Glu Glu Tyr Pro Asp Ala Ala Ile Leu Val
210 215 220
His Pro Glu Ser Pro Gln Ala Ile Val Asp Met Ala Asp Ala Val Gly
225 230 235 240
Ser Thr Ser Gln Leu Ile Ala Ala Ala Lys Thr Leu Pro His Gln Arg
245 250 255
Leu Ile Val Ala Thr Asp Arg Gly Ile Phe Tyr Lys Met Gln Gln Ala
260 265 270
Val Pro Asp Lys Glu Leu Leu Glu Ala Pro Thr Ala Gly Glu Gly Ala
275 280 285
Thr Cys Arg Ser Cys Ala His Cys Pro Trp Met Ala Met Asn Gly Leu
290 295 300
Gln Ala Ile Ala Glu Ala Leu Glu Gln Glu Gly Ser Asn His Glu Val
305 310 315 320
His Val Asp Glu Arg Leu Arg Glu Arg Ala Leu Val Pro Leu Asn Arg
325 330 335
Met Leu Asp Phe Ala Ala Thr Leu Arg Gly
340 345
<210> 78
<211> 368
<212> PRT
<213> Bacillus subtilis NadA (quinolate synthase)
<400> 78
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> 79
<211> 428
<212> PRT
<213> Corynebacterium glutamicum NadA (quinolate synthase)
<400> 79
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> 80
<211> 540
<212> PRT
<213> Escherichia coli NadB (L-aspartate oxidase)
<400> 80
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> 81
<211> 531
<212> PRT
<213> Bacillus subtilis NadB (L-aspartate oxidase)
<400> 81
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> 82
<211> 297
<212> PRT
<213> Escherichia coli NadC (quinolate phosphoribosyl transferase)
<400> 82
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> 83
<211> 289
<212> PRT
<213> Bacillus subtilis NadC (quinolate phosphoribosyl transferase)
<400> 83
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> 84
<211> 279
<212> PRT
<213> Corynebacterium glutamicum NadC (quinolate phosphoribosyltransferase)
<400> 84
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
<210> 85
<211> 20
<212> DNA
<213> Primer 10444
<400> 85
cggtaagtcc cgtctagcct 20
<210> 86
<211> 25
<212> DNA
<213> Primer 10447
<400> 86
atgtttgcaa aacgattcaa aacct 25
<210> 87
<211> 37
<212> DNA
<213> Primer 11222
<400> 87
ttacaccgaa tttctaataa taaccgggca ggccatg 37
<210> 88
<211> 37
<212> DNA
<213> Primer 11223
<400> 88
ggcctgcccg gttattatta gaaattcggt gtaagag 37
<210> 89
<211> 40
<212> DNA
<213> Primer 11226
<400> 89
cttttacacc gaatttttaa taataaccgg gcaggccatg 40
<210> 90
<211> 37
<212> DNA
<213> Primer 11227
<400> 90
ggcctgcccg gttattatta aaaattcggt gtaaaag 37
<210> 91
<211> 40
<212> DNA
<213> Primer 11230
<400> 91
cggatgaagc ggaatgttaa taataaccgg gcaggccatg 40
<210> 92
<211> 37
<212> DNA
<213> Primer 11231
<400> 92
ggcctgcccg gttattatta acattccgct tcatccg 37
