希瓦氏菌epa合成基因簇及含该基因簇的基因工程菌的制作方法

专利名称：希瓦氏菌epa合成基因簇及含该基因簇的基因工程菌的制作方法
技术领域：
本明涉及希瓦氏菌EPA合成基因簇，属于生物技术领域。本发明特别涉及获得包 含希瓦氏菌Shewanella piezotolerans WP3 EPA合成基因簇的大肠杆菌基因工程菌及WP3 中EPA合成基因簇，该基因簇包含有一个磷酸泛酰巯基乙胺转移酶(PPTase)基因和pfaA， B，C，D基因，并通过聚酮酶途径(PKS)合成EPA。
背景技术：
二十碳五烯酸(EPA)是大多数动物膜脂的重要组成成分，它对人体的健康很有好 处，它能预防多种疾病，具有很高的营养价值。早在60年代就有人证明EPA有抗实验性血 栓的效应，70年代便将其应用于临床。目前认为，EPA不仅与心血管病有关，也与炎症、神经 及其功能、免疫性疾病的防治有密切的联系。关于EPA生理作用的机制，近年来的研究进展 表明，可能是通过改变细胞膜的磷脂结构与功能，维持细胞膜磷脂和脂肪酸结构的完整性 来实现的。同时EPA也是一些荷尔蒙、类荷尔蒙和调节分子的重要前体。人们曾经认为细菌中不含EPA，但是现在越来越多的发现显示一些海洋细菌能够 合成EPA。而这些被发现的产EPA的细菌大多分布在高压、低温的深海环境或常年冰冻的极 地环境中。由于这些细菌的分布特性，人们推测EPA的合成可能和这些细菌对高压低温环 境的适应有很大关系。在对一些产EPA细菌的研究中表明，EPA的合成与温度有关，在一些 深海菌里，EPA的合成还和压力有关。一般都是当培养的温度降低，以及压力升高的时候， EPA的产量也会增加。这种增加被认为很可能是为了保持细胞膜在低温和高压下的流动性。 我们研究的菌株WP3的EPA合成也显示了相同的温度依赖性。脂肪酸系统对微生物适应深海极端生存环境起着举足轻重的作用。深海环境最 大的两个特点就是低温和高压，人们在很早就已经发现低温能够降低细胞膜流动性，使细 胞膜呈现出有序的晶体状态，从而使细胞膜无法行使正常的生理功能，最终阻碍细胞的正 常生长。最近，人们又发现高压能够对细胞膜产生相似的影响，同样能够降低细胞膜的流动 性，阻碍细胞膜的正常生理功能。为了应对细胞膜流动性的降低，生物体最常用的策略就是 通过调节细胞膜中脂肪酸的成分和含量来维持细胞膜的流动性，最直接的就是通过增加细 胞膜中低熔点脂肪酸的含量来达到增加细胞膜流动性的目的。在很多深海细菌当中发现存在一些长链的多不饱和脂肪酸EPA(20:5)和 DHA (22:6)，从包含这些多不饱和脂肪酸的细菌在环境中的分布来看，它们主要存在于那些 深海环境或者是极地寒冷环境中，因此人们推测这些多不饱和脂肪酸对这些细菌适应深海 和极地环境起了重要作用。在Shewanella piezotolerans WP3中，EPA作为一种细菌常见 的多不饱和脂肪酸，承担着增加细胞膜流动性的功能。Metz于2001年报道，在细菌中长链多不饱和脂肪酸(EPA 20:5, DHA 22:5)是通过 一种新型的聚酮酶途径(PKS)合成的。经分析发现，几乎所有已测序的希瓦氏菌株都包含 有完整的EPA合成基因簇，但是只有少数几种希瓦氏菌能够合成EPA。本发明将Shewanella piezotolerans WP3中EPA合成基因簇克隆到大肠杆菌，该基因簇对于研究深海细菌EPA的合成调控机制进而研究深海细菌的极端环境适应性机制有很大意义，并在应用上有很大的 潜力。

发明内容
本发明的目的在于提供一种希瓦氏菌Shewanella piezotolerans WP3中EPA合 成基因簇的核苷酸序列(SEQ ID NO 1)或互补序列，其包含有一个磷酸泛酰巯基乙胺转移 酶(PPTase)基因和pfaA, B, C，D基因，通过聚酮酶途径(PKS)合成EPA0本发明所提供的核苷酸序列可以被修饰或突变。包括碱基插入或置换，位点特异 性突变，不同序列的重新连接等。序列1 (SEQ ID NO 1)的互补序列可根据DNA碱基互补原则随时得到。序列1的 核苷酸序列或部分核苷酸序列可用过聚合酶链式反应(PCR)或用合适的限制性内切酶切 相应的DNA或其它合适的技术得到。本发明的另一目的在于提供一种包含希瓦氏菌Shewanella piezotolerans WP3 中EPA合成基因簇的基因工程菌。本发明的再一目的在于提供一种利用包含希瓦氏菌Shewanella piezotolerans WP3中EPA合成基因簇的大肠杆菌基因工程菌生产EPA的方法。本发明的目的通过以下技术手段实现一株从西太平洋深海沉积物中分离出来的希瓦氏菌WP3能够大量合成EPA作为 它体内唯一的多不饱和脂肪酸，有时EPA含量可高达整个细胞总脂肪酸含量的20%。该菌 株已公幵于 International Journal of Systematic and Evolutionary Microbiology, 2007，57，60-65.并保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号 CGMCC1. 6160。经Shewanella piezotolerans WP3全基因组测序分析，该菌株含有一个EPA合成 基因簇，其包括九个开放阅读框(ORF)，从ORFl到0RF9全长总共有28893bp。其中0RF2、5、 6、7、8是在Shewanella piezotolerans WP3中合成EPA所必须的，具体为ORFl 金属β -内酰胺酶基因，位于第1-1998碱基，编码metallo-beta-lactamas esuperfamily protein ( ^M β _ Μ月安_ )；0RF2 =PPTase基因，位于2062-2835碱基，编码磷酸泛酰巯基乙胺转移酶；0RF3 =TonB依赖受体基因，位于3912-6818碱基，编码TonB-d印endent receptor (TonB依赖受体蛋白)；0RF4 =Fis 调控因子基因，位于 7251-8117 碱基，编码 Helix-turn-helix， Fis-type, transcriptional regulator (Fis 转录调控因子)；0RF5 =PfaA 基因，位于第 8090-16279 碱基，编码 omega-3 polyunsaturated fattyacid synthase (omega-3多不饱和脂肪酸合成酶)；0RF6 =PfaB 基因，位于第 16279-8525 碱基，编码 omega-3 polyunsaturated fattyacid synthase (omega-3多不饱和脂肪酸合成酶)；0RF7 β -酮脂酰合成酶基因，位于第18525-24395碱基，编码 multi-domainbeta-ketoacyl synthase ( β -酮月旨酉先合成酶)；0RF8 =PfaD 基因，位于第 24588-26216 碱基，编码 polyunsaturated fattyacidsynthase (多不饱和脂肪酸合成酶)；0RF9 =GGDEF 基因位于第 27322-28893 碱基。WP3的EPA合成基因簇结构见附图1。一种获得含EPA合成基因簇的基因工程菌的方法，包括1)菌株培养采用2216E培养基，10 20°C常压下培养WP3菌株；cosmid文库的 克隆子于35 40°C在常压下培养，所用培养基为LB培养基加上终浓度为50 200ug/ml
的氨苄青霉素；2) WP3基因组cosmid文库的构建先按照标准的提总DNA的方法提取制备大分子 量的基因组DNA，并溶于一定量的TE缓冲液中。接着，将总DNA随机用枪头进行机械打断至 30-45kb，然后用脉冲电泳进行分离，并用电洗脱回收30-45kb的DNA片段；将回收到的DNA 进行末端补平反应以获得平末端的DNA片段，接着把补平后的DNA片段与pTOB载体进行连 接。连接后，产物用MaXPlaXTMX噬菌体包装蛋白进行包装，接着将包装产物去侵染大肠杆 菌EPI100，并用含氨苄青霉素抗性的LB平板进行筛选，完成WP3基因组cosmid文库的构 建；3)Cosmid文库的筛选选用两对引物对cosmid文库进行PCR筛选，以获得包含 EPA合成基因簇的克隆子；其中，第一对引物的扩增区域位于EPA基因簇的第一个开放性阅 读框，第二对引物的扩增区域位于第八个开放性阅读框；用分别位于ORFl和0RF8的两对引 物分别对文库进行PCR筛选，有扩增产物的为携带WP3EPA合成的基因簇的目的菌株。本发明所提供的核苷酸序列或氨基酸序列可用于合成EPA，低温和高压条件能导 致其提高EPA的合成。本发明提供了一种Shewanella piezotolerans WP3中EPA合成基因簇，相 比较于正常条件(20°C，0. IMPa)，低温(4 °C )或者高压(20MPa)条件下Shewanel Ia piezotoleransffP3中EPA必需基因的转录量没有变化，但是EPA的合成量能够提高2_3倍。 突变编码磷酸泛酰巯基乙胺转移酶(PPTase)的基因(swp3546)后，该基因簇丧失了在WP3 中合成EPA的功能(ΙΡ3δερα，表1)。突变位于pfaA基因上游针对DNA的Fis型转录调控因子 (swp3548)后，EPA合成必需基因转录量分别提高3_12倍，EPA的产量能够提高50%~60%o


图1为本发明EPA合成基因簇结构的示意图；图2为本发明实施例自杀载体PRE112质粒的结构示意图；图3为获得缺失目标基因DNA片段的示意图；引物2的5'端含有一段约16bp的 与引物3的5'端互补的序列，这样可以通过两轮PCR得到一段不含基因A的DNA片段。引 物1，引物2，引物3，引物4可根据目标基因两侧不同DNA序列来分别设计。引物1，引物2 和引物3，引物4可分别扩增出目标基因两端各500-1000bp的片段。
具体实施例方式实施例1 包含WP3EPA基因簇的大肠杆菌的筛选菌株及培养条件WP3 —般在15°C，常压下培养，所用的培养基为2216E培养基。 cosmid文库的克隆子一般在37°C，常压下培养，所用培养基为LB培养基加上终浓度为100ug/ml的氨苄青霉素。当在对cosmid文库克隆子进行GC-MS检测其脂肪酸含量时，克隆 子的培养温度为20°C。WP3基因组cosmid文库的构建所用试剂盒为EPICENTER公司的pWEBTM cosmid 克隆试剂盒。整个cosmid文库的构建都是按照试剂盒提供的说明书进行的。即先按照标 准的提总DNA的方法提取制备大分子量的基因组DNA，并溶于一定量的TE缓冲液中。接着， 将总DNA随机用枪头进行机械打断至30-45kb，然后用脉冲电泳进行分离，并用电洗脱回收 30-45kb的DNA片段。将回收到的DNA进行末端补平反应以获得平末端的DNA片段，接着把 补平后的DNA片段与pTOB载体进行连接。连接后，产物用MaxPlaxTMX噬菌体包装蛋白进 行包装，接着将包装产物去侵染大肠杆菌EPI100，并用含氨苄青霉素抗性的LB平板进行筛 选。构建的WP3基因组cosmid文库共包含了约2000个克隆子。经提质粒进行单酶切检测 可知每个克隆子的平均插入片段在38kb左右。Cosmid文库的筛选选用两对引物对cosmid文库进行PCR筛选，以获得包含EPA 合成基因簇的克隆子。其中，第一对引物的扩增区域位于EPA基因簇的第一个开放性阅读 框，第二对引物的扩增区域位于第八个开放性阅读框。这两对引物之间覆盖了大约24500bp 的范围，这个范围内包含了在大肠杆菌中合成EPA所必需的基因片段的绝大部分(0RF8还 有约500bp没包含在内)。因此，对于这两对引物都有扩增产物的克隆子就很可能包含了 合成EPA所必需的基因片段。用分别位于ORFl和0RF8的两对引物分别对文库进行PCR筛 选，在所有被分析的450个克隆子中，共有4个克隆子(2E4、3G3、4E1、5D5)对两对引物都有 扩增产物。2E4、3G3、4E1、5D5携带了 WP3EPA合成的基因簇。该携带有WP3 EPA合成基因簇的基因工程菌被命名为大肠杆菌4E1 (Escherichia coli4E 1)，并于2009年4月28日保藏于武汉大学的中国典型培养物保藏中心，编号CCTCC NO:M209092.实施例2 WP3 EPA合成基因簇功能的确定基因敲除的方法首先通过两轮PCR扩增得到目标基因缺失的DNA片段，将其克隆到自杀载体 PRE112中得到目的基因缺失突变载体pRE112-EPA。将pRE112_EPA通过化学转化的方法导 入二氨基庚二酸(DAP)营养缺陷型大肠杆菌WM3064中，随后利用结合转移的方法将质粒 pREl 12-EPA从供体菌WM3064导入WP3中，再通过两轮DNA同源重组筛选得到目的缺失的基 因工程菌株WP3"epa。一、材料l、pRE112质粒，结构见附图3，图中:Cm 氯霉素抗性基因；oriT 介导接合以及质粒转移；oriV 复制起始位点(只能在含有Π蛋白的宿主中复制)；sacBl 蔗糖毒性基因(包含有此质粒的菌株不能在含蔗糖的平板上生长)。2、供体菌株Dap营养缺陷菌株E. coli WM3046具体步骤(1)先通过两对引物，经一轮PCR扩增出目标基因两端各500_1000bp的片段。以去除Fis目标基因为例，先通过以下两对引物，经第一轮PCR扩增出目标基因Fis两端各500-1000bp的片段；引物对一引物 1 :AATCTAGACACAGCTAATGCAATGTATGACCTAGTTG ；引物 4 :AAGAGCTCAGCTGTGATGAGTCAGTCAACTCTAGG ；引物对二引物 2 CGGCGATTTTGCGTCGAATGAATCGACGCATCTCGGGAGTC引物 3 CGACGCAAAATCGCCGCACCTCTAC(2)再利用两内端引物上互补的16bp片段，通过第二轮PCR将第一轮PCR得到的 两片段连接起来得到一个目标基因缺失的DNA片段(见示意图)。以上述引物对为例，其内 端引物为引物对二。(3)分别利用SacI和XbaI内切酶处理pRE112自杀载体和两轮PCR所得的DNA片 段，并用T4DNALigaSe过夜连接，将目标片段克隆到自杀载体上，得到质粒pREl 12-EPA。(4)将质粒转化到Dap营养缺陷菌株E. coli WM3046。化学感受态细胞的制备挑取单菌落至5ml LB液体培养基中，37°C 200rpm/min摇 培过夜；按1 100接种至200ml LB液体培养基中，继续摇培至0D600nm约为0. 4 ；转移菌 液至250ml离心管，冰浴冷却IOmin ；4°C 3500rpm/min离心5min，去上清，倒置流尽液体；加 入IOml预冷的0. IM MgCl2,轻柔摇动重悬菌体，冰浴IOmin ；4°C 4000rpm/min离心5min，去 上清，倒置流尽液体；加入IOml预冷的0. IM CaC12/15%甘油，轻柔摇动重悬菌体，每个EP 管分装100-200 μ L，迅速转入-80°C超低温冰箱保藏。质粒的化学转化迅速融化E. coli 化学感受态细胞，加入适量质粒或连接产物混合均勻后，冰浴30min ；42°C水浴90s，迅速冰 浴冷却2min ；加入800 μ L LB液体培养基，于37°C摇床上低速摇动(< 120rpm) 45min进行 复苏。用截短的墙头吸取菌液，涂布到含有抗生素的LB平板上(含100yg/ml氨苄青霉素 或100ug/ml氯霉素)，37°C培养约14h。(5)将含有pRE 112-EPA质粒的E.coli菌株接入含有50mg/mL氯霉素和50mg/ mLDap的LB培养基中于37°C过夜培养，将野生型WP3接入2216E海洋培养基中于20°C过夜摇培。(6)将过夜培养的菌株分别以1 20比例转接到对应的新鲜培养基中，培养2-3 小时，至 0D600 = 0. 6。(7)将E. coli与WP3按1 4比例混勻，并取200uL点于2216E平板上(不含 任何抗生素，含50mg/mLDap)，待菌液干燥后用锡箔纸将平板包好，正置于28°C培养箱中 mating24h。(8)用3mL2216E培养基将菌苔洗下回收，并涂于含25ug/mL氯霉素，但不含Dap的 2216E平板上，置于20°C培养2-3天，直至单交换克隆子长出。(9)取PCR验证过的单交换克隆子接入不含任何抗生素的2216E培养基中过夜培 养进行松弛，后取适量菌液(20-80ul)涂布于含有10%蔗糖的2216E平板上，至于20°C培养。(10)待克隆子长出后用PCR筛选验证双交换突变子。EPA合成基因簇各开放读码框编码蛋白及功能名称碱基起止位点编码的蛋白ORFl1-1998Metallo-beta-lactamase superfamily protein (SEQ ID NO 2)(金属 β-内酰胺酶)0RF22062-28354' -phosphopantetheinyl transferase (SEQ ID NO 3)(磷酸泛酰巯基乙胺转移酶)0RF33912-6818TonB-dependent receptor (SEQ ID NO 4) (TonB依赖受体蛋白)0RF47251-8117Helix-turn-helix, Fis-type, transcriptional regulator (SEQ ID NO 5) (Fis转录调控因子)0RF58090-16279omega—3 polyunsaturated fatty acid synthase PfaA (SEQ ID NO 6) (omega-3 多 不饱和脂肪酸合成酶)0RF616279-8525omega-3 polyunsaturated fatty acid
synthase PfaB (SEQ ID NO 7) (omega-3 多 不饱和脂肪酸合成酶)0RF718525-24395multi-domain beta-ketoacyl synthase (SEQ ID NO 8) (β-酮脂酰合成酶)0RF824588-26216polyunsaturated fatty acid synthase PfaD (SEQ ID NO 9) (omega-3多不饱和脂肪酸合 成酶)0RF927322-28893GGDEF (SEQ ID NO 10)实施例3菌株培养及细菌脂肪酸含量的测定菌株及培养条件WP3 —般在15°C，常压下培养，所用的培养基为2216E培养基。 cosmid文库的克隆子一般在37°C，常压下培养，所用培养基为LB培养基加上终浓度为 100ug/ml的氨苄青霉素。当在对cosmid文库克隆子进行GC-MS检测其脂肪酸含量时，克隆 子的培养温度为20°C。2216E 培养基(1000ml)酵母提取物Ig
胰蛋白胨2gNaCl34gFe3PO40. Ig(1)不同温度生长条件下的WP3的培养在正常2216E培养基(盐度3.4%)过夜 培养后，将菌液烯释50倍至新鲜培养基中，分别置于低温(4°C )和常温(20°C )下培养。(2)高压力条件的WP3的培养用简易的压力模拟系统进行。过夜培养的菌液在 正常培养基(盐度3. 4% )生长至对数生长中期后，将菌液烯释100倍后装入巴斯德管，赶 走气泡并封口，放入压力阀，调至20MP培养2天。另留一份菌液在常压0. IMP下培养作为 对照。整个培养在20°C进行。细菌脂肪酸含量的测定(1)将细菌在不同条件下培养至指数生长期后期(保证细菌处于同一生长状态 下)，8000g，IOmin离心回收菌体。(2)尽量除尽培养基，并用3% NaCl清洗一遍。(3)将具体置于_70°C使菌体结冰，然后放入冷冻干燥机过夜冷冻干燥。(4)取冷冻干燥后的菌体(IOmg)置于IOmL水解管中，加入2mL浓度为lmol/L的 HC1-CH30H溶液(ν/ν = 1 11)，充氮气1分钟后旋紧螺帽，以防漏气。(5)将水解管置于超声波清洗仪中高功率档破碎30min后，在100°C沸水浴40_60 分钟。(6)待冷却后用ImL色谱纯正己烷萃取甲酯化的脂肪酸。(充分振摇水解管)(7)吸取上层有机相，储存于EP管中，置于-20°C保存。(8)进行GC-MS检测之前，先用干燥的硫酸铵(120°C烘4小时)吸掉样品中残留 水分。(9)用气相色谱与质谱联用仪(GC-MS)进行样品脂肪酸组成和含量的检测。结果 见表1 表1 :SWp3546突变株WP3"epa及野生型WP3在各条件下EPA含量
Culture conditionEPA含量WP3WPSaepa20。C6.5 ±1.104° C14.1 土 1.800. IMPa3·2 土 0.5020MPa9.1 士 1.30序列表<110>国家海洋局第三海洋研究所<120>希瓦氏菌EPA合成基因簇及含该基因簇的基因工程菌<160>10<210>1
