烟酰胺核糖苷的微生物生产的制作方法

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。本公开的一些实施方式涉及用于生产烟酰胺核糖苷(nicotinamideriboside)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，ec2.5.1.72)(flachmann,1988,europeanjournalofbiochemistry,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,biologicalchemistryhoppe-seyler,371(1),239-248)。已知esherichiacoli中的酶被下游产物nad+抑制(nasus,1982,jbiolchem,257(2),626-32)，但已经生成了反馈抗性突变体(hughes,1983,jbacteriol,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，ec2.4.2.19)；烟酸单核苷酸腺嘌呤基转移酶(nadd，ec2.7.7.18)；和nad+合成酶(nade，ec6.3.1.5)。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bacillussubtilis的酶对namn的vmax/km为其对nmn的vmax/km的104倍(olland,2002,jbiolchem,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bacillusanthracis中，vmax/km的差异>103倍(sorci,2009,jbiolchem,277(5),3698-3707)。与上述典型途径形成对比，francisellatularensis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,microbiolmolbiolrev,73(3),529-541)。nmn通过烟酰胺核苷酸酰胺酶(e.colipncc,b.subtiliscina,ec3.5.1.42)的作用被再循环为namn；nr通过烟酰胺核糖苷激酶(e.colinadr,ec2.7.1.22)被磷酸化成nmn或在可逆反应中通过嘌呤核苷磷酸化酶(e.colideod,b.subtilisdeod,pupg,pdp,ec2.4.2.1)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，ec3.5.1.19)被脱酰胺为na；na或nam通过烟酸磷酸核糖基转移酶(e.colipncb,b.subtilisyuekec6.3.4.21)分别被转变为namn或nmn。胞外nmn通过周质酸性磷酸酶(e.coliusha,b.subtilisyfkn,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.colipnuc,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,jbacteriol,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bacillussubtilis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,jbacteriol,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。技术实现要素：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seqidno：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seqidno：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seqidnos：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gonnet250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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidnos：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gonnet250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seqidno：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：seqidno：1是编码francisellatularensisnade*酶(ftnade*)的氨基酸序列，ftnade*是一种烟酸酰胺化蛋白。seqidno：2是编码francisellatularensisnade*酶(ftnade*)的开放阅读框的核苷酸序列。seqidno：3是编码francisellasp.fsc1006nade*酶(fspfnade*)的氨基酸序列，fspfnade*是一种烟酸酰胺化蛋白。seqidno：4是编码francisellaguangzhouensisnade*酶(fgnade*)的氨基酸序列，fgnade*是一种烟酸酰胺化蛋白。seqidno：5是编码francisellasp.tx077308nade*酶(fsptnade*)的氨基酸序列，fsptnade*是一种烟酸酰胺化蛋白。seqidno：6是编码francisellaphilomiragiasubsp.philomiragiaatcc25017nade*酶(fphnade*)的氨基酸序列，fphnade*是一种烟酸酰胺化蛋白。seqidno：7是编码francisellaphilomiragia菌株o#319-036[fsc153]nade*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。seqidno：8是编码francisellanoatunensissupbsp.orientalisstr.toba04nade*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。seqidno：9是编码francisellaphilomiragia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。seqidno：10是编码francisellapersicaatccvr-331nade*酶(fpenade*)的氨基酸序列，fpenade*是一种烟酸酰胺化蛋白。seqidno：11是编码francisellacf.novicida3523nade*酶(fnnade*)的氨基酸序列，fnnade*是一种烟酸酰胺化蛋白。seqidno：12是编码francisellatularensissubsp.novicidad9876nade*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。seqidno：13是编码francisellatularensissubsp.novicidaf6168nade*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。seqidno：14是编码francisellatularensissubsp.tularensis菌株nihb-38nade*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。seqidno：15是编码francisellatularensissubsp.holarcticaf92nade*酶的氨基酸序列，所述酶是一种预测的烟酸酰胺化蛋白。seqidno：16是编码dichelobacternodosusvcs1703anade*酶(dnnade*)的氨基酸序列，dnnade*是一种烟酸酰胺化蛋白。seqidno：17是编码mannheimiasuccinoproducensmbel55enade*酶(mnnade*)的氨基酸序列，mnnade*是一种烟酸酰胺化蛋白。seqidno：18是编码actinobacillussuccinogenesnade*酶(asnade*)的氨基酸序列，asnade*是一种烟酸酰胺化蛋白。seqidno：19是编码mannheimiasuccinoproducensmbel55enade*酶(mnnade*)开放阅读框的核苷酸序列。seqidno：20是编码dichelobacternodosusvcs1703anade*酶(dnnade*)开放阅读框的核苷酸序列。seqidno：21是编码actinobacillussuccinogenesnade*酶(asnade*)开放阅读框的核苷酸序列。seqidno：22是编码francisellaphilomiragiasubsp.philomiragiaatcc25017nade*酶(fphnade*)开放阅读框的核苷酸序列。seqidno：23是编码francisellacf.novicida3523nade*酶(fnnade*)开放阅读框的核苷酸序列。seqidno：24是编码francisellasp.tx077308nade*酶(fsptnade*)开放阅读框的核苷酸序列。seqidno：25是编码francisellasp.fsc1006nade*酶(fspfnade*)开放阅读框的核苷酸序列。seqidno：26是编码francisellaguangzhouensisnade*酶(fgnade*)开放阅读框的核苷酸序列。seqidno：27是编码francisellapersicaatccvr-331nade*酶(fpenade*)开放阅读框的核苷酸序列。seqidno：28是针对在e.coli中表达优化的编码mannheimiasuccinoproducensmbel55enade*酶(mnnade*)开放阅读框的核苷酸序列。seqidno：29是针对在e.coli中表达优化的编码dichelobacternodosusvcs1703anade*酶(dnnade*)开放阅读框的核苷酸序列。seqidno：30是针对在e.coli中表达优化的编码actinobacillussuccinogenesnade*酶(asnade*)开放阅读框的核苷酸序列。seqidno：31是针对在e.coli中表达优化的编码francisellaphilomiragiasubsp.philomiragiaatcc25017nade*酶(fphnade*)开放阅读框的核苷酸序列。seqidno：32是针对在e.coli中表达优化的编码francisellacf.novicida3523nade*酶(fnnade*)开放阅读框的核苷酸序列。