<210> 93
<211> 38
<212> DNA
<213> Primer 11232
<400> 93
gaaaggtggt gaactactat gaaaacagca gcatacgc 38
<210> 94
<211> 40
<212> DNA
<213> Primer 11233
<400> 94
gcgtatgctg ctgttttcat agtagttcac cacctttctc 40
<210> 95
<211> 40
<212> DNA
<213> Primer 11234
<400> 95
cacttacacc gaacttctaa taataaccgg gcaggccatg 40
<210> 96
<211> 37
<212> DNA
<213> Primer 11235
<400> 96
ggcctgcccg gttattatta gaagttcggt gtaagtg 37
<210> 97
<211> 33
<212> DNA
<213> Primer 11341
<400> 97
aagggaggtt tcatatgaaa attgttaaag att 33
<210> 98
<211> 37
<212> DNA
<213> Primer 11342
<400> 98
tttaacaatt ttcatatgaa acctccctta attctcg 37
<210> 99
<211> 35
<212> DNA
<213> Primer 11351
<400> 99
gtgaactact atgaaaattg taaaaaactt tattg 35
<210> 100
<211> 35
<212> DNA
<213> Primer 11352
<400> 100
ttacaatttt catagtagtt caccaccttt ctcta 35
<210> 101
<211> 35
<212> DNA
<213> Primer 11353
<400> 101
aagggaggtt tcatatgaaa atcgttaaag acttc 35
<210> 102
<211> 35
<212> DNA
<213> Primer 11354
<400> 102
taacgatttt catatgaaac ctcccttaat tctcg 35
<210> 103
<211> 36
<212> DNA
<213> Primer 11159
<400> 103
gctacttact ctcgagttac tttttccaga aatcat 36
<210> 104
<211> 33
<212> DNA
<213> Primer 11160
<400> 104
gctaacttag catatgatga cattgcaaca aca 33

Claims (26)

1.能够生产烟酰胺核糖苷(NR)的经遗传修饰的细菌，其中所述细菌包含至少一种选自以下的修饰：
a)添加异源烟酸酰胺化蛋白(NadE*)的活性；和
b)添加或提高烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白的活性，
其中与没有任何所述修饰的细菌相比，具有所述至少一种修饰的细菌产生提高量的NR。
2.权利要求1所述的细菌，其中所述细菌还包含一种或多种另外的选自以下的修饰：
a)阻断或降低通过抑制nadA基因、nadB基因、nadC基因或其组合的转录起抑制NAD+生物合成的作用的蛋白质的活性；
b)阻断或降低作为烟酰胺核糖苷转运蛋白起作用的蛋白质的活性；
c)阻断或降低作为烟酸单核苷酸腺嘌呤基转移酶起作用的蛋白质的活性；
d)阻断或降低作为烟酰胺单核苷酸酰胺水解酶起作用的蛋白质的活性；
e)阻断或降低作为嘌呤核苷磷酸化酶起作用的蛋白质的活性；
f)添加或提高作为烟酰胺单核苷酸水解酶起作用的蛋白质的活性；和
g)添加或提高作为L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶起作用的蛋白质或其组合的活性。
3.权利要求1或权利要求2所述的细菌，其中所述NadE*蛋白是包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：1和3至18之任一的同一性为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少97％或至少99％，其中所述多肽具有将烟酸单核苷酸转变为烟酰胺单核苷酸的烟酸酰胺化活性。
4.权利要求1或权利要求2所述的细菌，其中所述烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白是包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：66至70之任一的同一性为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少97％或至少99％，其中所述多肽具有将NAD+转变为烟酰胺单核苷酸和腺嘌呤的NAD+水解活性。
5.权利要求1-4中任一项所述的细菌，其中NAD+生物合成的负调控因子是包含SEQ IDNO：51、52或53的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有抑制NAD+生物合成的活性。
6.权利要求1-4中任一项所述的细菌，其中所述烟酰胺核糖苷转运体是包含SEQ IDNO：54、55或56之任一的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有导入烟酰胺核糖苷的烟酰胺核糖苷转运活性。