9
<211>28893<212>DNA<213> 希瓦氏菌(shewanella piezotolerans WP3)<400>1
0101]atgaaactctcagcccttactctcgccataataagcactgtcacactaggcttttcatcg600102]acgacggtcgcagcccagcatgaacatgatcacattaccgttcattatgaaggcaaggct1200103]gcgacccagcatacagcgca3. C 3. 3.3. C C 3.3.gctattgctgaaactctaaactttactgac1800104]aacaaggcttttgaacaatc3. C 3.3.3.3.3.3. Ccttgtcgcaaagtttgataaagcgactgcc2400105]gatatacttagggctgaatttgcttttattagcgagcaaacgcctgattcggttaaccca3000106]tcgctttatcgccaagcgcagctcaatatggtgccaaatggcttatacaaggtgtctgac3600107]gggatctatcaagtacgtggcacagacttatccaatctaaccttaataaagggtaaaacg4200108]ggttggatcgcctacgatgtattactcacaaaggaagcctctaaagcatcacttagcttt4800109]gcacttaaaaatctaccagaaggcagcgaccttcctgttgtggcgatgatctactctcat5400110]agccacgccgatcactttggtggtgcccgtggtattcaagagatgttccccaatgtcgaa6000111]gtttacggttC 3. C 3. 3.3. C 3. cactaaagagatcgttgatgaaaacgtacttgcaggtaac6600112]gcaatgagccgccgtgcagcgtatcaatatggtgcaaccttaggtaagcataaccacggt7200113]attgttgatgccgcgctcggtaaaggtttatcaaagggtgagatcacttatgtggctcca7800114]gattacaccctaaacggtaaaggaaagtgggaaaccatagaaatcgatggtctagagatg8400115]gtgtttatggatgcctctggcactgaagcagagtccgaaatgatcacctatattccgtct9000116]aaaaaagcgctttggactggtgagctgacttatcaggggatgcacaacatctacaccctg9600117]cgcggtgcaaaagtacgtgacgcgttaaaatggtcaaaggatatcaacgagatgatcagc10200118]gcctttggcactgatgttgaagtactgtttgcctctcattctgcgcctgtttggggtaat10800119]aaagatattaaccagtttttgcgtctgcaacgtgacaactatggtttggttC￡lC￡l￡lCC￡l￡l11400120]accctgaggttggccaataatggcgtcgggatccaagacattggtgatgcaattcaagac12000121]accattcctgaggcggtatacaaggcttggcataccaatggctatcacggcacctatagt12600122]cataatgccaaagccgtttataacaaatacttgggctatttcgacatgaacccggctaac13200123]ttaaacccactaccgacaaagctagagtcagctaaatttgtcgaatacatgggtggtgcc13800124]gatgcagcggtaaaacgcgcaatgggtgactttgctcaaggtgaatatcgctttgtcgct14400125]acagcactcaacaaagtcgtaacggcagatccgagtaacgatgccgcgcggcaacttctg15000126]gcagatacctatgagcaactaggctatcaatcagaaggtgcaggctggag aaatatctac1560
0127]ctaaccggcg cacaagaatt acgcgttggtattcaaaaaggcgcgcctaaaacagcttcg16200128]gctgatgtga tcagtgaaat ggatatgccaaccttgttcgacttcttggccgtcaaaatc16800129]gatagtcagc aagctgctaa acatggcttagtaaaaatgaacatcatcactcctgatacc17400130]aaacaagtgc tctacattga gcttagtaacggtaacttgagcaacaatctggttgacagt18000131]gagcaaactg cagacgctaa ccttattattaacaaggctgacgttacccaaattttactg18600132]ggtaagacca cgctaaaagc cctgctcgcatcaggtggtg0 ￡1￡1￡1￡Ι ￡10cggcgacaaa19200133]acagccttta gcaaaatagc aggcagtatggtggagtttgatccagcatttgaaatcgtg19800134]ccaataccgc aatcataaac gcttagccataagctagcacgaataaagggacgctctatg20400135]cgtcccttta tctattatta gctaataagctclclclclC Cclcltgcgttgatactgacgacttg2100
cgcttgataatcaatagaagctgcgtcaataggttgtccatccatactgaccgcaaaacg2160
gtactcgcgattcaaatgacccaaagcgcaatgccaattatcagctttactcgctgacag2220
gactttagcaggagtctcccccgttggcttatctgcaccaagcgcttggttatcagcctt2280
taaaccttgctctgcaaaaagttgcaaaccaacttgctgcttaaaatgcatctgctcagt2340
aaagcctactcggtgtagttctggcggcatattctcgctcgacttcaatactaaagtgct2400
ttgttgcatcacggagagatcaaatccaaatgatttcaacgacaaagccaagcctaagcc2460
cctagctttaatataagactctttcagtgcccaaagatcaaaaaatcggtcacgctgctg2520
ctcgcaaggcagtgcgagcagcgccgccgtttctggctcagaaaagtaatgatttagtat2580
ggaatgaatattggtgcgactgcggcaacgctcaatatcaacacccaactccaacggcag2640
catttcccccggctgctgaacaatagccagcaataaccagtcgccgctatggcttaaatt2700
aaaattgatgccactgtgtgcaaactgagctgcagttaaaCgCggtttgCctttttcacc2760
atattcaaactgccactcattaggtttaagtgtgccatgcagcgataatacagcccgcaa2820
atacccgcgcaccattaatccttgcacttgagctgatggctggatatagcgatccacttt2880
attaatctcgtcttgtggtaaccacgaacgaatcgttatcgccacgttatcgtcgagaga2940
cgatggcaatagcgggcagaaatagagtttgatactcggtgacgaaggggttagcagtgg3000
caaggcgaactcgaaacagtggcttattacacttattgtggcctcttttgCtclCclclclclCcl3060
acagagaagccaactgaattattcagtatttgcccttctgtgatttaaaaatgcaccacc3120
taatcagctgtagcgagtccatttactgcaactccttatacgctttgtcgggaataatgt3180
cttaacagcgaaaactcattgaggttagtccacctccaccggtcattgacttaagcctag3240
tagaatagtctgctacattttacagtggtgcctccatagcaactttcattttataatagt3300
cgccttttgctgagtaccacaggtatttttttacaatccagccacgctttttclclclCclCCcl3360
tttctataaatttatttttagtgaactttttccaaaactatgactcttacttaatagcag3420
aacgaaaacgctaacgccgttagcaacatgttaatcaaaacagttaaacacagtcgttta3480
tcgattaaaaatacagcaagcaagcctatttcaactactcagtgaaaaacagtgcaaatc3540
actacgtaaaagaaatagtacaatataattaccctccagcaaaaaatagcttcaacagta3600
cccaccttcactcacataaaaaaaatatcaagtaaaacaaataaatacagagcacaaaac3660
cgaatacttaataccgtataC 3.3.3.3.3.3. 3. aattccccatctataaccgcaacagcatta3720
aacaccacgaaaacacccagcaacaaacgaaagccctttagaaaggtatt ￡1￡1￡10￡1￡3780
atttgcctagccaaaacatgagtatatttcactaacagattgccaattaacaaccaaagt3840
attacaaaaagacaccaagcggttaacgtgttgttgcaatttgtatcaacgctttattta3900
agggaattattatgtttttgaattctaaactagcaagctcagtaaaactggctatatcag3960
taggcttaacagcctcactggcaatgcctgcttttgctgaagaagaaacaacagaagaac4020
aaatagaaagagttgcagttactggatctcgaatagcaaaagctgaacttactcaaccag4080
ctcctgttgttagcctttctgcagaagaactagccaaatttggtaattcagacattgcaa4140
gtgtcttggcggaactgccttcaattggtgC 3.3. C 3.3.3. 3. Ccatcattggt￡1￡10￡1￡Ι ￡1￡Ι ￡14200
gtaatgaaagtgctggtgttagttcagccgatttacgtcgactgggcgcgaacagaacgt4260
tagtattagtcaacggtaagcgtcatgtagctggtgagccaggttcagcgcaagttgatt4320
tatcaacaatcccaacaagtatggtatcacgaatagaaattgttactggtggtgcatcag4380
caatttatggttctgatgcggtatctggtgaacaattttg4440
aaggctttgaattcaatgcccgcacaagcggttcacttgaaggcgtaggcacgcaagatc4500
actcctttgatgtactaggtggtgcaaacgttgcagatggacgtggtaacgtaactttct4560
tcgctggttatgaacgaacaaaagaagtaatgtctaccgaaatccgtcaatttgatgcat4620
ggggaacaattgcaaacccagaaaatggcggtgaagaagacggcattccagatagacttc4680
gtgttccacgcgtttattcagaaatgatcaatgataccggtgttatcaatgcgtttggtg4740
gcggcattggtcgatcaacatttgaccaaaacggtaacccaattgcccaagcagaacgag4800
atggtagcaacagctttgcatttggtaatttccccgatggctgtgatacttgttttaaca4860
cagaatcatatgaaaactatattccagcagtagaaagaattaacgtcggttctacgttta4920
actacgactttactgatgaaattcaattctatactgattttagatatgtaaagtcagata4980
ttaaacaacaattccaaccatcattccgttttggtaacgtaagcatcaacgttcaagata5040
atgcatttttgaatgaagacttgcgccaatcgttattggatggcggccaaacaaacgcga5100
catttgctaagttcttcgatgagttaggtaaccgctctgcagaaaaccgtcgtgaacttt5160
tccgttatgtcggcggtttcaaaggtggcttcgagctaagcgaaactattttcgattacg5220
acctttactatatatatggtgaaacaaataaccgtcgtaagacacttaatgatctgatcc5280
cagataattttgttgcagcgcttgattcggttatcgatccagatacaggtttagctgcat5340
gtcgttcacaagttccaagtgcacaaggtgatgattatactgacccagcctctgttaacg5400
gcggtgactgtgttgcgtacaatcctttcggcttaggccaagcgtcagctgaagctcgcg5460
attgggtttctgcagatgtgactcgtgaagataaaattactcaacaagtgattggtggta5520
ctttagctggtgactctgaagagttatttgagcttcaaggtggtccagtagctatggtag5580
ttggttttgagtaccgtgaagaaacatcaggttcaacaactgatgaatttactaaagcgg5640
gcttcctaactaacgcagcaacacctgattcattcggcgaatatgatgttagcgaatact5700
ttgtcgaattgaatttaccaatccttaaagatctaccttttgcacatgaatttagtttag5760
acggtgcttaccgtaaagcagattactcacatgctggtaagactgaagcatggaaagtag5820
gtatgttttatgcgccattagagcaaatatctatccgtggtacaattggtgaagctgttc5880
gtgcaccaaacattgcagaagcattcagccctcgctcaccaggcttcggtcgagtttctg5940
atccatgtgatgcagataacattaatgacgatccagatcgtcctgcaaactgtgcggcac6000
taggtattccacctggattccaagctaatgataacgtaagtgttgataccatctctggtg6060
gtaaccctgaccttaagccagagtcatcgacttcatatactggtggtatagtctggacac6120
ctacatttgctgatagcctttcatttactgtcgattattatgacattgaaattgaagatg6180
ccatcatctctgtagctacacagacggttgcagataactgtgttgatgcaactggcgctc6240
cagacccagatttctgtagtcaagttgatcgtaaccctacaacctttgacattgaacttg6300
ttcgttctggttacctaaacgcagctgcaatttacaccagtggtattgagttccaaggtg6360
cttatgcattagacctcgagtctttcaatatgccaggtgagttccgctttaaccttcttg6420
ctaaccaactacttaaattagagcatcttgagttccagaatcgccctgatgaagtaaatg6480
atgaaaaaggtgaagttggcgaccctgagctgcaattccgcttcggtatggattatcgtt6540
tagacgatctaactgtaagttggaatactcgttatattgataatgtagtgacttatgatg6600
tgtctgaaaatggtggttctctagaagacctatcaccaggaactatcggttccatgacaa6660
ctcacgatctaagtgcgacttactatatctctgaaaatgtaatgattaatggtggcgtac6720
gtaacttgtttgatgctctacctccagggtacacagctaatgcaatgtatgacctagttg6780
tgcggcggatcaaattgttgattgcttcgccgcagacgcagctgtcgtcattaccgatga13860
tggtcataacgcaggcgtattagcagagaaactctcaaaaaaagcccttaaagtcgctgt13920
tgttcgtctgccaaaaggtgcagcgcaatcaccgctaaacagtgacgttgccagcttcca13980
agcagaaagtctcgatgaagcggggatcaatgctgttatcgctcaaattgagcaacaact14040
aggtgagattagtggctttattcacctacaaccagaagctgacagcaataacgttaatga14100
tgccattaacttgagcagcgaaagcttcactcacgttagccaagcattcctttgggctaa14160
gctgttacaaccaaaactaaacgttaccagtgataagcgtcgcagctttatctctgtcag14220
ccgaatcgatggtggctttggttacttagacactaaacagctgcaaaatgctgagctaaa14280
ccaagccgcattagcagggttaactaaaacactgagccatgaatggccaaCggttttttg14340
ccgcgcgttagatattgcaccgaatttagatgcaactcatttagccgacgccattactgg14400
cgagctgttcgataacgtgaccaatttgccagaggtcggtattgctagcgacacgaatgg14460
caacattgaacgttcaacattggttgcgggcaatgcacgttctaaacatgcaacggcaca14520
gctaaatagcgcagataaaatactggtgaccggcggcgcaaaaggtgtgacttttgaatg14580
tgccctagcacttgctaagcgcactcagtcgcatttcatccttgctggacgtagtgaact14640
acaagctattccagcttgggcggcagataaacagatcagcgaacttaaacctgcagctat14700
ttcacatattgtctctaccggtagtaagccgacacctaagcaggtagaagcattagtttg14760