seqidno：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。seqidno：34是针对在e.coli中表达优化的编码francisellasp.fsc1006nade*酶(fspfnade*)开放阅读框的核苷酸序列。seqidno：35是针对在e.coli中表达优化的编码francisellaguangzhouensisnade*酶(fgnade*)开放阅读框的核苷酸序列。seqidno：36是针对在e.coli中表达优化的编码francisellapersicaatccvr-331nade*酶(fpenade*)开放阅读框的核苷酸序列。seqidno：37是针对在e.coli中表达优化的编码francisellatularensisnade*酶(ftnade*)开放阅读框的核苷酸序列。seqidno：38是针对在b.subtilis中表达优化的编码francisellatularensisnade*酶(ftnade*)开放阅读框的核苷酸序列。seqidno：39是针对在b.subtilis中表达优化的编码mannheimiasuccinoproducensmbel55enade*酶(mnnade*)开放阅读框的核苷酸序列。seqidno：40是针对在b.subtilis中表达优化的编码francisellacf.novicida3523nade*酶(fnnade*)开放阅读框的核苷酸序列。seqidno：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。seqidno：42是针对在e.coli中表达优化的编码francisellatularensisnade*酶(ftnade*)开放阅读框并且编码y27t、q133g和r236v突变的核苷酸序列。seqidno：43是针对在e.coli中表达优化的编码mannheimiasuccinoproducensmbel55enade*酶(mnnade*)开放阅读框并且编码y22t、q128g和r231v突变的核苷酸序列。seqidno：44是针对在e.coli中表达优化的编码francisellacf.novicida3523nade*酶(fnnade*)开放阅读框并且编码y27t、q133g和r236v突变的核苷酸序列。seqidno：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。seqidno：46是编码e.colinade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。seqidno：47是针对在c.glutamicum中表达优化的编码francisellacf.novicida3523nade*酶(fnnade*)开放阅读框的核苷酸序列。seqidno：48是编码四环素抗性的核苷酸序列。seqidno：49是编码新霉素抗性的核苷酸序列。seqidno：50是编码壮观霉素抗性的核苷酸序列。seqidno：51是编码escherichiacolinadr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。seqidno：52是编码bacillussubtilisnadr(又名yxra)酶的氨基酸序列，所述酶是阻遏蛋白。seqidno：53是编码corynebacteriumglutamicumnadr(又名cgr_1153)酶的氨基酸序列，所述酶是阻遏蛋白。seqidno：54是编码acinetobacterbaylyipnuc酶的氨基酸序列，所述酶是nr转运蛋白。seqidno：55是编码corynebacteriumglutamicumpnuc酶的氨基酸序列，所述酶是nr转运蛋白。seqidno：56是编码escherichiacolipnuc酶的氨基酸序列，所述酶是nr转运蛋白。seqidno：57是编码escherichiacoliusha酶的氨基酸序列，所述酶是烟酰胺单核苷酸水解酶。seqidno：58是编码bacillussubtilisusha(又名yfkn)酶的氨基酸序列，所述酶是烟酰胺单核苷酸水解酶。seqidno：59是编码corynebacteriumglutamicumusha(又名cg0397)酶的氨基酸序列，所述酶是烟酰胺单核苷酸水解酶。seqidno：60是编码escherichiacolipncc酶的氨基酸序列，所述酶是烟酰胺单核苷酸酰胺水解酶。seqidno：61是编码bacillussubtilispncc(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。seqidno：62是编码corynebacteriumglutamicumpncc(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。seqidno：63是编码escherichiacolinadd酶的氨基酸序列，所述酶是烟酸单核苷酸腺嘌呤基转移酶。seqidno：64是编码bacillussubtilisnadd酶的氨基酸序列，所述酶是烟酸单核苷酸腺嘌呤基转移酶。seqidno：65是编码corynebacteriumglutamicumnadd(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。seqidno：66是编码acinetobacternudc酶的氨基酸序列，所述酶是nad+二磷酸酶。seqidno：67是编码escherichiacolinudc酶的氨基酸序列，所述酶是nad+二磷酸酶。seqidno：68是编码corynebacteriumglutamicumnudc(又名cg0888)酶的氨基酸序列，所述酶是nad+二磷酸酶。seqidno：69是编码burkholderiaceaenudc酶的氨基酸序列，所述酶是nad+二磷酸酶。seqidno：70是编码haemophilusinfluenzaenudc酶的氨基酸序列，所述酶是nad+二磷酸酶。seqidno：71是编码b.subtilisnupg蛋白的氨基酸序列，所述蛋白是nr转运蛋白。seqidno：72是编码b.subtilisdeod酶的氨基酸序列，所述酶是核苷磷酸化酶。seqidno：73是编码b.subtilispdp酶的氨基酸序列，所述酶是核苷磷酸化酶。seqidno：74是编码b.subtilispupg酶的氨基酸序列，所述酶是核苷磷酸化酶。seqidno：75是编码e.colideod酶的氨基酸序列，所述酶是核苷磷酸化酶。seqidno：76是编码c.glutamicumg18ng酶的氨基酸序列，所述酶是核苷磷酸化酶。seqidno：77是编码escherichiacolinada酶的氨基酸序列，所述酶是喹啉酸合酶。seqidno：78是编码bacillussubtilisnada酶的氨基酸序列，所述酶是喹啉酸合酶。seqidno：79是编码corynebacteriumglutamicumnada酶的氨基酸序列，所述酶是喹啉酸合酶。seqidno：80是编码escherichiacolinadb酶的氨基酸序列，所述酶是l-天冬氨酸氧化酶。seqidno：81是编码bacillussubtilisnadb酶的氨基酸序列，所述酶是l-天冬氨酸氧化酶。seqidno：82是编码escherichiacolinadc酶的氨基酸序列，所述酶是喹啉酸磷酸核糖基转移酶。seqidno：83是编码bacillussubtilisnadc酶的氨基酸序列，所述酶是喹啉酸磷酸核糖基转移酶。seqidno：84是编码corynebacteriumglutamicumnadc酶的氨基酸序列，所述酶是喹啉酸磷酸核糖基转移酶。seqidno：85：是引物10444。seqidno：86：是引物10447。seqidno：87：是引物11222。seqidno：88：是引物11223。seqidno：89：是引物11226。seqidno：90：是引物11227。seqidno：91：是引物11230。seqidno：92：是引物11231。seqidno：93：是引物11232。seqidno：94：是引物11233。seqidno：95：是引物11234。seqidno：96：是引物11235。seqidno：97：是引物11341。seqidno：98：是引物11342。seqidno：99：是引物11351。seqidno：100：是引物11352。seqidno：101：是引物11353。seqidno：102：是引物11354。seqidno：103：是引物11159。seqidno：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(uniquesequences)的邻接共同树。图7示出了细菌的根源系统发生树(bernm.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seqidno：1和3至18的多肽。seqidno：1来源于francisellatularensis，被称为ftnade*。ftnade*蛋白序列在genbank登录号yp_170217下提供。seqidno：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。seqidno：4来源于francisellaguangzhouensis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。seqidno：5来源于francisellasp.tx077308，被称为fsptnade*；蛋白质登录号可以在登录号wp_013922810.1下获得。seqidno：6来源于francisellaphilomiragiasubsp.philomiragiaatcc25017，被称为fphnade*；蛋白质登录号可以在登录号wp_004287429.1下获得。seqidno：7来源于francisellaphilomiragia菌株o#319-036[fsc153]，被称为nade*；蛋白质登录号可以在登录号wp_042517896.1下获得。seqidno：8来源于francisellanoatunensissubsp.orientalisstr.toba04，被称为nade*；蛋白质登录号可以在登录号wp_014714556.1下获得。seqidno：9来源于francisellaphilomiragia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。seqidno：10来源于francisellapersicaatccvr-331，被称为fpenade*；蛋白质登录号可以在登录号wp_064461307.1下获得。seqidno：11来源于francisellacf.novicida3523，被称为fnnade*；蛋白质登录号可以在登录号wp_014548640.1下获得。