7.权利要求1-4中任一项所述的细菌，其中所述烟酰胺单核苷水解酶是包含SEQ IDNO：57、58或59之任一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变为烟酰胺核糖苷的核苷水解酶活性。
8.权利要求1-4中任一项所述的细菌，其中所述烟酸单核苷酸腺嘌呤基转移酶蛋白是包含SEQ ID NO：63、64或65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变为烟酸腺嘌呤二核苷酸的烟酸单核苷酸腺嘌呤基转移酶活性。
9.权利要求1-4中任一项所述的细菌，其中所述烟酰胺单核苷酸酰胺水解酶蛋白是包含SEQ ID NO：60、61或62之任一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变为烟酸单核苷酸的烟酰胺单核苷酸酰胺水解酶活性。
10.权利要求1-4中任一项所述的细菌，其中所述嘌呤核苷磷酸化酶蛋白是包含SEQ IDNO：72至76之任一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核糖苷和磷酸转变为烟酰胺和核糖-1-磷酸的嘌呤核苷磷酸化酶活性。
11.权利要求1-4中任一项所述的细菌，其中所述喹啉酸合酶是包含SEQ ID NO：77、78或79的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有从亚氨基琥珀酸和磷酸二羟丙酮形成喹啉酸的活性，或者所述L-天冬氨酸氧化酶是包含SEQ ID NO：80或81的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有从天冬氨酸形成亚氨基琥珀酸的活性，或者所述喹啉酸磷酸核糖基转移酶是包含SEQ ID NO：82、83或84的氨基酸序列的多肽或所述多肽的变体，其中所述多肽具有从喹啉酸和磷酸核糖基焦磷酸形成烟酸单核苷酸的活性。
12.权利要求1-4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。
13.权利要求3-12中任一项所述的细菌，其中当与SEQ ID NO：1的参照氨基酸序列比较时，所述NadE*蛋白具有一个或多个以下保守氨基酸：a)第27位的酪氨酸，b)第133位的谷氨酰胺，和c)第236位的精氨酸，其基于ClustalW比对方法与SEQ ID NOS：1和3至18进行比较，使用缺省参数空位罚分＝10，空位长度罚分＝0.1和Gonnet 250系列蛋白质权重矩阵。
14.一种生产NR的方法，所述方法包括：在有效产生NR的条件下培养细菌细胞并从培养基中回收NR，从而生产NR，其中宿主微生物包含至少一种选自以下的修饰：
a)添加异源烟酸酰胺化蛋白(NadE*)的活性；
b)添加或提高烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白的活性；
c)阻断或降低通过抑制nadA基因、nadB基因、nadC基因或其组合的转录起抑制NAD+生物合成的作用的蛋白的活性；
d)阻断或降低作为烟酰胺核糖苷转运蛋白起作用的蛋白质的活性；
e)阻断或降低作为烟酸单核苷酸腺嘌呤基转移酶起作用的蛋白质的活性；
f)阻断或降低作为烟酰胺单核苷酸酰胺水解酶起作用的蛋白质的活性；
g)阻断或降低作为嘌呤核苷磷酸化酶起作用的蛋白质的活性；
h)添加或提高作为烟酰胺单核苷酸水解酶起作用的蛋白质的活性；和
i)添加或提高作为L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶起作用的蛋白质或其组合的活性。
15.一种生产NR的方法，所述方法包括：在有效产生NR的条件下培养细菌细胞并从培养基中回收NR，从而生产NR，其中宿主微生物包含至少一种选自以下的修饰：
a)添加异源烟酸酰胺化蛋白(NadE*)的活性；和
b)添加或提高烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白的活性。
16.权利要求15所述的方法，其中所述细菌细胞还包含至少一种选自以下的修饰：
a)阻断或降低通过抑制nadA基因、nadB基因、nadC基因或其组合的转录起抑制NAD+生物合成的作用的蛋白的活性；
b)阻断或降低作为烟酰胺核糖苷转运蛋白起作用的蛋白质的活性；
c)阻断或降低作为烟酸单核苷酸腺嘌呤基转移酶起作用的蛋白质的活性；
d)阻断或降低作为烟酰胺单核苷酸酰胺水解酶起作用的蛋白质的活性；
e)阻断或降低作为嘌呤核苷磷酸化酶起作用的蛋白质的活性；
f)添加或提高作为烟酰胺单核苷酸水解酶起作用的蛋白质的活性；和
g)添加或提高作为L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶起作用的蛋白质或其组合的活性。
17.从权利要求1至12中任一项所述的经遗传修饰的细菌获得的烟酰胺核糖苷化合物。
18.一种组合物，其包含从权利要求1至12中任一项所述的经遗传修饰的细菌获得的烟酰胺核糖苷化合物。
19.一种食品或饲料，其包含从权利要求1至12中任一项所述的经遗传修饰的细菌获得的烟酰胺核糖苷化合物。
20.一种经遗传修饰的细菌，其特征在于：由于遗传修饰，所述细菌产生NR并且能够在所述细菌生长的发酵液中积累产生的NR至至少100mg/L。
21.权利要求20所述的经遗传修饰的细菌，其中所述遗传修饰选自：
a)添加异源烟酸酰胺化蛋白(NadE*)的活性；和
b)添加或提高烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白的活性。
22.权利要求21所述的经遗传修饰的细菌，其中所述遗传修饰还包含一种或多种另外的选自以下的修饰：
a)阻断或降低通过抑制nadA基因、nadB基因、nadC基因或其组合的转录起抑制NAD+生物合成的作用的蛋白的活性；
b)阻断或降低作为烟酰胺核糖苷转运蛋白起作用的蛋白质的活性；
c)阻断或降低作为烟酸单核苷酸腺嘌呤基转移酶起作用的蛋白质的活性；
d)阻断或降低作为烟酰胺单核苷酸酰胺水解酶起作用的蛋白质的活性；
e)阻断或降低作为嘌呤核苷磷酸化酶起作用的蛋白质的活性；
f)添加或提高作为烟酰胺单核苷酸水解酶起作用的蛋白质的活性；和
g)添加或提高作为L-天冬氨酸氧化酶、喹啉酸合酶、喹啉酸磷酸核糖基转移酶起作用的蛋白质或其组合的活性。
23.权利要求22所述的经遗传修饰的细菌，其中所述NadE*蛋白是包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：1和3至18之任一的同一性为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少97％或至少99％，其中所述多肽具有将烟酸单核苷酸转变为烟酰胺单核苷酸的烟酸酰胺化活性。
24.权利要求23所述的细菌，其中当与SEQ ID NO：1的参照氨基酸序列比较时，所述NadE*蛋白具有一个或多个以下保守氨基酸：a)第27位的酪氨酸，b)第133位的谷氨酰胺，和c)第236位的精氨酸，其基于ClustalW比对方法与SEQ ID NOS：1和3至18进行比较，使用缺省参数空位罚分＝10，空位长度罚分＝0.1和Gonnet 250系列蛋白质权重矩阵。
25.权利要求20-24中任一项所述的细菌，其中所述烟酰胺腺嘌呤二核苷酸(NAD+)水解蛋白是包含以下氨基酸序列的多肽，所述氨基酸序列与SEQ ID NO：66至70之任一的同一性为至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少97％或至少99％，其中所述多肽具有将NAD+转变为烟酰胺单核苷酸和腺嘌呤的NAD+水解活性。
26.权利要求1-13和20-25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。
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112359082A (zh) * 2020-11-13 2021-02-12 南京工业大学 一种烟酰胺单核苷酸的制备方法
CN112795606A (zh) * 2021-04-14 2021-05-14 深圳瑞德林生物技术有限公司 一种β-烟酰胺单核苷酸的酶催化合成方法
CN112795582A (zh) * 2021-01-22 2021-05-14 江南大学 一种适合在微生物中高效合成nad衍生物的酶基因
CN113528562A (zh) * 2021-06-23 2021-10-22 苏州华赛生物工程技术有限公司 生产β-烟酰胺核糖的重组微生物及其构建方法和应用
CN113755415A (zh) * 2020-06-04 2021-12-07 苏州华赛生物工程技术有限公司 一种新的具有nmn合成路径的重组微生物及生产方法
CN113897382A (zh) * 2021-09-01 2022-01-07 浙江工业大学 辅酶自足型大肠杆菌及其构建方法与应用
CN114231477A (zh) * 2021-12-27 2022-03-25 天津科技大学 高产β-烟酰胺单核苷酸的基因工程菌株及其构建与应用
CN114854656A (zh) * 2022-05-05 2022-08-05 江南大学 一种产烟酰胺核糖苷的重组菌

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3374492A4 (en) * 2015-11-13 2019-07-24 DSM IP Assets B.V. MICROBIAL PRODUCTION OF NICOTINAMIDE RIBOSIDE
US20200255877A1 (en) * 2017-05-18 2020-08-13 Dsm Ip Assets B.V. Microbial production of nicotinamide riboside
CN117887788A (zh) 2017-09-29 2024-04-16 三菱化学株式会社 烟酰胺单核苷酸的制造方法
WO2020094828A1 (en) * 2018-11-09 2020-05-14 Wageningen Universiteit Microbial hosts engineered for increased tolerance to temperature shifts
US20220340945A1 (en) * 2019-10-11 2022-10-27 National Uninversity Corporation Shizuoka University Lactic acid bacteria that produce nicotinamide riboside, and lactic acid bacteria that produce nicotinamide mononucleotide and nicotinamide riboside
CN112501193B (zh) * 2020-12-09 2022-09-27 江南大学 一种烟酸及烟酰胺生物传感系统