gacagtgaaaagctctatcgagataaacactgctcttgcagcgtttagcgaagtgggcgc14820
aagtgccgagtatgtcagcatggatgtgaccgataccgccgcaattactgcagcgctaaa14880
cggtcgctcagctgagataactggccttatccacggcgctggcgttctggctgataaaca14940
tattcaagataagacgatagccgaattaggtcgcgtatacggcactaaagtcaatgggct15000
gaaagcgctccttgccgcacttgagccgagtaaagttaagctacttgcgatgttctcttc15060
ggctgcaggtttctacggtaataccgggcagagcgattacgccatgtctaacgacatcct15120
taacaaagcggcgttgcagttcactgcccgtcacccacaagctaaagtcatgagctttaa15180
ctggggcccttgggatggtggcatggttaccgatgcactgaagaagatgtttactgaacg15240
tggtgtttacgttattccacttaaagctggagcagaactttttgccactcagctattaag15300
tgaaacgggagcacaactgcttattggaacatctatgcaaggtggaactgacactcaaga15360
ggctgcatctgtaaaaaagcttaatgcgggtgaggtgcaaaacgcatcacgcccgctagc15420
accagtatctctaactagagtgttaacccctaatgcgctggtctttattcaagaccaccg15480
tattggtggtaacccagtgctaccgacggtatgtgcgattcagtggatgcgtgaggcggc15540
aagtgaaatgcttggcactcaagttaaagttatcgactataaattactgaaagggatcgt15600
ctttgacagcagtgatgctcaggagatgactcttgaattaacgccgagcggcaacgagca15660
aatgacggcattaatcagctgtcatggccgtccgcagtacaaggcgactttagctcgtaa15720
cgacaatgctgataatacgccatcaagggagttcaacagtcttggtaaagcagtgactac15780
agctgcagaactctatagcaacggcaccctgttccacgggcctcgtttgcaaggcataca15840
gcaggtgttcaagtttgatgatgcgggtctcatcgctcagtgccaattaccacaggtaag15900
tgatactgactgcggtcaatttgttgcaagccaacatcttggtggtagtcaaccttttgc15960
tgaagacttgctactgcaagcgatgctggtatgggcaaggcttaagtatggctcggcaag16020
cttaccatcgactatcggtgagtttatctcaaacaagccgctagcatttggtgataaggc16080
tgtgctggcgctagaggtggttaaaacaagtgcccgctcacttgaggctaacgttgcgct16140
gtggtggtggtgtaggaacacctgatgcggcgctagcaacctttaatatgggcgcagctt25560
atattgtgaccggttctgtcaaccaagcttgtgttgaagcgggtgcgagtgaacacactc25620
gtaaactactatcaaccactgagatggccgatgtgaccatggctccagctgcagatatgt25680
tcgaaatgggcgtgaagctgcaagtggttaagcgcggaaccttgtttccaatgcgcgcca25740
acaaactgtacgagctctacactcgttatgactcaatagaagcgattccagctgatgaac25800
gtgaaaaacttgagaaacaagtcttccgttcgacccttgatgatatttgggcaggaaccg25860
tggcgcactttaatgagcgcgatcctaagcaaattgaacgtgcaacagataaccctaagc25920
ggaaaatggcactgatttttcgctggtatctagggctttctagccgctggtcaaactctg25980
gggaaacaggtcgtgaaatggattaccaaatttgggctggtcctgctttaggcgccttta26040
accagtgggcgaaaggtagctatttagataattatcaagagcgtaacgccgtcgatgtgg26100
ctaagcatctgatgtatggggctgcgtaccttaaccgcgttaatagtttaacctcacaag26160
gtgtgaagttacccactcagctacttcgttggaagccaacgcaacgtatggcttaaatgc26220
catagcgcctaggcatgaatataaaaaccatcgagtgaattcgatggtttttttatgact26280
actattaccgagttgatctcatgactttcaccccaccaccacggctcaaaccataggctg26340
cgtgatcatacgtgcgaccgttgagctgttcagctctggtaatgacaccttcaagctcaa26400
agcctaagcgctcacagacactgcggctagccttattttcagtggcaaca26460
ctttctccatttgcagttcagtaaaggctaaatcaattaacttagccaccgatctgctaa26520
cgatccccttgccttgatgttcagcgctcaaccaatagcctatttctacttttttgagtg26580
attgatttattgtgttaaaacttacattccctaccaattcaccatcataaatcatgccgc26640
aaactaacgacttaccgatagcatagtcatgcagtgacttttcaataaagC C C C26700
aaacctcatcatgggcaagtagcggccataccagccactgacttaagtacgctctttgct26760
gtgagacaatttttaggtattttgcggcaaatgttttttctaccaaagtcagtgtcaaac26820
ctgaatctacttccaatgtaaacatagctctaccttcctaaaatggggcaacttcatact26880
tcctataaccctgcttcaattaacagtttaaccttttgggccttaacaaaatggtccaat26940
tgatgtgtttcaatccaccacttagcgacttccatcaatgactgcggctcatcattttta27000
ttagcaaaaaaggcataaaatgaaacgccaaccccctcgccttttgctgcaatttttttg27060
tcacagattgccactattctatctctcaatatttgtctcatcattcactcttatgcagtg27120
ttccctgcttttaaaatctcaaatatctgtggtttcaaacgttaacctatacaccgaagc27180
gaattaattaactactgctacaattaactcaacctcctctttgtacattacgaaaatgtc27240
caacttacaataggcgcctaattgacattgagaaccctcagctttaatca￡10 ￡10￡1￡1￡Ι 27300
agagtatttaccatcatcgtaatgttattcacagcggtgattttcagttatcgctttttt27360
attcaaattccgcaacttgaagctaccatgttaaagctttcagagagagagcttaatagt27420
atgatttttgctattaagcggatcaataagccgatggtaacccttaattatgactatgca27480
gtttgggatgaaacttttgaatatgtactacaaccgacctctaaacatagtttacgatat27540
ctggttgaaaactttttgaatgatacatttatccgtttagagattgatggagtttttatc27600
ttaaatagccaagcagaattattatattcaaaaggctttcSLCCSLCSLSLSLSLSLcgaacatgct27660
ttaacctttaacttacaccagtttggtacttttcctgagaatagagttctcatcgacact27720
ccttatgatggcaaaaaggttttagctaaaacaggagtggtatcaactttgtcaggtcca27780
gcactctatagctcaacccc3.3. 3. 3. 3.tcagataaaagtttaccctccaatggcttt27840
21
ctaatattcatacgcttgattaatcaagaa ctggttaatg aaatctcaca atttacggcc27900
acaccggttgtaatgacgcctatcagtagt caacaggaag caaaaggcct caaagattgg 27960
gatgctgaagtagaactgactcaaatattacccttttcccaaagagtcattcgtaacact28020
aaaggagaggcaatactgaaattgacgctatatcataccaatagccaagcgccatctata28080
tggggaataggcatgataagccttttcttcgccatgattatgctattgattacagtgtac28140
cttttactttcagggttcatcattcgcccagtccagcgccttgctcgaaacatcaaagca28200
atggacaaaaagcaaaaatataaagagcttcctaaaaactacatcattaccgaacttaga28260
aaaatatcatttcattttaatgctctgttggggacagtgcagcgacaaaacacactgctt28320
agccatcaagtttacgttgatgaactgacacaaatagccaaccgccgtgcgtttgaactc28380
catctagcaactcaaattcaattgttaaagcgacaaaatatcggttttacgctcatcttg28440
ggcgatatcgattactttaa3.3.3.3. 3. C 3.3. gataccttagggcatttagcgggggatgaa28500
gcattaaaagaggtcgcgaagatgttagaacaacactttaagcgagctgaagatatgtgc28560
gcacgctttggtggagaggagttcataatgctctacagtgatattccatttgccccgtta28620
gagcgtaaattgtcggagatattacagtcactacagcatagagctttgcctcacccaagc28680
tcagaagtcagcgattatatcaccgtcagctttggcgtgtgccaagttctacctatgccc28740
aaccactcttatcaagacaacagctttattacaggtaagcagatcaccgaaatggctgat28800
aaggcactttatcaagcaaaggaaagtgggcgaaaccaatattataaagtcactatcacg28860
gctaatgatatcccctcggataataaagactaa28893
<210>2
<211>665
<212>PRT
<213>希瓦氏菌(shewanella piezotolerans WP3) <400>2
Leu Ser Ala Leu Thr Leu Ala
Met 1
Leu
Lys Gly Phe
His lie Thr
Ser
20
Val
5
Ser
His 35
Ala Gln His Asn Gln 50
Asn Lys Ala Phe Glu 65
Asp Lys Ala Thr Ala 80
Ser Glu Gln Thr Pro 95
Ala Gln Leu Asn Met 110
Thr Thr Val Ala 25
Tyr Glu Gly Lys
lie 10 Ala
lie Ser Thr Val
Gln His
Glu His 30
Gln His
Ala Ala Thr 40
Ala lie Ala Glu Thr Leu Asn Phe Thr 55
Asn Leu Val Ala Lys 70
Ala Glu Phe Ala Phe 85 Ser Val Asn Pro Ser Leu Tyr Arg 100
Val Pro Asn Gly Leu Tyr Lys Val Ser 115
Gln Ser Ser Lys Asp lie Leu Arg Asp
Thr 15 Asp
Thr 45 Asp 60 Phe 75 lie 90 Gln 105 Asp 120Gly Ile Lys Pro Ser Phe Ile Ala Ile Thr Glu Ser Lys Asn Trp Val Lys Leu Ile Val
Ile Lys Glu Glu His Pro Val Ala Val Tyr Thr Gly Lys lie Ser Glu Asp Val Gln Tyr
Tyr Gly Ala Gly Ala Asn Asp Tyr Asp Val lie Thr Ala Tyr Lys Val lie His Asp Lys
Gln Lys Ser Ser Asp Val Glu Gln Ala Ala Glu Glu Leu Thr Asp Leu Asn Asn lie Ala
Val 125 Thr 140 Lys 155 Asp 170 His 185 Glu 200 Asn 215 Tyr 230 Ala 245 Pro 260 lie 275 Ala 290 Trp 305 Leu 320 lie 335 Phe 350 Gln 365 Gln 380 Gly 395 Trp
Arg Gly Ala Leu Phe Val Val Gly Leu Asp Asp Glu Thr Arg Asn Ala Phe Thr Asp His
Gly Trp Ser Pro Gly Tyr Leu Ala Gly Tyr Gly Ser Gly Gly Glu Ser Leu Leu Ala Thr
Thr lie Leu Val Gly Gly Ala Thr Lys Thr Leu Glu Glu Ala Met His Arg Arg lie Asn
Asp Ala Ser Val Ala Ser Gly Leu Gly Leu Glu Met Leu Lys lie Ser Leu Leu Gln Gly
Leu Ser
130 Tyr Asp
145 Phe 160 Ala 175 Arg 190 His 205 Asn 220 Gly 235 Leu 250 Asn 265 Met 280 lie 295 Thr 310 Val 325 Ser 340 Ala 355 Gln 370 Ala 385 Asp 400 Tyr
Ala Met Gly Asn Ala Lys Ser Gly Val Thr Tyr Arg Ala Pro Arg Asn Thr His
Asn Val Leu lie lie lie Met His Lys Lys Phe Tyr Gln Asp Phe Val Asp Asn lie Gly
Leu Leu Lys Tyr Gln Thr Ser Asn Gly Gly Met lie Gly Ala Gly Trp Asn Gly Pro Thr
Thr Leu Asn Ser Glu Lys Arg His Glu Lys Asp Pro Met Leu Thr Gly Tyr Val Glu Tyr
Leu 135 Thr 150 Leu 165 His 180 Met 195 Glu 210 Arg 225 Gly 240 lie 255 Trp 270 Ala 285 Ser 300 His 315 Lys 330 Asp 345 Asn 360 Gly 375 Gly 390 Ala 405 SerHis Met Ala Arg Thr Ala Ser Glu Ala Leu Val lie Glu Thr Ser lie Pro
Asn Ala Asn Pro Lys Phe Ala Met Ala Leu Arg Gln Glu Gly Leu Arg Asp Val Ala Val Lys Met Glu Leu Gln Thr Gln lie Gly Gly Ala Gly lie Pro
Lys Ala Val Gly Asn Leu Ala Val lie Lys Asn Ser Ala Leu Ala Ser Gln
410 Ala 425 Asn 440 Glu 455 Asp 470 Lys 485 Leu 500 Gly 515 Gly 530 Ser 545 lie 560 lie 575 Asn 590 Asp 605 Leu 620 Lys 635 Met 650 Ser 665
Val Leu Tyr Phe Val Ala Trp lie Glu Asp lie Gly Ala Gly lie Val
Tyr Asn Asn Pro Met Gly Ala Gln Val Thr Asp Thr Arg Asn Gln Lys Met Asp Ser Gln Thr Pro Asn Leu Asn Leu Lys Thr Thr Gly Glu Phe
Lys Leu Gly Gly Ala Tyr lie Gly Met Gln Asp Ser lie Thr Asp Asp
415 Tyr 430 Pro 445 Ala 460 Glu 475 Asp 490 Glu 505 Tyr 520 Ala 535 Pro 550 Ala 565 Thr 580 Asn 595 lie 610 Leu 625 Lys 640 Pro 655
Leu Thr Asp Tyr Pro Gln Leu Pro Thr Ala Lys Asn Asn Lys Thr Ala
Gly Tyr Lys Leu Ala Ala Arg Phe Ser Asn Leu Gly Thr Gly Lys Thr Leu Phe Lys His Gln Val Leu Val Lys Ala Ala Leu Ala Phe Phe Glu
420 Phe Asp 435 Glu Ser 450 Val Lys 465 Val Ala 480 Asp Ala 495 Tyr Gln 510 Ala Gln 525 Ala Ser 540 Asp Phe 555 Gly Leu 570 Leu Tyr 585 Asp Ser 600 Asp Val 615 Leu Ala 630 Ser Lys 645 lie Val 660
<210>3
<211>257
<212>PRT
<213>希瓦氏菌(shewanella piezotolerans
WP3)0682]
0683]
0684]
0685]
0686]
0687]
0688]
0689]
0690]
0691]
0692]
0693]
0694]
0695]
0696]
0697]
0698]
0699]
0700]
0701]
0702]
0703]
0704]