seqidno：12来源于francisellatularensissubsp.novicidad9876，被称为nade*；蛋白质登录号可以在登录号wp_003037081.1下获得。seqidno：13来源于francisellatularensissubsp.novicidaf6168，被称为nade*；蛋白质登录号可以在登录号wp_003034444.1下获得。seqidno：14来源于francisellatularensissubsp.tularensis菌株nihb-38，被称为nade*；蛋白质登录号可以在登录号wp_003025712.1下获得。seqidno：15来源于francisellatularensissubsp.holarcticaf92，被称为nade*；蛋白质登录号可以在登录号wp_010032811.1下获得。seqidno：16来源于dichelobacternodosusvcs1703a，被称为dnnade*；蛋白质登录号可以在登录号wp_011927945.1下获得。seqidno：17来源于mannheimiasucciniciproducensmbel55e，被称为msnade*；蛋白质登录号可以在登录号wp_011201048.1下获得。seqidno：18来源于actinobacillussuccinogenes130z，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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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:theeuropeanmolecularbiologyopensoftwaresuite,rice等人,2000,trendsgenet.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x100)/(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francisellatularensis经由替代途径合成nad+，其中nmn是通过f.tularensisnmn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seqidno：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seqidno：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seqidno：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seqidno：1编号。参见图5中的seqidno：1和3至18的clustalw比对。因此，在一些实施方式中，当与seqidno：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seqidnos：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gonnet250系列蛋白质权重矩阵。seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：18或seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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seqidno：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,molecularcloning:alaboratorymanual,coldspringharborlabspress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,corynebacteriumglutamicum,escherichiacoli,acinetobacterbaylyi和ralstoniaeutropha。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，seqidno：1和3至18的功能性变体分别与seqidno：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seqidno：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seqidno：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seqidno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(internationalunionofbiochemistryandmolecularbiology)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、candidaglabrata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francisellatularensis的基因组编码的酶ftnade*(seqidno：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(sorcil.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(seqidno：2)、从2016年9月14日的ncbinr/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(seqidno：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(seqidno：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.colik-12密码子使用表，稀有阈值设定为0.4。优化的序列(seqidno28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.molecularcloning:alaboratorymanual.coldspringharborlaboratorypress:newyork；和f.m.ausubel,r.brent,r.e.kingston,d.d.moore,j.g.seidman,j.a.smith,k.struhl(编).1998.currentprotocolsinmolecularbiology.wiley:newyork)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25μmiptg的m9nc培养基，并在用airpore胶带片(qiagen)密封的24孔深孔板(whatmanuniplate，10ml，圆底)中生长3天(inforsmultitron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25μmiptg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iptg25umiptgme407ftnade*<loq0.11me409无<loq<loqme644dnnade*<loq0.28me645asnade*0.080.31me646fphnade*0.02<loqme647fnnade*<loq1.91me648fsptnade*0.031.29me649fspfnade*<loq0.82me650fgnade*<loq0.64me651fpenade*0.141.09me652mnnade*0.12.73实施例4e.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francisellatularensisy27、q133、r236残基对齐的残基并用在bacillusanthracisnade中编码的氨基酸残基置换(分别为t、g和v；seqidno：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5e.colinade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(seqidno：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.colinade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、corynebacteriumglutamicum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2mlm9nc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(rochehighpurepcr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，seqidno：48至50)被并入pcr产物中(phusionhotstartflexdna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(phusionhotstartflexdna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(“molecularbiologicalmethodsforbacillus”.1990.crharwood和s.m.cutting编.johnwileyandsons)，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::spepupg::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::spepupg::neodeod::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.subtilis168密码子使用表，稀有阈值设定为0.4)，优化的序列(seqidno：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(bacillusgeneticstockcenter)和gblocks作为模板，如上所述扩增含有amye5’区域、cat(氯霉素抗性)、pvegi启动子的侧翼区和含有amye3’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