CN112852678B (zh) * 2021-03-08 2021-11-05 泓博元生命科技（深圳）有限公司 一株产烟酰胺单核苷酸的成都肠杆菌及其应用
JP7111878B1 (ja) 2021-10-27 2022-08-02 旭化成ファーマ株式会社 ニコチンアミドモノヌクレオチドの製造方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090202680A1 (en) * 2004-02-10 2009-08-13 Charles Brenner Yeast strain and method for using the same to produce nicotinamide riboside
CN102154339A (zh) * 2011-02-16 2011-08-17 南京工业大学 一种产丁二酸大肠杆菌基因工程菌株的构建方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015069860A1 (en) * 2013-11-06 2015-05-14 President And Fellows Of Harvard College Biological production of nad precursors and analogs
EP3374492A4 (en) * 2015-11-13 2019-07-24 DSM IP Assets B.V. MICROBIAL PRODUCTION OF NICOTINAMIDE RIBOSIDE

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090202680A1 (en) * 2004-02-10 2009-08-13 Charles Brenner Yeast strain and method for using the same to produce nicotinamide riboside
CN102154339A (zh) * 2011-02-16 2011-08-17 南京工业大学 一种产丁二酸大肠杆菌基因工程菌株的构建方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GAZZANIGA F等: "Microbial NAD metabolism: lessons from comparative genomics", 《MICROBIOL MOL BIOL REV》 *
SORCI L等: "Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis", 《PROC NATL ACAD SCI U S A》 *
WANG L等: "Identification of UshA as a major enzyme for NAD degradation in Escherichia coli", 《ENZYME MICROB TECHNOL》 *
丁文成: "烟酸及异烟酸衍生物的合成及其缓蚀性能的研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113755415A (zh) * 2020-06-04 2021-12-07 苏州华赛生物工程技术有限公司 一种新的具有nmn合成路径的重组微生物及生产方法
CN113755415B (zh) * 2020-06-04 2024-03-29 苏州华赛生物工程技术有限公司 一种新的具有nmn合成路径的重组微生物及生产方法
CN112359082A (zh) * 2020-11-13 2021-02-12 南京工业大学 一种烟酰胺单核苷酸的制备方法
CN112359082B (zh) * 2020-11-13 2023-02-21 南京工业大学 一种烟酰胺单核苷酸的制备方法
CN112795582B (zh) * 2021-01-22 2022-12-13 江南大学 一种适合在微生物中高效合成nad衍生物的酶基因
CN112795582A (zh) * 2021-01-22 2021-05-14 江南大学 一种适合在微生物中高效合成nad衍生物的酶基因
CN112795606A (zh) * 2021-04-14 2021-05-14 深圳瑞德林生物技术有限公司 一种β-烟酰胺单核苷酸的酶催化合成方法
CN113528562A (zh) * 2021-06-23 2021-10-22 苏州华赛生物工程技术有限公司 生产β-烟酰胺核糖的重组微生物及其构建方法和应用
CN113897382B (zh) * 2021-09-01 2023-10-20 浙江工业大学 辅酶自足型大肠杆菌及其构建方法与应用
CN113897382A (zh) * 2021-09-01 2022-01-07 浙江工业大学 辅酶自足型大肠杆菌及其构建方法与应用
CN114231477A (zh) * 2021-12-27 2022-03-25 天津科技大学 高产β-烟酰胺单核苷酸的基因工程菌株及其构建与应用
CN114231477B (zh) * 2021-12-27 2024-03-15 天津科技大学 高产β-烟酰胺单核苷酸的基因工程菌株及其构建与应用
CN114854656A (zh) * 2022-05-05 2022-08-05 江南大学 一种产烟酰胺核糖苷的重组菌
CN114854656B (zh) * 2022-05-05 2023-07-18 江南大学 一种产烟酰胺核糖苷的重组菌

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