0705]
0706]
0707]
0708]
0709]
0710]
0711]
0712]
0713]
0714]
0715]
0716]
0717]
0718]
0719]
0720]
<400>3 Met Val
1
Leu Pro Asn Gln Arg Phe Gln Tyr Phe Lys Thr Ala Lys Ala Arg lie
Lys Arg
Arg
Pro
Leu
Pro Cys Ser Gln lie Gly
Glu Glu Pro Asp Phe Asp
Gly Asn Thr
Leu Ser His
Gly Arg Glu Arg Lys Phe
Ser Ser
Gln Gln Thr Asn Ala Tyr
Glu Ser Pro Asp Ala Asp Glu Met Gly Gly Trp Val Gln
Tyr
5 Glu 20 Ala 35 Ser 50 Met 65 Arg 80 Glu 95 Arg 110 Arg 125 Leu 140 Asn 155 His 170 Leu 185 Glu 200 His 215 Ser 230 Ala 245
Leu Arg Ala Val Trp Gln Phe Glu Ala Gln Phe Ala Gly Asp Trp Leu Leu Pro Leu Glu Thr Asn lie His Thr Ala Ala Leu Phe Phe Asp Leu Gly Leu Gly Leu Val Met
Ser
Met Pro Pro
Phe Lys Thr Cys Met Gln
Lys Gln Ala Asp Pro Ala Ala Leu Asp Gly Val Val
Gln Glu Gln Asn Lys Gly Gln Ser
Leu 10 Tyr 25 His 40 Leu 55 Leu 70 Ser 85 Leu 100 Trp 115 Ala 130 Gln 145 Leu 160 Val 175 Gln 190 Val 205 His 220 Pro 235 lie 250
Ser Leu His
Gly Ser Leu Gly lie Ala Ala
Glu Gly Ala Val Leu
Ser His Gly Ala
Leu lie Asn
Lys lie lie Asp Asn
Leu Pro
Leu Lys
Leu Ser Leu
Thr Arg Leu Leu
Leu Ser
Asn Asp Ala
Leu Val Gln Gly Ala Arg Ala Leu
Gly Thr 15
Gly Lys 30
Asn Phe 45
Val Gln 60
lie Glu 75
His Tyr 90
Cys Glu 105 Glu Ser 120 Lys Ser 135 Val Leu 150 Gly Phe 165 Leu Phe 180 Ala Asp 195 Ser Lys 210 Glu Tyr 225 Ala Ser 240 Val Leu 255
Ala Tyr <210>4 <211>968 <212>PRT0721]<213>希瓦氏菌(shewanella piezotoleransWP3)0722]<400>40723]MetPheLeuAsnSerLysLeuAlaSerSerValLysLeuAlalie0724]1510150725]SerValGlyLeuThrAlaSerLeuAlaMetProAlaPheAlaGlu0726]2025300727]GluGluThrThrGluGluGlnlieGluArgValAlaValThrGly0728]3540450729]SerArglieAlaLysAlaGluLeuThrGlnProAlaProValVal0730]5055600731]SerLeuSerAlaGluGluLeuAlaLysPheGlyAsnSerAsplie0732]6570750733]AlaSerValLeuAlaGluLeuProSerlieGlyAlaThrAsnThr0734]8085900735]lielieGlyAsnAsnAsnSerAsnGluSerAlaGlyValSerSer0736]951001050737]AlaAspLeuArgArgLeuGlyAlaAsnArgThrLeuValLeuVal0738]1101151200739]AsnGlyLysArgHisValAlaGlyGluProGlySerAlaGlnVal0740]1251301350741]AspLeuSerThrlieProThrSerMetValSerArglieGlulie0742]1401451500743]ValThrGlyGlyAlaSerAlalieTyrGlySerAspAlaValSer0744]1551601650745]GlyVallieAsnlielieLeuLysAsnAsnPheGluGlyPheGlu0746]1701751800747]PheAsnAlaArgThrSerGlySerLeuGluGlyValGlyThrGln0748]1851901950749]AspHisSerPheAspValLeuGlyGlyAlaAsnValAlaAspGly0750]2002052100751]ArgGlyAsnValThrPhePheAlaGlyTyrGluArgThrLysGlu0752]2152202250753]ValMetSerThrGlulieArgGlnPheAspAlaTrpGlyThrlie0754]2302352400755]AlaAsnProGluAsnGlyGlyGluGluAspGlylieProAspArg0756]2452502550757]LeuArgValProArgValTyrSerGluMetlieAsnAspThrGly0758]2602652700759]VallieAsnAlaPheGlyGlyGlylieGlyArgSerThrPheAsp0760]
0761]
0762]
0763]
0764]
0765]
0766]
0767]
0768]
0769]
0770]
0771]
0772]
0773]
0774]
0775]
0776]
0777]
0778]
0779]
0780]
0781]
0782]
0783]
0784]
0785]
0786]
0787]
0788]
0789]
0790]
0791]
0792]
0793]
0794]
0795]
0796]
0797]
0798]
Gln Ser Asn Asn Phe Phe Asp Gly Gly Gly Tyr Thr
Asn Phe Thr Val Tyr Gln Asn Gly Asn Gly Asp
Leu Asn Ser Val Ile Pro Ser
Val Asn Gly Ala Ser Glu Asp Asp Ser Val Val
Gly Asn Ala Phe Glu Ser Gly Ser Thr Asp Pro Ser Ala Phe Gln Thr Arg Ser Phe Lys Tyr Asp Asp Ala Asp Glu lie Glu Phe
Leu
Gly Ala Lys Glu Gly
275 Pro 290 Gly 305 Tyr 320 Thr 335 Phe 350 Phe 365 Leu 380 Asn 395 Ala 410 Gly 425 Tyr 440 Leu 455 Pro 470 Gln 485 Cys 500 Ala 515 Thr 530 Leu 545 Glu 560
lie Asn Glu Phe Arg Arg Asn Ala Glu Gly
Ala Phe Asn Asn Tyr Phe Glu Thr Asn Phe
Gln Ala
Pro Asp
lie Tyr lie Pro Asp Thr Gly Asp Val Ala Arg Asp Gln Gln Phe Glu Tyr Arg
Tyr Tyr Val Gly Asp Phe Arg Glu Gly Asp Gly Asp Tyr Trp Val
lie Asp Lys Asn Leu Ala Arg Leu Glu Asn Leu Tyr Asn Val lie
Leu Gln
Glu Glu
280 Glu 295 Gly 310 Pro 325 Phe 340 Ser 355 Val 370 Arg 385 Lys 400 Glu 415 Ser 430 Thr 445 Phe 460 Ala 475 Thr 490 Pro 505 Ser 520 Gly 535 Gly 550 Thr 565
Arg Cys Ala Thr Asp Ser
Asp Asp Val Asp lie lie
Gly Thr Glu Glu Lys Asn
Leu Glu Asn Val Ala Asp Phe Ala Gly Gly
Phe Phe Thr Asn Ala Cys Pro
Ser Cys Arg lie Gln Val
Gln Ser Leu Leu Phe
Asp Arg lie Arg Ala Arg Ala
Gly Leu Asp Val Thr Leu Pro Val
Ser Gly Ser
Glu Tyr Phe Arg Leu Ser Ser Gly Thr Ala Ala Thr
285 Asn 300 Phe 315 lie 330 Gln 345 Gln 360 Gln 375 Asp 390 Leu 405 Val 420 Asp 435 Lys 450 Asp 465 Gln 480 Val 495 Gln 510 Arg 525 Gly 540 Met 555 Thr 570
27Asp Glu Phe Thr Lys Ala Gly Phe Leu Thr Asn Ala Ala Thr Pro 575580585
Asp Ser Phe Gly Glu Tyr Asp Val Ser Glu Tyr Phe Val Glu Leu 590595600
Asn Leu Pro lie Leu Lys Asp Leu Pro Phe Ala His Glu Phe Ser 605610615
Leu Asp Gly Ala Tyr Arg Lys Ala Asp Tyr Ser His Ala Gly Lys 620625630
Thr Glu Ala Trp Lys Val Gly Met Phe Tyr Ala Pro Leu Glu Gln 635640645
lie Ser lie Arg Gly Thr lie Gly Glu Ala Val Arg Ala Pro Asn 650655660
lie Ala Glu Ala Phe Ser Pro Arg Ser Pro Gly Phe Gly Arg Val 665670675
Ser Asp Pro Cys Asp Ala Asp Asn lie Asn Asp Asp Pro Asp Arg 680685690
Pro Ala Asn Cys Ala Ala Leu Gly lie Pro Pro Gly Phe Gln Ala 695700705
Asn Asp Asn Val Ser Val Asp Thr lie Ser Gly Gly Asn Pro Asp 710715720
Leu Lys Pro Glu Ser Ser Thr Ser Tyr Thr Gly Gly lie Val Trp 725730735
Thr Pro Thr Phe Ala Asp Ser Leu Ser Phe Thr Val Asp Tyr Tyr 740745750
Asp lie Glu lie Glu Asp Ala lie lie Ser Val Ala Thr Gln Thr 755760765
Val Ala Asp Asn Cys Val Asp Ala Thr Gly Ala Pro Asp Pro Asp 770775780
Phe Cys Ser Gln Val Asp Arg Asn Pro Thr Thr Phe Asp lie Glu 785790795
Leu Val Arg Ser Gly Tyr Leu Asn Ala Ala Ala lie Tyr Thr Ser 800805810
Gly lie Glu Phe Gln Gly Ala Tyr Ala Leu Asp Leu Glu Ser Phe 815820825
Asn Met Pro Gly Glu Phe Arg Phe Asn Leu Leu Ala Asn Gln Leu 830835840
Leu Lys Leu Glu His Leu Glu Phe Gln Asn Arg Pro Asp Glu Val 845850855
Asn Asp Glu Lys Gly Glu Val Gly Asp Pro Glu Leu Gln Phe ArgThr Val Ser Trp Asn 885
Asp Val Ser Glu Asn 900
Thr lie Gly Ser Met 915
lie Ser Glu Ash Val 930
Asp Ala Leu Pro Pro 945
Val Gly Arg Arg Ala 960
860865
Phe Gly Met Asp Tyr Arg Leu Asp Asp Leu 875880
Thr Arg Tyr lie Asp Asn Val Val Thr Tyr 890895
Gly Gly Ser Leu Glu Asp Leu Ser Pro Gly 905910
Thr Thr His Asp Leu Ser Ala Thr Tyr Tyr 920925
Met lie Asn Gly Gly Val Arg Asn Leu Phe 935940
Gly Tyr Thr Ala Asn Ala Met Tyr Asp Leu 950955
Phe Phe Gly Leu Lys Val Met Met 965
<210>5 <211>288 <212>PRT
<213>希瓦氏菌(shewanella piezotolerans <400>5
Met Ala Glu Gln Val Asn Thr Ser Leu Asn 1510
Pro Asn Lys Cys Thr Gln Ser Ala Thr Glu
2025
Ala Thr Thr Thr Pro Glu Met Arg Arg Phe 3540
Phe Ser Val Ser Gln Leu Ser Lys Val Leu 5055
Thr Val Arg Lys Trp Arg Lys Arg Asp Ser
6570
Asn Thr Pro His His Leu Asn Thr Thr Leu
8085
Tyr Val Val Val Gly Leu Arg Tyr Gln Leu 95100
Arg Leu Leu Thr Ala Thr Gln Lys Phe lie 110115
Arg Ser Gly Leu Ala Arg Cys Leu Lys Arg Tyr Gly Val
125130
Val Ser Asp lie Glu Ser Pro Asp Val Pro Met Arg Tyr
WP3)
Ser Ala Ala Ala Arg His lie Gln Glu Asn lie Ser lie Glu Asn Thr Pro Leu Lys Met Pro Asn Pro Asn
Thr Ser Ser Glu Thr Gln Leu Val Ser Phe
Asn 15 Asn 30 Asp 45 Ala 60 Pro 75 Glu 90 Asp 105 Ser 120 Arg 135 Asn
140145150
Gln Leu Pro I le Thr Gln Gly Set Asp Val Gln Thr Tyr Thr Leu
155160165
Hi S Tyr Glu Thr Leu Ala Lys Thr Leu Ala Leu Pro Set Thr Gly
170175180
Ala Asp ASh Val Val Gln Val Val Set Leu Thr I le Pro Pro Gln
185190195
Leu Thr Asp Glu Gln Pro Set Set Ile Leu Leu Gly Ile Asp Pro
200205210
HiS Set Asp Trio Ile Tyr Leu Asp Ile Tyr Gln Asp Gly ASh Thr
215220225
Gln Ala Thr ASh Arg Tyr Met Ala Tyr Val Leu Lys Hi S Gly Pro
230235240
Phe HiS Leu Arg Lys Leu Leu Val Set ASh Tyr HiS Set Phe Ile
245250255
Glu Arg Phe Pro Gly Ala Thr Gln ASh Arg Arg Thr Set Thr Asp
260265270
Lys Pro Lys Thr ASh ASh Lys Thr Pro Glu Cys Gln Ala Set Thr
275280285
Gly Asp Set
<2 10)6
<211>1305
<2 l 2>PRT
(213)希瓦氏菌(shewanella pieZ。t。leranS WP3)
(400)6
Asp Leu Ala Glu Cys Arg Thr Leu Gly Glu I le Val Set Tyr Met
l5lo15
ASh Set Lys Leu Set Thr ASh Asp Ala Ala Gly Ala Set Set Glu
202530
Set Ala Ala Pro Gln Val Ala Thr Ala Lys Thr Ala Leu Set Ala
354045
Gln Lys Val Gln Ala Thr Met Met Set Val Val Ala Glu Lys Thr
505560
Gly Tyr Pro Thr Glu Met Leu Glu Leu Glu Met Asp Met Glu Ala
]657075
Asp Leu Gly Ile Asp Set Ile Lys Arg Val Glu Ile Leu Gly Thr
808590
Val Gln Asp Glu Leu Pro Gly Leu Pro Glu Leu ASh Pro Glu Asp
95100105
Leu Ser Gly Thr Val Met Glu Leu Val Thr Val Thr Met Ser Pro Thr Phe Ser Lys Glu
Ala Lys Ala Ala Ala Asp lie Asn Ser Asn Ala Met Leu lie Gly Leu Glu Ala Thr Ala
Glu Leu Ser Leu Glu Met Leu Pro Tyr Asp Thr Met Glu Lys Leu Gly Ala Glu Gly Asp
Cys Ala Ser Ser Lys Glu Gly Glu Met Ala Ala Ser Leu Arg Pro Glu Ala Gln Tyr Leu
Arg 110 Asp 125 Glu 140 Ala 155 Thr 170 Ala 185 Thr 200 Asp 215 Asn 230 Ala 245 Lys 260 Val 275 Ser 290 Val 305 Glu 320 lie 335 Ala 350 Val 365 Pro 380 Gly
Thr Gly Asp Gln Gly Asp Val Leu Ser Gly Thr Val Met Glu Leu Val Pro Gln Thr lie
Leu Ser Ala Lys Tyr Leu Gln Ala Lys Ala Ala Ala Asp lie Asn Ser Ala Gly Glu Asp
Gly Lys Ala Val Pro Gly Asp Glu Leu Ser Leu Glu Met Leu Pro Tyr Ala Thr Met Ser
Glu Leu Pro Gln Thr lie Glu Cys Ala Ser Ser Lys Glu Gly Glu Met Thr Met Leu lie
lie Val
115 Ser Thr
130 Gln 145 Ala 160 Glu 175 Asp 190 Leu 205 Arg 220 Asp 235 Glu 250 Ala 265 Thr 280 Ala 295 Thr 310 Asp 325 Asn 340 Gln 355 Met 370 Glu 385 Lys
Val Thr Met Ser Pro Thr Gly Asp Gln Gly Asp Val Leu Ser Val Ser Leu Arg
Ser Asn Ala Met Leu lie Gly Leu Ser Ala Lys Tyr Leu Gln Ala Lys Ala Val Ser Val
Tyr Ala Thr Met Glu Lys Leu Gly Lys Ala Val Pro Gly Asp Glu Leu Asn Val Met Glu
Met Ala Ala Ser Leu Arg Pro Glu Leu Pro Gln Thr lie Glu Cys Ala Gly Ala Asp lie
Asn 120 Ala 135 Lys 150 Val 165 Ser 180 Val 195 Glu 210 lie 225 Ser 240 Gln 255 Ala 270 Glu 285 Asp 300 Leu 315 Arg 330 Thr 345 Leu 360 Glu 375 Met 390 Leu
31Gly Thr Val Glu Asp Leu Met Asn Ser lie Ala Asp Leu Glu Ala Ala Val Ile
Gly Ala Ala Glu lie Glu Pro Glu Ser Glu
Gln Ala Lys Ala Leu Pro
Asp Thr Leu Ser Lys Ala
Gln Ala
Gln Asp Lys Gln
Ser Leu
Gln Asp Ser Phe
Leu Leu Gln Pro Phe lie Ser Val Thr Lys Gln Leu Gly Leu Thr Lys Arg Ala Leu Asp Asp Ala lie Thr
395 Asp 410 Glu 425 Leu 440 Val 455 Pro 470 lie 485 Asp 500 Ala 515 Ser 530 Asp 545 Leu 560 Ser 575 Thr 590 Lys 605 Ser 620 Gln 635 Thr 650 lie 665 Gly 680
Glu Leu Pro Gly Cys Arg Thr Leu Thr Lys
His Val Gly Leu Pro Glu Gly Asn His Leu Arg Asn
Arg Thr Ser Val Ser Val Ser Glu Asp Cys His Asn Lys Val Leu Asn Ala Gly Glu lie Asn Val Val Ser Asn Val lie Asp Ala Glu
Gly Ala Val Phe Ala Ala Ser lie Ser Asn Gln Thr Gly Leu
Glu Ala Thr
400
Leu Pro 415 Gly 430 Ser 445 Glu 460
Ala Leu 475 Ala 490 Gly 505 Val 520 Asp 535 Asn 550 Gly 565 Asp 580 Ala 595 Ser 610
Gly Phe
625 Asn Gln
Ala Val Val Val Ala Phe Ala Phe Asp
640
Leu Ser His Glu Trp Pro 655
Ala Pro Asn Leu Asp Ala 670
Glu Leu Phe Asp Asn Val 685
Glu Leu lie Val Ala Thr Val Thr Lys Lys Asp Ala Leu Ala Arg Leu Ala Ser Val lie lie His lie Asn Leu Trp Lys Arg Gly Tyr Ala Ala Thr Val Thr His Thr Asn
Asn Ala Ala Ser Leu Ala Glu Pro Phe Ala Leu Leu Ala Arg Leu Leu Phe Leu Leu
405 Pro 420 Tyr 435 Val 450 Ser 465 Ser 480 Val 495 Lys 510 Lys 525 Gln 540 Gln 555 Gln 570 Ser 585 Lys 600 Ser 615 Asp
630 Ala 645 Cys 660 Ala 675 Pro 690Glu Thr Leu Val His Trp Ser Glu Ala Ser Gly Leu Arg Ala Ala Ser His Gly Gly Thr
Val Leu Asn Thr Phe Ala His Ala Leu Met Arg Ala Val Leu Ala Asn Pro Gly Val Gln
Gly Val Ser Phe lie Ala lie Leu Ala Asp Ser Asp Tyr Glu Gly Asp Gln Met Tyr Leu
lie Ala Ala Glu Leu Asp Val Val Ala Val Ala Lys Gly
Ala 695 Gly 710 Asp 725 Cys 740 Ala 755 Lys 770 Ser 785 Trp 800 Phe 815 Thr 830 Glu 845 His 860 Thr 875 Pro Ser
Ser Asn Lys Ala Gly Gln Thr Thr Ser Asp lie lie Lys Lys
Phe lie Ala Val Val Leu
890
Tyr Gly
905 Leu 920 Lys 935 Thr 950 lie 965 Ser
Asn Val Asp Pro Glu
Asp Ala lie Leu Arg lie Gly Val Glu Thr Thr Gln Val Val Asn Lys Met Ala Leu Thr
Thr Arg Leu Ala Ser Ser Ser Lys Val Ala Gly Asp Asn Lys Thr Ala Ser Leu Lys Gly
Asn Ser Val Leu Glu Glu Lys Ser Gly Ala Leu Lys Gly Leu Gly Ala Phe Lys Ala Ala
Gly 700 Lys 715 Thr 730 Ala 745 Leu 760 Leu 775 Pro 790 Ser 805 Ala 820 lie 835 lie 850 Thr 865 Leu 880 Leu 895 Gln 910 Leu 925 Asn 940 Lys 955 Gly 970 Gln
Asn His Gly Lys Gln Lys Thr lie Ser Thr His lie Lys Ala Ser Gln Trp Met Ala Leu
lie Ala Gly Arg Ala Pro Pro Glu Ala Ala Gly Ala Ala Met Asp Phe Gly Phe Glu Leu
Glu Thr Ala Thr lie Ala Lys lie Glu Ala Ala Glu Leu Phe Tyr Thr Pro Thr Leu lie
Arg Ser 705 Ala Gln 720 Lys Gly 735 Gln Ser 750 Pro Ala
765 Ala lie
Gln Asn Tyr Leu Gly Leu Leu Ser Ala Ala Trp Glu Phe Gly
780 Val 795 Thr 810 Val 825 Asn 840 Val 855 Gly 870 Ala 885 Ser 900 Met 915 Arg 930 Asp 945 Arg 960 Ala 975 Thr1033]
1034]
1035]
1036]
1037]
1038]
1039]
1040]
1041]
1042]
1043]
1044]
1045]
1046]
1047]
1048]
1049]
1050]
1051]
1052]
1053]
1054]
1055]
1056]
1057]
1058]
1059]
1060] 1061] 1062]
1063]
1064]
1065]
1066]
1067]
1068]
1069]
1070]
1071]
Ser Met Gln Lys Leu Asn
Pro lie Cys Thr Phe Ser Pro Thr Ala Leu lie Gln Glu Tyr Asn Val
Val Gln Ala Gln Asp Gly Gln
Ser Asp lie Val Ser Ser Asn Glu
Tyr Lys Pro Ser Arg
Ala Gln Ala Phe Asp Gly Lys Val
Glu Gly Gln Val
Leu Leu
Ser Ala
Pro Leu
Lys Tyr His Glu
980 Gly Gly Thr
995 Ala Gly Glu 1010 Leu Thr Arg
1025 His Arg lie
1040 Gln Trp Met
1055 Lys Val lie 1070 Asp Ala 1085 Gln Met 1100 Ala Thr 1115 Glu Phe 1130 Leu Tyr Ser 1145 Gln Gln 1160 Gln Leu 1175 Ser Gln 1190 Leu Gln 1205 Ser Leu 1220 Ala Phe 1235 Ser Ala 1250 Gly Glu 1265
lie Cys Ala
Asp Thr Val Gln Val Leu Gly Gly Arg Glu Asp Tyr Gln Glu Thr Ala Leu Ala Asn Ser Asn Gly Val Phe Pro Gln
Gln
Asn
Thr
Asn
Ala
Lys
Met
Leu
Arg
Leu
Thr
Lys
Val
Thr Asn
His Leu Gly Ala Met Leu Pro Ser Thr Gly Asp Lys Arg Ser Leu Leu Ser Ala
985
Glu Ala 1000 Ala Ser 1015 Pro Asn 1030 Pro Val 1045 Ala Ser 1060 Leu Leu 1075 Thr Leu 1090 lie Ser 1105 Asn Asp 1120 Gly Lys 1135 Leu Phe 1150 Phe Asp 1165 Ser Asp 1180 Gly Ser 1195 Val Trp 1210 lie Gly 1225 Ala Val 1240 Glu Ala 1255 Met Met 1270
Ala Ser Val Arg Pro Leu Ala Leu Val Leu Pro Thr
Glu Lys Glu Cys Asn Ala His Asp Thr Gln Ala Glu
Met Leu Gly lie Leu Thr His Gly Ala Asp Val Gly Ala Asp Pro Phe
Arg Phe Leu Ala Asn Val Lys Ala
990 Lys 1005 Ala 1020 Phe 1035 Val 1050 Gly
1065 Val 1080 Pro 1095 Arg 1110 Asn 1125 Thr Thr 1140 Pro Arg 1155 Gly Leu
1170 Cys Gly 1185 Ala 1200 Leu Lys 1215 lie Ser 1230 Leu Glu 1245 Ala Leu 1260 Ala Lys 1275Val Thr lie Ser Lys Asn Leu Asn Ser Ala Phe Leu Thr
12801285
Ala Glu Ser Asp Lys Asp Lys Thr Leu Ser Thr Gly Glu 12951300
<210>7
<211>727
<212>PRT
<213>希瓦氏菌(shewanella piezotolerans WP3) <400>7
Ala Ser Ala lie Ser Ser
Glu Thr 1290 Gln Ser 1305
Met Val Ser Leu Thr
15
lie Lys Ser Glu Met 20
Ser Pro Leu Asp Leu 35
lie Ser Val Leu Leu 50
Ser Ser Glu Leu Val 65
Leu Thr Ala Thr lie 80
lie Ser Thr Ala Thr 95
Ala Ala Gln Asn Arg 110
Gln lie Ser Ser Phe 125
Ser Cys Met Asn Lys 140
Ser Val Ser Lys Arg 155
Gln Phe Asp Thr Val 170
Thr His Gln Arg Asp 185
Asn His Tyr Trp Phe 200
lie Asn Leu Thr Asn 215
Pro
Ser
Pro
Ser
Asp
Leu Arg Ala Asn Glu Leu Val Gln Ala lie
lie Thr Ala Phe Cys Ala Thr Thr
lie Val His Val Glu
Ser Ser Leu Leu
His Pro His
Gln Ser Leu Gly Asp Leu Lys Leu Ser Ser Glu His Val Glu
Glu Gln Pro lie lie His Ser
10
Ala Leu 25
Glu Gln 40
Ser Ser 55
Asp Asp 70
His Gly 85
Met Met 100
Ala Gln 115
Ser Thr 130
Ala Lys 145
Gln Leu 160 Asn Asp 175
Ser Ala 190
Gln Ala 205
Thr Thr 220
Ser Asp Leu Val Thr Ser Lys Leu Phe Glu Lys lie His Ala Leu Ala Thr Leu Arg His Asn Ser Leu Ala Lys Gln Arg Val Ser Cys
Gln Val 15
Leu Pro 30 Glu Leu 45 Leu Gln 60 Ala Asn
75 Val Gln
90 Leu Lys 105 Ala Met 120 Ala Asp 135 Ala Asp 150 Thr Gln 165 Cys Arg 180 Ala Ala 195 Met Ala 210 lie Ser 2251111] 1112]
1113]
1114]
1115]
1116]
1117]
1118]
1119]
1120] 1121] 1122]
1123]
1124]
1125]
1126]
1127]
1128]
1129]
1130]
1131]
1132]
1133]
1134]
1135]
1136]
1137]
1138]
1139]
1140]
1141]
1142]
1143]
1144]
1145]
1146]
1147]
1148]
1149]
Phe Ile Ala Ser Asp Ile Met Ile Thr Val Ala Lys Ser Arg Pro Gly Gln Ser Gln Val
lie Asp Glu Lys Asn Asp Gln Ser Ala Tyr Pro Val Ser Gln Thr Lys Asn Gly Leu Ser
Val Ser Leu Gln Leu Lys Gln Glu Asn Pro lie Asp Ala Gln Phe Ser Gly Ser Cys Thr
Ser Lys Leu Ala Leu Tyr Glu Gln Ala Trp Phe Ala Cys Leu Arg Trp Ser Ser Gly Arg
Gln 230 Arg 245 Asn 260 Ala 275 Ala 290 Ala 305 Leu 320 Gly 335 Leu 350 Gly 365 Pro 380 Ser 395 Tyr 410 Leu 425 Ser 440 Cys 455 Thr 470 Ser 485 Ser 500 Leu
Gly Leu Glu Ala Tyr lie Lys Gln Gly Arg Phe Gly Ala Ala Arg Met Phe Leu Leu Pro
Thr Gln Leu Pro Glu Thr Ala Tyr Thr Tyr Ser Arg Pro Asp Leu Glu Tyr Ala Arg Cys
Gly Phe Ala Ser Lys Leu Met Arg Val Ser Leu His Trp Ser Leu Arg Gln Ala Cys His
Phe lie Arg Ser Gln Gln Ala Thr Glu Leu Cys Tyr Arg Val His Pro Cys His Lys Cys
lie 235 Val 250 Leu 265 lie 280 His 295 Ala 310 Asp 325 Pro 340 Gln Asn
Ala Ser Asn Leu Asn Asn Ala Ala
355 Trp 370 Gln 385 Leu 400 Pro 415 Thr 430 Gly 445 Thr 460 His 475 Gln 490 Ser 505 Ser
Arg Ala Pro Ser Gly Arg Arg Phe Pro His Arg
Ala Ala Ser Thr Asp Ser Leu Gly Gly Tyr Ala Phe Tyr Ser Ser Leu Arg Tyr Ser Arg
Lys Asn Lys Leu Lys Val Pro Ser Leu Val Arg Gly Cys lie lie Asn Gln Cys Thr Tyr
Ser Ser Leu Met Val Glu Lys Tyr Ala Ser Trp Leu Trp Cys Asn Lys Ala Leu Ser Leu
lie 240 Glu 255 Glu 270 Arg 285 Ala 300 Thr 315 lie 330 Phe 345 Phe 360 lie 375 Phe 390 Lys 405 Cys 420 His 435 Val 450 Asp 465 Asn 480 Glu 495 Ala 510 Cys1150]
1151]
1152]
1153]
1154]
1155]
1156]
1157]
1158]
1159]
1160] 1161] 1162]
1163]
1164]
1165]
1166]
1167]
1168]
1169]
1170]
1171]
1172]
1173]
1174]
1175]
1176]
1177]
1178]
1179]
1180] 1181] 1182]
1183]
1184]
1185]
1186]
1187]
1188]
Asp Glu Pro Thr Leu Tyr Tyr Arg Thr His Gln Ala Leu Asn
Cys Ala Arg Ala Trp Cys Ser Ala Arg Cys Gly Thr Cys lie
Trp Trp Asn Thr Gln Ala Ala Arg Ser Ala lie Tyr Ser Thr
515 Leu Asn 530 His Cys 545 Gly Arg 560 Arg lie 575 His Gly 590 Leu His 605 Tyr Pro 620 Arg Arg 635 Pro Met 650 Asn Lys 665 Arg Pro 680 Trp lie 695 Leu Arg 710 Arg Gly 725
Pro Ser Thr Thr lie Cys Arg Ala Arg Cys Ala Lys Glu
Val Pro Lys Gln Ser Gln Ala His Glu His Arg Tyr Ser Lys Lys Leu Gln Gln Gln Arg
Arg Ser Cys Tyr Asn Phe Ala His Val Arg
His Ser Ser Pro Ser Asn Thr Asp Lys
Ser Leu
520525
lie lie Ser Ala Asp Glu 535540
Asp Arg Tyr Ala Tyr Cys
550555
Arg Ala Val Leu Arg Ser
570 Cys Cys 585 Ser Asn 600 Arg Leu 615 lie Cys 630 Cys Gln 645
Leu Ser Leu Leu 660
His His Pro Ala 675 Cys lie 690
Gly Ala lie Ser 705
Asp Val Glu Pro 720
565 Gln 580 His 595 Leu 610 Arg 625 Thr 640 Phe 655 Arg 670 Ser 685 Ser 700 Arg 715
Ser Ala Tyr Ala Val Leu Tyr Gln His Gln His Phe Asp lie Arg Asn
Val Gly Gln Arg Tyr Arg Tyr Ser Pro His Ala Thr
<210>8
<211>4107
<212>PRT
<213>希瓦氏菌(shewanella piezotolerans <400>8
Met Pro Gly lie His Trp Arg Leu lie Met 1510
Pro Gln Gln Ser Ser Ala Ser Glu Gln Ala
2025
Asp Thr Arg Leu Asn Lys Arg Leu Lys Asp
WP3)
Ser Gln Thr Ser Lys 15
Gln Asp Ser Gln Ala 30
Met Pro ValAla lieVal Gly Met Ala Phe Trp Asp Leu Pro Ala Thr His Ser Thr Pro Asp Asp Val Asp Phe Leu Glu Leu Thr Lys Glu Asp Lys
Val Leu Ala Asp Ala 140
lie Gly lie Thr Leu
Ser His Ser Leu
Val lie Phe Arg Ala Glu Asn Thr
Phe Ala Asn Lys Lys Phe Pro Gly Ser Asp Phe
Ser Pro Ser
Thr Asn Glu Met Met lie Gly Glu Asp Ala Glu
35 Ser 50 lie 65 Trp 80 Lys 95 Asn 110 Asp 125 Ala 140 lie 155 Thr 170 Ser 185 Gln 200 Leu 215 Gly 230 Ala 245 Glu 260 Met 275 Thr 290 Glu 305 Arg 320
lie Phe Ser Glu Gln Lys Ser Tyr Pro Met Ser Ser
Ala Gly Asp Gly Gly
Met
Phe
Gly Ser Leu Gly Arg Ser Glu Met Met
Ala Met
Ala Lys Glu Cys Glu Gln Asn Gly
40
Asn Ser Arg 55
lie Asp Ala 70
Tyr Phe 85
Arg Gly 100
Phe Gly Leu
Asp Lys
Leu
Leu
Val
Arg Leu Gln lie Ser Asp Gln Asn
Tyr Val Asn Arg lie
Val
lie
Cys
Met
Thr
Tyr Met Ser Phe
lie Gln Pro Phe Gly lie Asp Gly
115 Leu 130 Pro 145 Gly 160 Tyr 175 Glu 190 His 205 Ala 220 Val 235 Ala 250 Gly 265 Ser 280 Asp 295 lie 310 lie 325
Val
Tyr Leu Asn lie Asp Gly
Thr Ala Phe
Glu Val Gly Gly Pro Val Asp Ser Trp Gly
Glu Arg Asp
Leu Ser
Gly Met Asp Arg lie Tyr Ser
Glu
Asn
Leu
Pro Pro Asn
Ser Leu lie Val Tyr Gln
Asp Lys Leu Lys Glu Met Glu Asn lie Ala Ala Ala Glu Leu
Gly Val Cys Thr Lys Thr Pro Ala lie Asp Ser
Lys
Ala Leu Lys Arg Val lie
45 Lys 60 Leu 75 Lys 90 Pro 105 lie 120 Ala 135 Arg 150 lie 165 Lys 180 Leu 195 Ser 210 Asn 225 Cys 240 Val 255 Asp 270 Phe 285 Gly 300 Leu 315 Lys 3301228]
1229]
1230]
1231]
1232]
1233]
1234]
1235]
1236]
1237]
1238]
1239]
1240]
1241]
1242]
1243]
1244]
1245]
1246]
1247]
1248]
1249]
1250]
1251]
1252]
1253]
1254]
1255]
1256]
1257]
1258]
1259]
1260] 1261] 1262]
1263]
1264]
1265]
1266]
Gly Pro Asp Gly Cys Leu Gly Val Asp Trp Ser Tyr Arg Lys Ser Val Asn Lys Ile Phe
Ile Arg Ala Thr Ser Gly Thr Leu Ile Leu Phe Lys Gln Asp Ala Arg Ser Leu Ser Ala
Gly Pro Gly Gly Val Ser Ala Pro Glu Pro Gly Ala Arg Ala Ala Glu Ala Thr Phe Gly
Ser Glu Phe Thr Phe Val Gly Ala Asn Arg Phe Glu Gln Leu Glu Leu Ala Ala Arg Gln
Ser Ser 335
Gly Gln 350
Ala Pro 365
Ala Ala 380
Ser Asp 395
Lys Ser 410
Leu lie 425 Thr lie
440 Ser 455 Val 470 Gly 485 His 500 Val 515 lie 530 Phe 545 Ala 560 Glu 575 Ser 590 Ala 605 Gly
Pro Asp Gly Ser Ala Lys Asn Thr Leu Asp Ala Ser
Asp Ala His Gly Gly Gln Lys Asn Phe Gly Thr Arg Gln Glu Leu Asp Ala Asp Ala Gln
Gly Lys Thr Asp Asn lie Ala Val Tyr Thr Asn Glu Ser Leu Lys Ala Ser Thr lie Tyr
Lys Ala Leu Ala Asp Gly Ala
Ser
445 Leu Asn 460 Pro Arg 475 Phe His 490 Asp Ser 505 Phe Leu 520 Asn Thr 535 Asp Ala 550 Pro Arg 565 Phe lie 580 Gln Trp 595 Glu Gly 610 Leu Asn
Phe Lys 340
Leu Asn 355
Gly Leu 370
Ala Glu 385
Thr Lys 400
His Thr 415
Leu Ala
430 Gln Pro
Thr
Arg
Phe
Glu
Val
Leu
Ala
lie
Lys
Gln
Lys
Met
Ser Arg lie Phe Gln Lys Leu Ser Glu Ala Val Lys Ser Ala Ala Gly Gln Leu Val Gly
lie Ala Glu Ala His Ser His Pro Thr Gly Leu Ala Ala Ala Asn Leu Ala Pro Ala Arg
Tyr Tyr Ala Gly lie Thr His Lys Arg lie Glu Lys Thr Ala Tyr Val Leu Gly Ala Asp
Ala 345 Asp 360 His 375 Leu 390 Ala 405 Ala 420 Lys 435 Leu 450 Pro 465 Ser 480 Glu 495 Tyr 510 Ser 525 Ser 540 Ala 555 Ala 570 Ser 585 Gly 600 Leu 615 LeuAla Lys Tyr Ala Ile Ala Cys Phe Gly Ala Gly Ala Asp
Cys Val
Tyr Phe Pro Lys Pro
His
His Thr Asp Ala
Tyr Ala
Val Leu Ser Met Asp Met Ala Ala
Ala Arg Asp Asp Lys
Glu Val Gly Pro
Thr Gly Val Gly Gly Ala Leu
Val Thr
lie Asn
Ala Ser Gln Leu Phe Gly Pro
Ser Phe Glu
Ala Gly Lys Ala Lys
620 Pro 635 Ala 650 Val 665 Asn 680 Gln 695 Ala 710 Val 725 Asp 740 Asp 755 Ser 770 Ala 785 Ala 800 Lys 815 Val 830 Lys 845 Ala 860 Lys 875 Met 890 Asn 905
Glu Met Arg Lys Lys Asn
Asn
Phe
Thr
Asp Asn Ala Ala
Leu Phe
Tyr
Gly His Ser Phe
lie Ser
Ala Met
Cys Gly
Thr Leu
Asn Tyr Ser Thr Tyr Lys Ala lie
Leu Gly
Glu Leu Val Gly His Ala
Phe Thr
Leu Tyr
Thr Ala Ala Glu Asn Thr Asn Ala Lys Glu
His Met Leu Gln
Phe Asp Ala lie Leu Gln
625 Gln Gln 640 Thr Pro 655 Asp Asp Val
670 Ala Gln Ser 685
Ser Ala 700 Gly 715 Asp Asp Tyr
730 Thr Lys Ala
745 Met Phe Ala
760 Ser Thr lie
775 Ala Pro Thr
790 Asp Ala Ala
805 Leu Pro 820 Gln Ala Pro
835 Pro Ser Arg
850 Ser Thr Ala 865
Ser Val 880
Ala Arg 895
Leu Val 910
Leu Val Leu Ser Lys Ala Ala
Gly Lys
Thr Ala Gln Thr Ala Gln lie Gly Gly Phe
Glu Leu Ser Ala
Tyr Lys Val Ala Gln Lys
Lys Leu Thr Lys lie His Lys Phe Ser Val
Ala Leu Val Ser Gly Ala Ala
Phe Ala Ala Arg Val Gln
Lys Leu Tyr Lys Val Phe Thr Phe Val Gly Thr
630 Asp 645 Leu 660 Glu 675 Ala 690 Asn 705 Leu 720 Ala 735 Asp 750 Lys
765 Asp 780 lie 795 Ser 810 Phe 825 Ala 840 Ser 855 Lys 870 Ala 885 Glu 900 Leu 915Ala Pro Leu Ala Leu Ala Glu Glu Lys Pro Val Ala Gly Gly Gln Phe Leu Ala Ala Pro
Ser Lys Ala Asp Asn Lys Glu Lys lie Thr Ser Ala Glu Ser Phe Leu Asn Val Pro Val
Lys Gly Val lie Ala Ser Lys lie Val Ala Thr Glu Thr Gly His Glu Gly Gln Ala Ala
Leu Asp Ala Ala Ala Leu lie Val Glu Pro Asp Leu Phe Val Gln Met Ala
Glu Asp Val Pro His Thr Glu
Asp 920 Ser 935 Gly 950 Ala 965 Asn 980 Ala 995 Val 1010 Glu Val 1025 lie Ala 1040 Val Gln 1055 Ala Leu 1070 His Gln 1085 Thr Thr 1100 Ala Leu 1115 Leu Gln 1130 Gln Ala 1145 Ala Thr 1160 Arg Pro Gln
1175 Met Ser Ala
1190 Pro Val Val
Leu Ser Leu Gln Gln Leu Ala Lys lie Ser Gly Thr Lys Val Pro Thr Val Pro Asn Asn Ser Gln Gln Phe Leu Met Pro Glu Ala Gln Gly Asn Val Leu Gln Ala Pro Val Asn Asn
Val Leu Asp Arg Pro Val Val lie Met Thr Phe Leu Thr Ser Thr Asn Thr
Ser Gln lie Pro Thr Ser
lie 925 Lys 940 Asn 955 Ser 970 Ala 985 Lys 1000 Glu Lys 1015 lie Glu 1030 Ser Asn 1045 Ala Ala 1060 Phe Ala 1075 Ser lie
1090 Glu Gln 1105 Leu Gln 1120 Val Gln 1135 Asn Ala 1150 Ala Gln 1165 Val Gln Gln 1180 lie Thr Thr
1195 Thr Pro Ala
lie Asn Ala Ala Asp Pro Met Ser Arg Ala Ala Thr Val Val Lys Glu Glu His Pro Ser Ala Gln Pro Gln Thr Lys Arg Ser Ser His Ala Leu Pro Leu Val Ala Gln Ala Pro Val
Ser 930 Gln 945 Gln 960 Thr 975 Met 990 lie 1005 Val 1020 Glu
1035 Val 1050 Ser 1065 Gln 1080 Tyr
1095 Ala 1110 Glu 1125 Gln 1140 Met 1155 Ser 1170 Val Ser 1185 Ala Ala 1200 Gln Gln
Pro Leu Tyr Leu Lys lie Glu Val Val Val Gln Gln Leu Met Thr Asn His
41Ala Val Ala Thr Ala Pro Val Val Ser Asn Ser Ala Thr Met Leu Glu Met Leu Glu Leu Ser lie Lys Arg Pro Gly Leu Pro
Thr Leu Gly Ser
Gly Lys Ser Ala Ala Val
Gln Gly Asp Val Val Lys
Lys Tyr
Leu Gly Gln Asp
Gln Pro Thr lie Glu
Ser Leu
Ser Glu Pro Leu Asp Ser Val Leu Leu Ser Glu Leu Val
1205 Gln lie 1220 Ala Ala 1235 Val Ala 1250 Val Val 1265 Glu Met 1280 Val Glu 1295 Glu Leu 1310 Glu lie Val
1325 Leu Ser Thr
1340 Pro Gln Val 1355 Gly Thr 1370 Glu Met 1385 Asp Ser 1400 Leu Pro 1415 Thr Ala Ser
1430 Met Pro Leu
1445 Leu Ser Ala 1460 Pro Glu 1475 Ser Val 1490
Glu Ala Val Pro Ser Ser Val Leu Ala Glu Asp Met lie Leu
Ser Pro Ser Tyr Thr Ala Ala Thr
Lys Glu Gly Glu
Met Met Ser Leu Glu lie Lys Gly Leu Ala lie Arg lie Asn Val
Leu His Ser
Gln Val Asp
1210 Thr Lys 1225 Ala Pro 1240 Ser Ala Glu 1255 Thr Gly 1270 Ala Asp 1285 Thr Val 1300 Asp Leu 1315 Met Asn Ser 1330 Val Gly 1345 Lys Thr 1360 Val Val 1375 Leu Glu Met
1390 Arg Val Glu
1405 Pro Glu Leu
1420 Ser Ser Ser 1435 Leu Leu 1450 Gln Thr 1465 Ser Lys 1480 Asp Phe 1495
Ala Ala
Ala Glu
Ala Ala
Gln Ala Lys Ala Ala Ala Asp lie
Ala Val Val
Lys Tyr Pro Leu Gly Asp Glu
Leu
Gln Thr lie Glu Cys Ala
Ser Ser
Leu Ser
Glu Lys Met Glu Leu Gly Ser Pro Glu Asp Gln Val Val Leu Pro Ser Glu Leu Leu Leu Gln Glu Ala Asn
1215 Glu Ala 1230 Pro Val 1245 Ala 1260 Glu 1275 Asp 1290 Leu 1305 Arg 1320 Asp 1335 Glu 1350 Ala
1365 Thr 1380 Ala 1395 Thr 1410 Met 1425 lie 1440 Ser 1455 lie 1470 Ser 1485 Leu 15001385]
1386]
1387]
1388]
1389]
1390]
1391]
1392]
1393]
1394]
1395]
1396]
1397]
1398]
1399]
1400]
1401]
1402]
1403]
1404]
1405]
1406]
1407]
1408]
1409]
1410]
1411]
1412]
1413]
1414]
1415]
1416]
1417]
1418]
1419]
1420]
1421]
1422]
Thr Ala Ser Thr Ala Gln lie Ser Cys Met Val Ser Phe Asp His Gln His Tyr Asn Leu lie Val Asp Ser Glu Leu Lys Gln Asn Leu Asp Lys Gln Gln Ser Glu Ala Asn Tyr Pro
Thr Ala Asn Ser Asn Lys Thr Arg Trp Thr Ser Lys Leu Ala Leu Tyr Glu Gln Ala Trp