1mlbhi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2mlm9nc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53.2me517无42.2me517无38.8me517无37.6me795msnade*72.3me795msnade*75.5me814fnnade*102.9me814fnnade*98.0me820fspnade*99.1me820fspnade*94.8me824ftnade*76.8me824ftnade*79.9实施例8表达nade*的corynebacteriumglutamicum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(seqidno：47)由idt合成为gblock，所述gblock含有在编码ecori限制位点和corynebacteriumglutamicum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2mlvy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2mlaz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，corynebacteriumglutamicum摇板培养物中的烟酰胺核糖苷浓度(mg/l)菌株0mmiptg0.25mmiptgatcc130320.020.03me7630.030.16me7630.010.11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900μlms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hilicuplc柱(watersbeh酰胺，2.1x75mmp/n1860005657)上。保留1分钟后，使用从99.9％(ph9.0的10mm乙酸铵，95％乙腈/5％水)流动相d至70％(ph9.0的10mm乙酸铵，50/50的乙腈/水)流动相c的线性梯度，以400ulmin-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)、5gbacto酵母提取物(difco)。1升m9nc培养基含有50g葡萄糖、6gna2hpo4、3gkh2po4、0.5gnacl、1gnh4cl、2mmmgso4、15mgna2edta、4.5mgznso4*7h2o、0.3mgcocl2*6h2o、1mgmncl2*4h2o、4.5mgcacl2*2h2o、0.4mgna2moo4*2h2o、1mgh3bo3和0.1mgki。1升az培养基含有20g葡萄糖、2gnacl、3g柠檬酸钠、0.1gcacl2*2h2o、4gk2hpo4、2gkh2po4、7.5gnh4so4、3.75g尿素、0.5gmgso4*7h2o、450μg硫胺素、450μg生物素、4mg泛酸盐/酯、15mgna2edta、4.5mgznso4*7h2o、0.3mgcocl2*6h2o、1mgmncl2*4h2o、4.5mgcacl2*2h2o、0.4mgna2moo4*2h2o、1mgh3bo3和0.1mgki。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>patentinversion3.5<210>1<211>249<212>prt<213>francisellatularensis<400>1metlysilevallysasppheserprolysglutyrserglnlysleu151015valasntrpleuseraspsercysmetasntyrproalagluglyphe202530valileglyleuserglyglyileaspseralavalalaalaserleu354045alavallysthrglyleuprothrthralaleuileleuproserasp505560asnasnglnhisglnaspmetglnaspalaleugluleuileglumet65707580leuasnilegluhistyrthrileserileglnproalatyrgluala859095pheleualaserthrglnserphethrasnleuglnasnasnarggln100105110leuvalilelysglyasnalaglnalaargleuargmetmettyrleu115120125tyralatyralaglnglntyrasnargilevalileglythraspasn130135140alacysglutrptyrmetglytyrphethrlyspheglyaspglyala145150155160alaaspileleuproleuvalasnleulyslysserglnvalpheglu165170175leuglylystyrleuaspvalprolysasnileleuasplysalapro180185190seralaglyleutrpglnglyglnthraspgluaspglumetglyval195200205thrtyrglngluileaspasppheleuaspglylysglnvalserala210215220lysalaleugluargileasnphetrphisasnargserhishislys225230235240arglysleualaleuthrproasnphe245<210>2<211>750<212>dna<213>francisellatularensis<400>2atgaaaatagttaaagattttagtcctaaagaatattcacaaaagttagttaattggcta60agtgatagttgtatgaattatcctgctgaaggatttgtgattggtcttagtggcggtata120gattcagcagttgcggcttctttagctgtcaaaactggattaccaactacagctttaata180ctaccttcagataataatcaacaccaagatatgcaagatgctctagagcttattgaaatg240cttaatattgaacattataccatttcgattcaaccagcttatgaggcttttcttgcttca300acgcaaagctttacaaatctacaaaacaatagacaacttgtgatcaagggaaatgctcaa360gcacgtttaaggatgatgtatttgtatgcctatgcgcaacaatataacagaatagttata420ggtactgataatgcttgtgagtggtatatgggatattttacaaaattcggtgatggggct480gccgatatacttccactagttaatctcaaaaaatctcaagtttttgaattaggcaaatac540ctagatgtccctaaaaacatacttgataaagctccatctgcaggactatggcaaggacaa600actgatgaggatgaaatgggtgtaacttatcaagaaattgatgatttcttagatggtaaa660caagtttcagcaaaagctctagaaagaataaatttctggcataatcgtagtcaccataag720agaaaattagctttaactcctaatttctag750<210>3<211>249<212>prt<213>francisellasp.fsc1006<400>3metservalvallysasnphelysproasnglutyralaasnlysile151015thrglutrpleulysaspsercysleuasntyrproalagluglyphe202530valvalglyileserglyglyileaspseralavalalavalserleu354045alavalasnthrglyleuprovalthrglyleuilemetproserlys505560asnasnaspasplysaspthrleuaspalailegluleualalyslys65707580leuasnileglutyrhisleuileproileglnprovaltyrgluthr859095pheleuaspseralagluaspilelysasnseralaasnasparggln100105110hisvalilelysglyasnalaglnalaargpheargmetiletyrleu115120125tyralatyralaglnglnasnasnargmetvalileglythraspasn130135140alacysglutrptyrmetglytyr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ccaaattcggcgacggcgcagcggacgtactgcct480ttagcgggattgcgcaaagagcaggtatttgaactcggacgttatcttggcgtaccgcaa540agcgtgctggataaaaaaccgagcgccggtttatgggcaggacaaacggacgaagctgaa600atgggtgttacttatgcggaaatcgacgcttatctgcgcggcgaaaccgttagcccgcag660gcattgcaacaaatccgtttctggcacaaccgttctcatcacaaacgtatgttgccacct720aaaccgaaatcacccgatgaagcggagtgttaa753<210>20<211>747<212>dna<213>dichelobacternodosusvcs1703a<400>20atgaccgttcatcaatacatcgattatttactcgtgtggttagaagagcagcgcgctcat60ctttatgcatcagatggttatacgttgggcgtcagcggcggcatcgattccgccgtttgt120ctgcatttactcgccaaaacgggaaaacccgtgcaagcgttagttttgccgatcaatgcg180aacgcgaacgattgtgaagatgccgaattagtgttaaaaaatgctaatatttccggcaat240attatcgcgctcgatgatgtttataccgccgcacaaaacaccttggcgcctgttttaaat300cgcgattatgaacgtatgcccgtattaaacggcaatttaatggcgcggctgcgtatggtt360atgctttataccgtggcgcaaagtcatcgttcggtggtcgtgggaacggataacgcggtg420gaatattatttaggttactttacaaaatttggcgacggcgcctgcgatattttgccgctg480gcaaaactgacaaaatcagaagtaggacaattggcaaaagcgttaggcgttccgaaaaaa540atccgagaaaaagcgccgagcgcaggcttgtggcaagggcaaaccgatgaaaacgaaatc600ggcgtatcgtacgcggatttagatgcttttttgtgcggtaaaaccgttgatgatgccgtc660agagaaaaaattgcttattggcatcaacgctcgcatcataaaagaatgttgccgccgatg720ccggaaatcggattatctttggcgtaa747<210>21<211>750<212>dna<213>actinobacillussuccinogenes130z<400>21atgagaacggcagcatacgtagattatttagtgcgatggctggaaacccagcgtaccgaa60ttatacggtatggacggctacacgctgggggtcagcggcggtatcgacagtgccgtttgc120gcccatttagcggcacgcaccggcgcccccgtacaggcattgattttacccgccgaagtc180accagccctgaagatgtggcggatgctcagattaccttggaaagtgcaggtattgacggg240cggattatttctatcgctccttggtacgatttaattatgctacaacttacccccgcatta300aatgcggaatctgaacgcattaacgtattgaaaggtaacttaatggcgcgcttacgtatg360atcgcattatttaccacggcgcaaagccaccgttctatcgtattgggtacggataacgcc420gccgaaatgttaacgggctatttcaccaaattcggcgacggtgcggcggacgtattgccg480ttagcgaggttgcgcaaagaacaggtattcgaattagggcgttatcttggcgtaccgaaa540tccgtgctggagaaaaaaccgagtgcgggcttatgggcggggcaaacggacgagggggaa600atgggtgtcagttatgcggaaatcgacgcctatctgcgcggcgaaaccgtcagtccgcag660gcgttaaagcagattcaattctggcacaaccgttctcatcacaaacgtatgctgccgccg720acgccagaaccgccggatgaaatcgattaa750<210>22<211>750<212>dna<213>francisellaphilomiragiasubsp.philomiragia<400>22atgaaaataataaaaaattttattgcagaagagtattctaaaaaattaatagaatggtta60aaaaaaatttgtataaactatcctgcagaaggttttgttattggtattagtggtggcata120gattcagcagtagcggcatctttagcggttaaaactggattacctacaacagcactaata180ctaccatcaaaaaataatcaagatcaagatatgaaagatggactagagctcattaaaaat240cttgatatagaacatcatattgttccaatacaacctgcttatgatacatttatagagtca300actcttaactttacaaactcacaaaatgaccgccaacatgtcatcaaaggtaatgctcaa360gctcgtcttaggatgatgtatctatatgcctatgctcaacaaaataacagaattgtaata420ggcacagataatgcctgcgaatggtatatgggttatttcacaaaatttggcgatggtgca480gcagatatactaccccttgttaatctaaagaaatcacaagtctttgaaatgggcaagtat540ctcaaagtgccgcaaaatattatagataaagctccatctgctggtctatggcaaggtcaa600actgatgaagatgaaatgggtgtcacatatcaagaaattgataactttttagatggtaaa660gaagtctcagccaaagctcttgagagaataaacttttggcataatcgtagtcatcacaaa720agatctatggcttttactccaaacttttaa750<210>23<211>750<212>dna<213>francisellacf.novicida3523<400>23atgaaaatagttaaagattttagtcctaaagaatattcacaaaatttagttaattggcta60agtgatacttgtataaattatcctgctgaaggatttgtaatcggcattagcggtggtata120gattcagctgttgcagcttctttagctgtcaaaactggattaccaactacagctttaata180ctaccttcaaaaaacaatcaacaccaagatatccaagatgctctagaacttgttgagaaa240cttaatattgaacatcatattgttacaattcaaccagcatacgaaaattttctagcatca300acacaggaatttataaatacagataataatagacaacttgtgatcaagggaaatgctcaa360gcacgtttaaggatgatgtatttatatgcctatgcccaacaatataacagaatagttata420ggtactgataatgcttgtgagtggtatatgggatattttacaaaatttggtgatggcgct480gctgatatatttccgctaattaatcttaaaaaatcacaagtttttgaattaggtaaatac540ttagatgttccgaaaaatataattgataaagctccgtctgctggactatggcaaggacaa600actgatgaggatgaaatgggcgtaacttatcaagaaattgatgatttcttagatggtaaa660caaatttcagcaaaagccctagaaagaataaacttctggcataatcgtagtcatcataag720agaaaactagctttaactcctaatttctaa750<210>24<211>750<212>dna<213>francisellasp.tx077308<400>24atgaaaatagtaaaaaactttattgtagaacagtattctaataatttaataaaatggtta60aaagagaattgcataaaatatcctgctgaaggttttgtgattggtattagtggtggtatc120gattcggcagtagccgcatctttagcagtcaaaacaggattacctacaactgctctaata180ttgccatcgaagaacaatcaagaccaagatatgcgagatggaatagaactaatcgaaaat240cttaatatagagtatcatactgtttcaatacaacctgcttatgacacgtttatagagtca300acatttaactttacaaactcacaaaatgatcgccaacatgttatcaaaggaaatgcccaa360gcgcgtcttagaatgatgtatttatatgcttatgctcagcaaaataatagaattgttata420ggtacagataacgcatgtgaatggtacatgggatatttcactaaatttggtgatggtgca480gcagatatattaccacttattaatctcaaaaaatctcaagtttttgaactaggtaaatac540ttaaaagtgccaaaaaacattatccaaaaagatccttctgccggtctatggcaaggtcaa600actgatgaggatgaaatgggtgtcacatacaaagaaattgatgacttcttagacggtaaa660gaagtctcagaaaaagctctcgaaagaataagcttctggcataatcgtagtcaccataaa720agatccatggcttttacccctaatttttaa750<210>25<211>750<212>dna<213>francisellasp.fsc1006<400>25atgagtgtagtaaaaaattttaaacctaatgaatatgccaataaaattactgaatggctg60aaagactcttgtttaaattatcccgctgaaggttttgtggtaggtattagtggaggtata120gattcagcagtagcagtctctttagcagtaaatactggactacctgttacagggctaata180atgccatcaaaaaataatgatgataaagataccttagatgctatagaattagctaaaaaa240ttaaatatagaatatcatctcatacccattcaaccagtatatgaaacatttctagattca300gctgaagatatcaaaaacagtgctaatgaccgtcaacatgtaatcaaaggaaatgcacaa360gctcgttttagaatgatatacttgtatgcttacgctcagcaaaataatagaatggtaatt420ggtacagataatgcttgtgaatggtatatgggctattttacaaaatttggagatggagcc480gctgatatactgcctcttataaaattaaaaaaatcacaagtttttgaattaggtagctat540cttaatgtacccaataacatcctcacaaaagctccttccgcaggactttggcttggacaa600actgatgaagcagagatgggggtttcatatcaagaaatagatgatttccttgatggtaaa660catgtctcagattatgctcttaatcaaataaaattctggcataaccgtagtcatcataaa720agaatcatggctaaggctccagatttttaa750<210>26<211>753<212>dna<213>francisellaguangzhouensisstrain08hl01032<400>26atgaacgtagtaaaaaatttcactcctgaaaaatattcagaaaaacttatacaatggctc60actaatagctgtataaaatatcctgcagaaggtttcgtaattggtgtaagtggtggtata120gattctgcagtatgtgcatcacttttatccaaaactgatcttcctacaacagcttttata180ctaccatcaaaaaataactctgatcaagatatgatcgatgcattagaacttataaataaa240ttaaatattccataccatataataccaatccagccagtttatgaaagttttctaaagtcc300acacagctatttacaaatccacaaaatgacagacaaaatgtcataaaaggtaacgctcaa360gctcgttttagaatgatgtatttatatgcttatgcacaacaaaataatcgtatagtagtt420ggaacagataatgcttgtgaatggtatatgggttatttcaccaaatttggcgatggagct480gctgatatactaccattaataaatcttaaaaagtcccaggtatttgagttaggtaaatac540ttagatgttccaaggaatatcctaactaaggcaccctctgctggtctttggcaaggccaa600actgatgaaggtgaaatgggagttacttatcaggaaatagataattttctcgacggtaaa660gaagtatcgccagcaacttttgaaaaaataagctactggcataatcgctctcaccacaaa720agaaagatggctttaacgccagattttaactaa753<210>27<211>750<212>dna<213>francisellapersicaatccvr-331<400>27atgaaaatagttaaagatttcaacatcaaagaatattcacaaaagttaattgattggcta60agtgatacttgtatgaattaccctgctgaaggatttgtcattggtcttagcggtggtata120gattcggcagttgcagcttctttagctgtcaaaactggattatcaactacagctttaata180ttaccatcaaaaaacaatcaacaccaagatatacaagatgctctagaacttgcagataaa240attaatattgaacatcatactattacaattcaaacagtatacgaaacttttcttgcgtca300ataaaaaaaattacaaataccgaacgtgatagacaacttgtcattaaaggaaatgctcaa360gctcgtttgaggatgatgtatttatatgcctatgctcaacaatataatagagtggttatt420ggtactgataatgcttgtgaatggtatatgggatattttacaaagtttggtgatggtgct480gctgatattcttccactagttaatctcaaaaaatctcacgtttttgaattaggtaaatac540ttaggtgttcctaaaaatatacttgataaagctccatctgctgggctatggcaaggacaa600actgatgaagatgaaatgggcgtaacttatcaagaaattgatgatttcttagatggtaag660caagtttcagcgaaagctctagaaagaataaatttctggcataatcgtagtcatcataag720agaaaactagctttaattcctaatttctaa750<210>28<211>753<212>dna<213>mannheimiasucciniciproducens<400>28atgaaaacggcagcatacgcagattatctgattcaatggctggaaaaccaacgcaccgaa60ctgtacggcatggacggctataccctgggcgtcagcggcggtattgacagcgccgtctgc120gctcatctggcagcgcgcaccggcgcgccggtacaagccctgattctgccggcggaagta180accagtccgtcagatgtggcggatgcgcaagccaccctggaaagcgccggtattgacggc240caaattatttccattgcaccgtggtacgatctgattatgcaacaactgtccccggtactg300aatagcgaaccggagcgcgttaacgtactgaaaggtaatctgatggcacgcctgcgtatg360attgcgctgtttaccacggcacaaagccatcgttctattgtgctgggcaccgataatgcg420gcggaatggctgacgggttattttaccaaattcggcgacggcgcagcggacgtactgccg480ctggcgggcctgcgcaaagagcaggtatttgaactgggccgttatctgggcgtaccgcaa540agcgtgctggataaaaaaccgagcgccggtctgtgggcaggccaaacggacgaagctgaa600atgggtgttacctatgcggaaatcgacgcttatctgcgcggcgaaaccgttagcccgcag660gcactgcaacaaatccgtttctggcacaaccgttctcatcacaaacgtatgctgccgccg720aaaccgaaatcaccggatgaagcggagtgttaa753<210>29<211>747<212>dna<213>dichelobacternodosusvcs1703a<400>29atgaccgttcatcaatacattgattatctgctggtgtggctggaagagcagcgcgctcat60ctgtatgcatcagatggttatacgctgggcgtcagcggcggcattgattccgccgtttgt120ctgcatctgctggccaaaacgggcaaaccagtgcaagcgctggttctgccgatcaatgcg180aacgcgaacgattgtgaagatgccgaactggtgctgaaaaatgctaatatttccggcaat240attatcgcgctggatgatgtttataccgccgcacaaaacaccctggcgccggttctgaat300cgcgattatgaacgtatgccggtactgaacggcaatctgatggcgcgcctgcgtatggtt360atgctgtataccgtggcgcaaagtcatcgttcggtggtcgtgggcacggataacgcggtg420gaatattatctgggttactttaccaaatttggcgacggcgcctgcgatattctgccgctg480gcaaaactgaccaaatcagaagtaggccaactggcaaaagcgctgggcgttccgaaaaaa540atccgcgaaaaagcgccgagcgcgggcctgtggcaaggccaaaccgatgaaaacgaaatc600ggcgtatcgtacgcggatctggatgcttttctgtgcggtaaaaccgttgatgatgccgtc660cgcgaaaaaattgcttattggcatcaacgctcgcatcataaacgtatgctgccgccgatg720ccggaaatcggcctgtctctggcgtaa747<210>30<211>750<212>dna<213>actinobacillussuccinogenes130z<400>30atgcgcacggcagcatacgtagattatctggtgcgctggctggaaacccagcgtaccgaa60ctgtacggtatggacggctacacgctgggcgtcagcggcggtatcgacagtgccgtttgc120gcccatctggcggcacgcaccggcgccccggtacaggcactgattctgccggcggaagtc180accagcccggaagatgtggcggatgctcagattaccctggaaagtgcaggtattgatggc240cgcattatttctatcgctccgtggtacgatctgattatgctgcaactgaccccggcactg300aatgcggaatctgaacgcattaacgtactgaaaggtaacctgatggcgcgcctgcgtatg360atcgcactgtttaccacggcgcaaagccaccgttctatcgtactgggtacggataacgcc420gccgaaatgctgacgggctatttcaccaaattcggcgacggtgcggcggacgtactgccg480ctggcgcgcctgcgcaaagaacaggtattcgaactgggccgttatctgggcgtaccgaaa540tccgtgctggagaaaaaaccgagtgcgggcctgtgggcgggccaaacggacgagggcgaa600atgggtgtcagttatgcggaaatcgacgcctatctgcgcggcgaaaccgtcagtccgcag660gcgctgaaacagattcaattctggcacaaccgttctcatcacaaacgtatgctgccgccg720acgccggaaccgccggatgaaattgattaa750<210>31<211>750<212>dna<213>francisellaphilomiragiasubsp.philomiragia<400>31atgaaaattattaaaaattttattgcagaagagtattctaaaaaactgattgaatggctg60aaaaaaatttgtattaactatccggcagaaggttttgttattggtattagtggtggcatt120gattcagcagtagcggcatctctggcggttaaaaccggcctgccgaccaccgcactgatt180ctgccgtcaaaaaataatcaagatcaagatatgaaagatggcctggagctgattaaaaat240ctggatattgaacatcatattgttccgattcaaccggcttatgatacctttattgagtca300accctgaactttaccaactcacaaaatgaccgccaacatgtcatcaaaggtaatgctcaa360gctcgtctgcgcatgatgtatctgtatgcctatgctcaacaaaataaccgcattgtaatt420ggcaccgataatgcctgcgaatggtatatgggttatttcaccaaatttggcgatggtgca480gcagatattctgccgctggttaatctgaaaaaatcacaagtctttgaaatgggcaaatat540ctgaaagtgccgcaaaatattattgataaagctccgtctgctggtctgtggcaaggtcaa600accgatgaagatgaaatgggtgtcacctatcaagaaattgataactttctggatggtaaa660gaagtctcagccaaagctctggagcgcattaacttttggcataatcgtagtcatcacaaa720cgctctatggcttttaccccgaacttttaa750<210>32<211>750<212>dna<213>francisellacf.novicida3523<400>32atgaaaattgttaaagattttagtccgaaagaatattcacaaaatctggttaattggctg60agtgatacctgtattaattatccggctgaaggctttgtaatcggcattagcggtggtatt120gattcagcggttgcagcttctctggctgtcaaaaccggcctgccgaccaccgctctgatt180ctgccgtcaaaaaacaatcaacaccaagatattcaagatgctctggaactggttgagaaa240ctgaatattgaacatcatattgttaccattcaaccggcatacgaaaattttctggcatca300acccaggaatttattaataccgataataatcgccaactggtgatcaaaggcaatgctcaa360gcacgtctgcgcatgatgtatctgtatgcctatgcccaacaatataaccgcattgttatt420ggtaccgataatgcttgtgagtggtatatgggctattttaccaaatttggtgatggcgct480gctgatatttttccgctgattaatctgaaaaaatcacaagtttttgaactgggtaaatac540ctggatgttccgaaaaatattattgataaagctccgtctgctggcctgtggcaaggccaa600accgatgaggatgaaatgggcgtaacctatcaagaaattgatgatttcctggatggtaaa660caaatttcagcaaaagccctggaacgcattaacttctggcataatcgtagtcatcataaa720cgcaaactggctctgaccccgaatttctaa750<210>33<211>750<212>dna<213>francisellasp.tx077308<400>33atgaaaattgtaaaaaactttattgtagaacagtattctaataatctgattaaatggctg60aaagagaattgcattaaatatccggctgaaggttttgtgattggtattagtggtggtatt120gattcggcagtagccgcatctctggcagtcaaaaccggcctgccgaccaccgctctgatt180ctgccgtcgaaaaacaatcaagaccaagatatgcgcgatggcattgaactgatcgaaaat240ctgaatattgagtatcataccgtttcaattcaaccggcttatgacacgtttattgagtca300acctttaactttaccaactcacaaaatgatcgccaacatgttatcaaaggcaatgcccaa360gcgcgtctgcgcatgatgtatctgtatgcttatgctcagcaaaataatcgcattgttatt420ggtaccgataacgcatgtgaatggtacatgggctatttcaccaaatttggtgatggtgca480gcagatattctgccgctgattaatctgaaaaaatctcaagtttttgaactgggtaaatac540ctgaaagtgccgaaaaacattatccaaaaagatccgtctgccggtctgtggcaaggtcaa600accgatgaggatgaaatgggtgtcacctacaaagaaattgatgacttcctggacggtaaa660gaagtctcagaaaaagctctggaacgcattagcttctggcataatcgtagtcaccataaa720cgcagcatggcttttaccccgaatttttaa750<210>34<211>750<212>dna<213>francisellasp.fsc<400>34atgagtgtagtaaaaaattttaaaccgaatgaatatgccaataaaattaccgaatggctg60aaagactcttgtctgaattatccggctgaaggttttgtggtaggtattagtggcggtatt120gattcagcagtagcagtctctctggcagtaaataccggcctgccggttaccggcctgatt180atgccgtcaaaaaataatgatgataaagataccctggatgctattgaactggctaaaaaa240ctgaatattgaatatcatctgattccgattcagccggtatatgaaacctttctggattca300gcggaagatattaaaaacagtgctaatgaccgtcaacatgtaatcaaaggcaatgcacaa360gctcgttttcgcatgatttacctgtatgcttacgctcagcaaaataatcgcatggtaatt420ggtaccgataatgcttgtgaatggtatatgggctattttaccaaatttggcgatggcgcc480gctgatattctgccgctgattaaactgaaaaaatcacaagtttttgaactgggtagctat540ctgaatgtaccgaataacatcctgaccaaagctccgagcgcgggcctgtggctgggccaa600accgatgaagcagagatgggcgtttcatatcaagaaattgatgatttcctggatggtaaa660catgtctcagattatgctctgaatcaaattaaattctggcataaccgtagtcatcataaa720cgcatcatggctaaagctccggatttttaa750<210>35<211>753<212>dna<213>francisellaguangzhouensisstrain08hl01032<400>35atgaacgtagtaaaaaatttcaccccggaaaaatattcagaaaaactgattcaatggctg60accaatagctgtattaaatatccggcagaaggtttcgtaattggtgtaagtggtggtatt120gattctgcggtatgtgcatcactgctgtccaaaaccgatctgccgaccaccgcttttatt180ctgccgtcaaaaaataactctgatcaagatatgattgatgcactggaactgattaataaa240ctgaatattccgtaccatattattccgatccagccggtttatgaaagttttctgaaatcc300acccaactgtttaccaatccgcaaaatgaccgccaaaatgtcattaaaggtaacgctcaa360gctcgttttcgcatgatgtatctgtatgcttatgcacaacaaaataatcgtattgtagtt420ggcaccgataatgcttgtgaatggtatatgggttatttcaccaaatttggcgatggcgct480gctgatattctgccgctgattaatctgaaaaaatcccaggtatttgagctgggtaaatac540ctggatgttccgcgcaatatcctgaccaaagcaccgtctgctggtctgtggcaaggccaa600accgatgaaggtgaaatgggcgttacctatcaggaaattgataattttctggacggtaaa660gaagtatcgccggcaacctttgaaaaaattagctactggcataatcgctctcaccacaaa720cgcaaaatggctctgacgccggattttaactaa753<210>36<211>750<212>dna<213>francisellapersicaatccvr-331<400>36atgaaaattgttaaagatttcaacatcaaagaatattcacaaaaactgattgattggctg60agtgatacctgtatgaattacccggctgaaggctttgtcattggtctgagcggtggtatt120gattcggcagttgcagcttctctggctgtcaaaaccggcctgtcaaccaccgctctgatt180ctgccgtcaaaaaacaatcaacaccaagatattcaagatgctctggaactggcagataaa240attaatattgaacatcataccattaccattcaaaccgtatacgaaacctttctggcgtca300attaaaaaaattaccaataccgaacgtgatcgccaactggtcattaaaggcaatgctcaa360gctcgtctgcgcatgatgtatctgtatgcctatgctcaacaatataatcgcgtggttatt420ggtaccgataatgcttgtgaatggtatatgggctattttaccaaatttggtgatggtgct480gctgatattctgccgctggttaatctgaaaaaatctcacgtttttgaactgggtaaatac540ctgggtgttccgaaaaatattctggataaagctccgtctgctggcctgtggcaaggccaa600accgatgaagatgaaatgggcgtaacctatcaagaaattgatgatttcctggatggtaaa660caagtttcagcgaaagctctggaacgcattaatttctggcataatcgtagtcatcataaa720cgcaaactggctctgattccgaatttctaa750<210>37<211>750<212>dna<213>ftnade_star_optimized<400>37atgaaaatcgtcaaagacttctccccgaaagaatattcccaaaaactggtgaactggctg60agcgactcgtgtatgaactatccggcagaaggctttgtcattggtctgagtggcggtatc120gattccgctgtggcggcctcactggccgttaaaaccggcctgccgaccacggcactgatt180ctgccgtctgacaacaatcagcatcaagatatgcaggacgcgctggaactgattgaaatg240ctgaacatcgaacactacaccatttccatccagccggcgtatgaagcgtttctggcgagc300acccaatctttcacgaacctgcagaacaatcgtcaactggtgatcaaaggcaatgcgcag360gcccgtctgcgcatgatgtatctgtacgcgtatgcgcagcaatacaaccgcattgttatc420ggcaccgataatgcctgcgaatggtacatgggttattttacgaaattcggcgatggtgca480gctgacattctgccgctggtcaacctgaaaaaatcgcaggtgtttgaactgggtaaatac540ctggatgttccgaaaaatatcctggacaaagcaccgagcgcaggtctgtggcagggtcaa600accgatgaagacgaaatgggcgttacgtatcaggaaattgatgacttcctggatggtaaa660caagtcagcgcgaaagccctggaacgtatcaacttctggcacaaccgctcacatcataaa720cgcaaactggcactgaccccgaacttctaa750<210>38<211>782<212>dna<213>optimized_ftnade_star_bacillus<400>38gctacctgagaagcttatgaaaatcgttaaagacttctctccgaaagaatattctcaaaa60acttgtgaactggcttagcgactcttgtatgaactatccggcagaaggctttgttattgg120tctttctggcggtatcgactctgctgtggcggcttcacttgctgttaaaacaggtcttcc180gacaacagcacttattcttccgtctgacaacaatcagcatcaagatatgcaggacgcgct240tgaacttattgaaatgcttaacatcgaacactacacaatttctatccagccggcgtatga300agcgtttcttgcgagcacacaatctttcacaaaccttcagaacaatcgtcaacttgtgat360caaaggcaatgcgcaggctcgtcttcgcatgatgtatctttacgcgtatgcgcagcaata420caaccgcattgttatcggcacagataatgcttgcgaatggtacatgggttattttacaaa480attcggcgatggtgctgctgacattcttccgcttgttaaccttaaaaaatctcaggtgtt540tgaacttggtaaataccttgatgttccgaaaaatatccttgacaaagcaccgagcgcagg600tctttggcagggtcaaacagatgaagacgaaatgggcgttacatatcaggaaattgatga660cttccttgatggtaaacaagttagcgcgaaagctcttgaacgtatcaacttctggcacaa720ccgctcacatcataaacgcaaacttgcacttacaccgaacttctaagcatgcagtaagta780gc782<210>39<211>785<212>dna<213>optimized_ae016827_bacillusmannheimiasucciniciproducensmbel55e<400>39gctacctgagaagcttatgaaaacagcagcatacgcagattatcttattcaatggcttga60aaaccaacgcacagaactttacggcatggacggctatacacttggcgttagcggcggtat120tgacagcgctgtttgcgctcatcttgcagcgcgcacaggcgcgccggtacaagctcttat180tcttccggcggaagtaacatctccgtcagatgtggcggatgcgcaagctacacttgaaag240cgctggtattgacggccaaattatttctattgcaccgtggtacgatcttattatgcaaca300actttctccggtacttaatagcgaaccggaacgcgttaacgtacttaaaggtaatcttat360ggcacgccttcgtatgattgcgctttttacaacagcacaaagccatcgttctattgtgct420tggcacagataatgcggcggaatggcttacaggttattttacaaaattcggcgacggcgc480agcggacgtacttccgcttgcgggccttcgcaaagaacaggtatttgaacttggccgtta540tcttggcgtaccgcaaagcgtgcttgataaaaaaccgagcgctggtctttgggcaggcca600aacagacgaagctgaaatgggtgttacatacgcggaaatcgacgcttatcttcgcggcga660aacagttagcccgcaggcacttcaacaaatccgtttctggcacaaccgttctcatcacaa720acgtatgcttccgccgaaaccgaaatcaccggatgaagcggaatgttaagcatgcagtaa780gtagc785<210>40<211>782<212>dna<213>optimized_cp002558_bacillusfrancisellacf.novicida3523<400>40gctacctgagaagcttatgaaaattgttaaagatttttctccgaaagaatattcacaaaa60tcttgttaattggctttctgatacatgtattaattatccggctgaaggctttgtaatcgg120cattagcggtggtattgattcagcggttgcagcttctcttgctgttaaaacaggccttcc180gacaacagctcttattcttccgtcaaaaaacaatcaacaccaagatattcaagatgctct240tgaacttgttgaaaaacttaatattgaacatcatattgttacaattcaaccggcatacga300aaattttcttgcatcaacacaggaatttattaatacagataataatcgccaacttgtgat360caaaggcaatgctcaagcacgtcttcgcatgatgtatctttatgcttatgctcaacaata420taaccgcattgttattggtacagataatgcttgtgaatggtatatgggctattttacaaa480atttggtgatggcgctgctgatatttttccgcttattaatcttaaaaaatcacaagtttt540tgaacttggtaaataccttgatgttccgaaaaatattattgataaagctccgtctgctgg600cctttggcaaggccaaacagatgaagatgaaatgggcgtaacatatcaagaaattgatga660tttccttgatggtaaacaaatttcagcaaaagctcttgaacgcattaacttctggcataa720tcgttctcatcataaacgcaaacttgctcttacaccgaatttctaagcatgcagtaagta780gc782<210>41<211>782<212>dna<213>optimized_cp002872_bacillusfrancisellasp.tx077308<400>41gctacctgagaagcttatgaaaattgtaaaaaactttattgtagaacagtattctaataa60tcttattaaatggcttaaagaaaattgcattaaatatccggctgaaggttttgtgattgg120tatttctggtggtattgattctgcagtagctgcatctcttgcagttaaaacaggccttcc180gacaacagctcttattcttccgtctaaaaacaatcaagaccaagatatgcgcgatggcat240tgaacttatcgaaaatcttaatattgaatatcatacagtttcaattcaaccggcttatga300cacatttattgaatcaacatttaactttacaaactcacaaaatgatcgccaacatgttat360caaaggcaatgctcaagcgcgtcttcgcatgatgtatctttatgcttatgctcagcaaaa420taatcgcattgttattggtacagataacgcatgtgaatggtacatgggctatttcacaaa480atttggtgatggtgcagcagatattcttccgcttattaatcttaaaaaatctcaagtttt540tgaacttggtaaataccttaaagtgccgaaaaacattatccaaaaagatccgtctgctgg600tctttggcaaggtcaaacagatgaagatgaaatgggtgttacatacaaagaaattgatga660cttccttgacggtaaagaagtttcagaaaaagctcttgaacgcattagcttctggcataa720tcgttctcaccataaacgcagcatggcttttacaccgaatttttaagcatgcagtaagta780gc782<210>42<211>750<212>dna<213>revertedftnade_star<400>42atgaaaatcgtcaaagacttctccccgaaagaatattcccaaaaactggtgaactggctg60agcgactcgtgtatgaacactccggcagaaggctttgtcattggtctgagtggcggtatc120gattccgctgtggcggcctcactggccgttaaaaccggcctgccgaccacggcactgatt180ctgccgtctgacaacaatcagcatcaagatatgcaggacgcgctggaactgattgaaatg240ctgaacatcgaacactacaccatttccatccagccggcgtatgaagcgtttctggcgagc300acccaatctttcacgaacctgcagaacaatcgtcaactggtgatcaaaggcaatgcgcag360gcccgtctgcgcatgatgtatctgtacgcgtatgcgggtcaatacaaccgcattgttatc420ggcaccgataatgcctgcgaatggtacatgggttattttacgaaattcggcgatggtgca480gctgacattctgccgctggtcaacctgaaaaaatcgcaggtgtttgaactgggtaaatac540ctggatgttccgaaaaatatcctggacaaagcaccgagcgcaggtctgtggcagggtcaa600accgatgaagacgaaatgggcgttacgt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