lie Thr Arg Phe Lys Arg Val Asp Phe Asn Gln Arg Asn Ala Ala Ala Leu Gly Leu Gly
Asp Ala 1505 Ser Ser 1520 lie His 1535 Thr Gln 1550 Ala Leu 1565 Phe Asp 1580 Cys Lys 1595 Ala Ser 1610 Thr Glu 1625 Thr Val 1640 Gly Thr 1655 Leu Gln 1670 Glu Leu 1685 Ala Pro 1700 Tyr Glu 1715 lie Thr 1730 Lys Ala 1745 Gln Tyr 1760 Gly Thr 1775 Arg Tyr
lie Glu Leu Leu Pro His Ser Glu Gly Gln Leu Pro Leu lie Ser lie His His Glu Ser Gly Phe Phe lie Ala Arg Ser Ser Lys Gln Leu Gln Met Ala Arg Thr Val Glu Ser Leu
His Met Ala Ser Ala Gln Asn Ser Gln Thr lie Val Leu lie His Ala Asp Pro Gln Trp
Gly Lys 1510 Met His 1525 Gln Leu 1540 Thr Thr 1555 Lys Arg 1570 Leu Asn 1585 Asp Leu 1600 Ala Lys 1615 Ala Arg 1630 Thr Ser 1645 Ala Ala 1660 Ser Ala 1675 Asn Ser 1690 Leu Thr 1705 Asn Asp 1720 Asn Ser 1735 Ala Leu 1750 Ala Gly 1765 Asn Gly 1780 Arg Tyr
lie Ala Ala Leu His Ser Ala Gln Val Cys Lys Asn Lys Leu Lys Val Pro Ser Leu Val
Val Leu Ala Ala Ala Thr Cys Ala Met lie Ser Ser Leu Met Val Glu Lys Tyr Ala Ser
Gln Lys Met Asp Asp Gln Arg Ala Ala Ser lie Glu Glu Arg Ala Thr lie Phe Phe lie
lie 1515 Ala 1530 Gln 1545 Ser 1560 Ser 1575 Gln 1590 Thr 1605 Asn 1620 lie 1635 Phe 1650 lie 1665 Ala 1680 Ser 1695 Asp 1710 lie 1725 Met 1740 lie 1755 Thr 1770 Val 1785 Ala
431423]
1424]
1425]
1426]
1427]
1428]
1429]
1430]
1431]
1432]
1433]
1434]
1435]
1436]
1437]
1438]
1439]
1440]
1441]
1442]
1443]
1444]
1445]
1446]
1447]
1448]
1449]
1450]
1451]
1452]
1453]
1454]
1455]
1456]
1457]
1458]
1459]
1460]
1461]
Pro lie Phe Pro Val Asp Ala Ser Ser Ala Cys Tyr Gln Leu Leu Phe
Gln Thr Lys Asn Gly Leu Ser Cys Ala Arg Ala Trp Cys Ser
Arg Ser Ser Trp Cys Gly Ser Thr Ser Ser Ser
Cys Thr Trp Trp Asn Thr Gln Ala Ala Arg
Gly Ser Arg Leu Leu Asn His Cys Arg lie His Gly
Gly Arg
Leu His Tyr Pro Arg Arg
Ala
Arg Ser Pro Met
1790 Phe Ser 1805 Gly Arg 1820 Ala Pro 1835 Ala Asp 1850 Arg Leu 1865 Met Glu 1880 Phe Tyr 1895 Leu Ala 1910 Leu Arg 1925 Pro Cys 1940 Pro Val 1955 Ser Lys 1970 Thr Ser 1985 Thr Ala 2000 lie Glu 2015 Cys Arg 2030 Arg Ser 2045 Ala Lys 2060 Arg Gln 2075
Leu Cys Gln His Tyr Leu Trp Arg Pro Ser Val Thr Leu His Gly Arg Pro Thr Gln Cys His Ala His Gln Cys Lys Ser His Cys Ser Pro Arg lie
Gln Gln
Ser Asp Arg
Cys His Tyr Gln His Asn His Phe Leu Ala Arg
Tyr Lys
Leu His Thr
Gln Val Phe
1795 Ala Ala 1810 Pro Phe 1825 Ser Tyr 1840 Gly Ser 1855 Arg Ser 1870 Arg Leu 1885 Phe Arg 1900 Pro Tyr 1915 His Ser 1930 Arg Arg 1945 lie Ser 1960 Arg Tyr 1975 Ala Val 1990 Val Tyr 2005 Gly His 2020 Gln His 2035 Arg Asp 2050 Tyr Arg 2065 Arg Leu 2080
Arg Gly Cys lie lie Asn Gln Cys Thr Tyr Ala Ala
Trp Phe Leu Lys Trp Cys Cys His Asn Val Lys Asp Ala Asn Leu Glu Ser Ala Leu Cys
Asp Glu Tyr Cys Leu Arg Ser Gln Cys Cys Gln Ser Asn Phe lie Asn
Arg lie Cys
Leu
Cys
Gln
Ser Leu Leu
1800 Lys 1815 Ser 1830 Arg 1845 Pro 1860 Gly 1875 Gln 1890 Ser 1905 Gln 1920 Val 1935 Asp 1950 Glu 1965 Pro 1980 Thr 1995 Leu 2010 Tyr 2025 Tyr 2040 Arg 2055 Thr 2070 His 2085
441462]
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Cys Gly Thr Cys Ile Ser Pro Lys Asn Tyr Ser Ser Ala Val Leu Asp Ala Gly Gly Ser
Ala Ile Tyr Ser Thr Lys Asp Arg Ser Cys Gly Phe Gly Ile Phe Lys Ser Ala Leu Val
Asn Arg Trp Leu Arg Gln Ala Asp Ala Asp Tyr Leu Ile Met Leu Leu Pro lie Gly Tyr
Lys Pro lie Arg Gly Ser Asn Ser Asp Lys Lys Trp Ala Gly Pro Gly Arg Ala Ser Ser
Cys Gln 2090 Ala His 2105 Lys Pro 2120 Glu Thr 2135 Ser Leu 2150 Lys lie 2165 Ser Pro 2180 Arg Ser 2195 Tyr Gln 2210 Gly Gly 2225 Leu Ala 2240 Ala Leu 2255 Leu Asn 2270 Ala Leu 2285 lie Tyr 2300 Ser Gln 2315 Ala Thr 2330 His Asn 2345 Ala Gln 2360 Leu Lys
Arg Ser Ser Asp Met Ala Gln Thr Gly Tyr Ala Asp Asn Ser His Asn Asn Thr Leu Leu
Arg Ser Asn Lys Ser lie Gln Leu Val lie Asp Thr Asp Phe Ser Phe Glu Ser Ser Ala
Arg Ser Ser Arg Ser Val Phe Thr Gln Glu Ser Ser Asp Pro Ala Lys Ser Lys Leu Cys
Tyr His 2095 Ser Pro 2110 Ala Gly 2125 Thr Asp 2140 Ser Asp 2155 Gly Leu 2170 Trp Gln 2185 Asp Lys 2200 Gly Glu 2215 Asn Phe 2230 Leu Glu 2245 Arg Lys 2260 Leu Leu 2275 Thr Glu 2290 Val Glu 2305 Leu Ser 2320 Thr Leu 2335 Val Val 2350 Asp Ala 2365 Asp Tyr
His His Ala Val Arg Ala Asn Lys Ser Ser Ser Ala Ala Arg Lys Ala Asn Ala Ala Leu
Pro Cys lie Glu Asn Thr Leu Leu Asp Phe Leu Leu Arg Ser Ala Thr Thr Asp Cys Ser
Ala lie Ser Pro Thr Leu Leu Gly Arg Asp Asp Asp Thr Asn Leu Asn Ala Ala Ala Thr
Gln 2100 Ala 2115 Leu 2130 Asn 2145 Ser 2160 Tyr 2175 Glu 2190 Ala 2205 Phe 2220 Pro 2235 Asn 2250 Asp 2265 Gly 2280 Asp 2295 Gln 2310 Ala 2325 Asn 2340 Leu 2355 Ser 2370 GlyLys Phe His Ala Ala Gly Pro Asp Gly Phe Ser Pro
Ala Phe Gly Gly Glu
Asp lie Val Glu Arg
Leu Ser
Lys lie Thr Phe Ala Gly
Gly Glu
Val Asn Ser Gly Asp Asn Gln Pro
Pro Leu
942469 A说明书44/53页
2380 Gly Ala Val Ser
2395 Ser lie Phe His
2410 Asp Gly Asn Ser
2425 Leu Val Leu Lys
2440 lie Tyr Ala Val
2455 Gly Gln Phe Val
2470 Phe Glu Arg Ala
2485 Glu Val lie Glu
2500 lie Glu Leu Thr
2515 Asp Ser Ser Ala
2530 His Leu Leu Thr
2545 Phe Ala Met Lys
2560 Asp Ala lie Ser
2575 Pro Asn Leu Val
2590 Phe Gly Val Lys
2605 Gly Gly Cys Asn 2620 Ser Ala Pro Ser
2635 Pro Ala Pro Leu
2650 Leu Ser Thr lie 2665
Glu Lys lie
Pro Pro Ser
Leu Phe Gly Asp Lys Val Glu Val
Asp Ser Met lie Gln Pro Ser
Gly
Leu
Ser
Ser
Ser
Val
Val Tyr Thr
Leu Ser Ser His
2375 Met Leu 2390 Met Gly 2405 Val Pro 2420 Ala Gly 2435 Asn Asp 2450 Asp Gly 2465 Val Lys 2480 Lys Asp 2495 Gly Asp 2510 Lys Leu 2525 Asn Leu 3540 Lys Met 2555 Asn lie 2570 Ala Thr 2585 Asn Asn 2600 Phe Gly 2615 Ser Gln 2630 Ala Ala 2645 Phe Gly 2660
Ala Phe Phe Val Lys Lys Ala lie Lys Gln Gly lie Ser Met His Phe Lys Thr Pro
2385 Gly Ala Asp Pro 2400 Ala Tyr Pro Asp 2415 Lys Gly Leu Phe 2430 Arg Leu Glu Asp 2445 Val Ser Gly Val
2460 Leu Ser Pro Asn 2475 Tyr Ala Ala Ser 2490 Cys His Ala Thr 2505 Ser Met Glu Thr 2520 Pro Leu lie Gly 2535 Ala Ala Gly Met 2550 Glu Gly Leu Leu 2565 Ser Pro Asn Gln 2580 Gln Gly Trp Pro 2595
Thr Arg His Ala 2610 Ala His Leu Leu 2625 Gln Ala Gln Ser 2640 Lys lie Val Gly 2655 Asn Gln Leu Asp 2670Asn Lys Phe Glu Arg Ala Leu Arg Gly Ala Ser Asp Val Asn Phe Thr Glu Gly Gln Thr
Ala Arg Gly Leu Leu Ile Val Val Val Met Phe Phe Ser Leu Glu Pro Gly Arg Gln Asn
lie Trp Leu Asp lie Arg Ala Asn Asn Asp lie Asn Arg Asp Gln Lys Ala Ala Leu Ser
Ala Lys Asn Phe Ser Asp Met Leu Leu Ser Gly Gly Cys Ala Val Gly Gly Tyr Pro Thr
Ser Asn 2675 Gly Leu 2690 Ala Ala 2705 Leu Arg 2720 Gln Gln 2735 Ala Lys 2750 Glu Thr 2765 His Thr 2780 Ser lie 2795 Val Leu 2810 Asn lie 2825 Pro Ala 2840 lie Asp 2855 Val Val 2870 lie Leu 2885 Asn Thr 2900 Ala Val 2915 Gly Gln 2930 Gln Val 2945 Ser lie
Ser Glu Pro Phe Leu Leu Glu Gln Asp Asp Met Phe Val lie Lys Ala Val lie Thr Asn
Asp Lys Lys Lys Met Gln Leu Leu Glu Ala Ala Thr Ala Ala Asn Ala Leu Asp Asp Val
Ala His Gly Leu Leu Ala Glu Gln Tyr Ala Ser lie Gln Ala Ser Gln Val Ala Lys Val
Phe lie 2680 Pro Glu 2695 Ala Tyr 2710 Pro Pro 2725 Met Arg 2740 Gly Gln 2755 Leu His 2770 Gln Ser 2785 Gln Ala 2800 Lys Leu 2815 Arg Val 2830 Ser Ala 2845 Asn Leu 2860 Val Asp 2875 lie Ala 2890 Ala Ser 2905 Lys Asp 2920 Leu Ala 2935 Leu Leu 2950 Glu Thr
Glu Leu Val Asn Val Lys Gln Leu Leu Asn Ser Ala lie Leu Pro Trp Glu Phe Thr Asn
Leu Leu Asp Glu Thr Val Phe Ala Glu Gln Ser Glu Ala Ser Val Asn Ser Gly Gln lie
Pro Ala Asn Asp Asp Ala Arg Ala Ala Tyr Leu Gln Glu Gly Ala Val Thr His Thr Ala
Glu 2685 Gln 2700 Phe 2715 Asp 2730 Glu 2745 Val 2760 Gly 2775 Leu 2790 lie 2805 Thr 2820 Trp 2835 Ser 2850 Asp 2865 Ser 2880 Met 2895 Gly 2910 Ser 2925 Ala 2940 Ala 2955 ProGly Ser Ala Val Ala Ala Arg Asn Glu Asn Gln Gln Ala Lys lie Tyr lie Lys Gln Pro Leu Ser Thr Ala Thr Ser Val Val Phe Gln Leu Lys Lys Gln Thr Val lie Ala
Leu Thr Thr
Ala Gly Ala Gln Ala
Met Ala Ala Leu
Lys Lys
His Gln Gln His Asn Asn Thr Leu Ala Val Ser Met Ser Thr Gln Asn Ser Arg Gln Leu Thr Thr Ala Ala
2960 Thr Asn 2975 Ser Ala 2990 Ala Ser 3005 Ala Thr 3020 Ser Gln 3035 Ala Ala 3050 Leu Val 3065 lie Tyr 3080 Met Ala 3095 Pro Met 3110 Pro Met 3125 Ala Thr 3140 Thr Thr 3155 Thr lie 3170 Gln Gln 3185 Ser Ala 3200 Ala Gln 3215 Thr Ala 3230 Val Asn 3245
Val Thr Leu Asp Asp Arg Leu Leu His Asn Lys Ala Leu Leu lie Ala Val
Arg Leu Lys Gln Asp Thr Asn Gly Val Val Ser Ala Gln Thr Gln Val Asp Val Leu Ala Gly Met Ala Thr Pro Ala Val lie
Pro Ala Arg Val Leu Lys Ser Gln Gln Gly Ala Ala
2965 Asn Asp 2980 lie Gly 2995 Gly Leu 3010 Leu lie 3025 Ser Gln 3040 Asn Glu 3055 Thr Leu 3070 Leu Ala 3085 Val Ser 3100 Ala Gln 3115 Ser Ala 3130 Ser Thr 3145 Pro Thr 3160 Ala Gly 3175 Gln Ala 3190 Val Ala 3205 Gln Ala 3220 Val Ala 3235 Pro Asp 3250
Ser Phe Val His Cys Phe Leu Ser Val Thr Asn Val Ser Gln Cys Leu Lys Ser Gly Gly Arg Ser Leu Ala Thr Leu Pro Gln
Thr Ser Asp Thr Ala
Gln Gln Asp Leu
Asp Val Gln Pro
Ala lie Leu
Asn Ala His His Ala
Ala Leu Ala Pro
Ala His Ala Asn
2970
Asn
2985
Ala
3000
Ser
3015
Ser
3030
Glu
3045
Asp
3060 Asp
3075 Ala 3090 Leu 3105 Gln 3120 Asn 3135 Gly 3150 Pro 3165 Ala 3180 Phe 3195 Leu 3210 Gln
3225 Pro
3240 Val 32551618]
1619]
1620] 1621] 1622]
1623]
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1625]
1626]
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1650]
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1653]
1654]
1655]
1656]
Pro Asn Val Val Lys Thr Gln Met Arg Leu Asn Glu Cys Val Lys Ser Cys Phe Glu Lys
Pro Tyr Phe Arg Leu Glu lie Leu Val Pro Tyr Cys Ala lie Lys Tyr Phe Ala Arg Gln
Tyr Ala Gly Leu Asp Tyr Pro lie Tyr Arg Ala Phe Gly Arg Thr Pro Gly Ser Asn Leu
lie Asp Ser Pro Ala Asp Trp Ser Arg Gly His Val Phe Thr Phe Glu Ala Glu Gly Glu
Ala Pro 3260 Leu Val 3275 Asp Tyr 3290 Thr Thr 3305 Thr lie 3320 lie Pro 3335 Ala Val 3350 Tyr Leu 3365 Leu Leu 3380 Gly Asp 3395 Asn Gly 3410 Gly Asp 3425 Phe Thr 3440 Glu Asp 3455 Asn Pro 3470 lie His 3485 Ser His 3500 Lys Phe 3515 Gly Thr 3530 Ala Asn
Thr Glu Ala Asp Asn Val Ala Gly Asp Thr Glu Thr Asp Glu Leu Lys Lys Leu Trp His
Pro Tyr lie Tyr Glu Asp Val lie Cys Leu Thr Met Glu lie Leu Leu Gly Met Gly Trp
Ala Leu Lys
3265 Ala Glu Gly
3280 lie Asp Ser
3295 Leu Leu Val
3310 Phe Lys Pro 3325 Pro Tyr 3340 Ser Gly 3355 Phe Glu 3370 Leu Thr 3385 Tyr Asp 3400 Leu Phe 3415 Leu Lys 3430 Leu Ala 3445 Ala Arg 3460 Cys Pro 3475 Thr Ala 3490 Ala Gln 3505 Glu Gln 3520 Gly Leu 3535 Phe Pro
Ala Glu Asp Thr Arg Leu lie Glu Lys Asp Leu Asn lie Leu Tyr
Pro Asp Tyr Ser Cys Leu Gln Asn Phe lie Phe Met Asp Ser Lys Asp Pro Val lie Cys
Cys lie Ser Arg Ser Val Cys Lys Leu Lys Phe Asp Gly Leu Thr lie Ser Ser Glu His
lie Ala Arg Val Met Asp Asp Gly Gly lie Ser Gly Lys Ala Ser Glu Leu Lys Gly Phe
Trp
3270
Asn
3285
Arg
3300
Thr
3315
Thr
3330
Gly
3345
Leu
3360
Glu
3375
Asp
3390
Asn
3405
Tyr
3420
Gly
3435 Gly 3450 Val 3465 Phe 3480 Gly 3495 Cys 3510 Val 3525 His
3540 Gln
49Gly Gln Thr Val Tyr Ala Asp Thr Thr Asn Asn Pro
Asp Gln
Leu Leu
Lys Arg Arg Lys Phe Arg Ala Ala Lys Gly
Tyr Gly Pro
Pro Gly Tyr Gln Pro
Cys Lys Glu Ala Asn
Asn
Cys
Met
Val Gln Gly Arg Glu Asn Asn
Ala Gln Tyr Pro Thr Cys Pro Leu Gly Val Tyr Pro
His Met Phe Phe Asp Gly
Glu Gly Arg Val Val Gly
Pro
Met
Phe
3545 Met Ala 3560 Phe Tyr 3575 Arg Phe 3590 Gly Gln 3605 Val Thr 3620 lie Asp 3635 Leu Gly 3650 Leu Leu 3665 Asn Ala 3680 Met Ala 3695 lie Pro 3710 Asn Arg 3725 Glu Phe 3740 Ser lie 3755 Leu Gln 3770 Asp Phe 3785 Thr Asp 3800 Pro Tyr 3815 lie Ser 3830
Gly Ser Leu Met Leu His Gln Pro Leu Val Leu Pro Glu lie Gly lie Leu Leu Val Met lie Thr Ser Ser His Lys Glu Gln lie Pro lie Val Ala Tyr Val Lys Ala
Ser His
Ser
Gly
Pro Thr Arg Thr Lys Trp lie Tyr
Asp Gly Gly Thr Pro Tyr Leu Met
3550 Met Ala 3565 Leu Gly 3580 Glu Asn 3595 Gln Ser 3610 Phe Asn 3625 Asn Gly 3640 Lys Glu 3655 Thr Thr 3670 Tyr Gln 3685 Asp Leu 3700 Val Glu 3715 Thr Val 3730 Asn lie 3745 Met lie 3760 Arg Val 3775 Ser Ser 3790 Phe Asp 3805 Met Glu 3820 Gly Thr 3835
Glu Gly Cys Met His Thr Ser Gln
Ala Gly Pro Lys Glu Asn Ala Thr Ala
Val Gln Ala Asp Glu Ala Lys Pro
Pro Phe Glu Asn Pro Pro
Glu lie Asn
Leu Pro Val Glu Pro Ser Ala lie Thr Cys Arg Val Ala
lie Cys Lys lie Ser Leu Thr Leu Gly
Ser
3555 Gly
3570 Gln 3585 Gln 3600 Thr 3615 Tyr 3630 Val 3645 Cys 3660 Ser 3675 Val 3690 Pro 3705 Thr 3720 Pro 3735 Phe
3750 Thr 3765 Asn 3780 Glu 3795 His 3810 Gln 3825 Phe 38401696]
1697]
1698]
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1700]
1701]
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1733]
1734]
Pro Leu Ser Ser Gly Gln Ala Ser Leu Asp Thr Asp Thr Lys Tyr Val Thr Val
Gly Leu Leu Arg Arg Leu Phe Ser Ser Ala Leu Gly Asn Asp Cys Arg Ala Gly Asn Gly lie Asp Pro Val Met Gln Asn Pro Arg Gly Ser lie Gly Asn Phe Asp
Glu Leu Phe Phe Arg Asn Leu Asp Gly Ser Gly Glu 384538503855
Glu Val Asp Leu Arg Gly Lys Thr lie Arg Asn Asp 386038653870
Leu Ser Thr Val Met Ala Gly Thr Asn lie lie Gln 387538803885
Phe Glu Leu Ser Thr Asp Gly Glu Pro Phe Tyr Arg 389038953900
Val Phe Gly Tyr Phe Lys Gly Glu Ala Leu Lys Asp 390539103915
Leu Asp Asn Gly Arg Val Thr Gln Pro Trp His Val
392039253930
Val Ala Pro Thr Thr Thr Val Asn Leu Leu Asp Lys 393539403945
His Phe Asn Ala Pro Ala Ser Gln Pro His Tyr Arg 395039553960
Gly Gln Leu Asn Phe lie Asp Ser Val Glu lie Val
396539703975
Gly Thr Glu Gly Leu Gly Tyr Leu Tyr Ala Glu Arg 398039853990
Pro Ser Asp Trp Phe Phe Gln Phe His Phe His Gln 399540004005
Met Pro Gly Ser Leu Gly Val Glu Ala lie lie Glu 401040154020
Thr Tyr Ala lie Ser Lys Asp Leu Gly Ala Gly Phe 402540304035
Lys Phe Gly Gln lie Leu Ser Asn lie Lys Trp Lys 404040454050
Gln lie Asn Pro Leu Asn Lys Gln Met Met Asp 405540604065
Thr Ser Val Lys Asp Glu Asn Gly Lys Lys Val lie 407040754080
Ala Ser Leu Ser Lys Asp Gly Leu Arg lie Tyr Glu 408540904095
lie Ala lie Ser lie Glu Glu Ser Val 41004105
<210>9
<211>542
<212>PRT1735]
1736]
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1740]
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1773]
<213>希瓦氏菌(shewanella piezotolerans <400>9
Met Asn Pro Thr Thr Thr Asn Glu Met Leu 15
Leu Val Thr Asp Thr Asn 20
Asp Phe 35
Val Gly 50
Asn Ser 65
Thr Glu 80
Lys Tyr 95
Glu Glu 110
Phe Gly 125
Asn Arg 140
Leu lie
155 Glu Leu
Gln Asn Asp Pro Val Gly lie Glu Tyr Arg Glu Arg Ala Lys Asp Val His
Asn Val Gln Glu Gln Ala His lie Leu Lys Met Gly Ala Ala Asn Phe Glu Pro Thr
Pro Thr Leu Lys Ala Leu Gly Ser Cys Ala Phe Ser Ser Ala Lys Met Gly Met Asp
WP3) Ser Pro
Asp Arg Gly Ala Gly Val Ser Ser lie Asn Val Ala Gly Val Met Ser Ala Asn
170 Phe 185 Leu 200 lie 215 Pro 230 lie 245 Asp 260 Arg
Leu Ser Ala Ala Thr Asp Pro
lie Ser lie Asn Ser Ala Leu Ala lie His Phe Gly Arg Lys Pro Pro lie Leu
Ser Arg Ala Ser Leu Tyr Val Ala Gln Ser Leu Leu Asp Val Ala Glu Thr Val
10
Phe Asp 25 Gly Cys 40 Gln Thr
55 Leu Pro 70 Gly Asp 85 Tyr Ala 100 lie Ala
Gly Ala Pro Lys Thr Ala Ser Lys Gln Ala Thr
115 Leu 130 Ala 145 Ser 160 His Lys
Val Tyr Ala Val Ser Gly Leu lie Leu Glu
175 Pro 190 Gln 205 Arg 220 Met 235 Met 250 Glu 265 Leu
Gln Gly Thr Leu Glu Ala Leu
Thr Val Glu Ala Asn Ala Gly Pro Pro Pro Val lie Asn Glu Gln Leu Asp Pro
Trp Pro Trp 15
Val Met Glu 30
Val Asn His 45
Leu Val Ala 60
Ala Phe Thr 75
Phe Arg Arg 90
Met Ala Asn 105
Gln Ala Gly 120
Ser Arg Val 135
Asn Gly Pro 150
Ala Leu Glu 165
Arg Thr Val 180
Val Tyr Tyr 195
lie Val Val 210
Val Ala Glu 225
Lys Leu Val 240
Ala Gln Leu 255
Ser Gly Gly 270
Thr lie Leu
521774]
1775]
1776]
1777]
1778]
1779]
1780]
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1789]
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1797]
1798]
1799]
1800] 1801] 1802]
1803]
1804]
1805]
1806]
1807]
1808]
1809]
1810] 1811] 1812]
Ala Ile Leu Val Lys Ala Arg Tyr Glu Trp Ile Phe Glu Leu Tyr Gly Val
Leu Lys Arg Val Ala Thr Asn Gln
Glu Gly Phe Ala
Leu Leu Ser
Ala Asp Gly Thr Thr Arg Lys Leu Ala Gly Glu Arg Arg Trp Thr Gly Gly Ala Gln Glu Ala Ala Lys Leu
Met Leu Tyr Glu Thr Ala Tyr Arg Phe Arg Tyr Pro
275 Glu 290 Cys 305 Asn 320 Cys 335 Thr 350 Phe 365 Phe 380 Asp 395 Lys 410 Val 425 Thr 440 Leu 455 Glu 470 Asn 485 Asn 500 Leu 515 Thr 530
lie Gly Met Val Thr Glu Pro Ser Gln Ala Asp Gly Met Gln Ala Asn Gln
Gln Thr Gly Gly Gly Ala Glu Ala Glu Met Met Gly Met Arg lie Glu Val Phe His Phe Asn Pro
Lys Val Ala Gly Ala Val Ala Ala Arg Asn Lys
Leu Ser Ser
Asp Tyr Trp Ala Val Asp Arg Val Leu Leu
Gln Lys Val Asn Arg
280 Tyr 295 Gly 310 Tyr 325 Ala 340 Asp 355 Lys 370 Asn 385 lie 400 Ser 415 Glu 430 Arg 445 Arg 460 lie 475 Gly 490 Ala 505 Ser 520 Trp 535
Gln Thr lie Ser Val Leu
Tyr Pro Asp
Pro Val Glu Thr Gln
Lys Leu Pro Ala
Thr Arg Lys Trp Trp
Thr
His
Met
Val
Tyr Asp Asp Asp Pro
Leu
Met
Ser
Ala
Ala Asn Gly
Thr Ala Gly Thr Ala Val Glu Glu Asp Lys Leu Ser Pro
Ser Tyr Leu Asp Lys His Leu Met
Leu Thr Ser Lys Pro Thr
Gln Gln
285 Pro 300 Ala 315 Ser 330 Arg 345 Pro 360 Lys 375 Leu 390 Arg 405 lie 420 Gln 435 lie 450 Gly 465 Ala 480 Asn 495 Tyr 510 Gly 525 Arg 540
Met Ala <210>10 <211>523 <212>PRT
53[1813 [1814 [1815 [1816 [1817 [1818 [1819 [1820 [1821 [1822 [1823 [1824 [1825 [1826 [1827 [1828 [1829 [1830 [1831 [1832 [1833 [1834 [1835 [1836 [1837 [1838 [1839 [1840 [1841 [1842 [1843 [1844 [1845 [1846 [1847 [1848 [1849 [1850 [1851
<213>希瓦氏菌(shewanella piezotolerans WP3) <400>10
Met 1
lie Leu Val Tyr Glu Val Phe Phe Asp Ser Lys Asn Val Lys Ser Leu lie Thr
Leu Pro Asn Thr Val Asn Phe His Gly Gly Gly Ser Gln Val Asp Gln Thr Gly Val
Phe Gln Ser Leu Leu Phe lie His Thr Lys Pro Leu Glu Met Trp Arg Leu Met Tyr
Thr Leu Met Asn Gln Leu Leu Lys Phe Lys Ala Pro Leu Thr Asp Val Tyr lie Leu
Ala
5 Glu 20 lie 35 Tyr 50 Pro 65 Asn 80 Asn 95 Asn 110 Pro 125 Val 140 Leu 155 Ser 170 Val 185 Pro 200 Ala 215 lie 230 His 245 Ser 260 Leu
Val Ala Phe Asp Thr Asp Ser Glu Glu Leu Tyr Asn Asn lie Glu Arg Thr Leu Leu
lie Thr Ala Tyr Ser Thr Gln His Asn Ala Ser Gly Glu Ser Val Asn Asn Phe Ser
Phe Met lie Ala Lys Phe Ala Ala Arg Lys Ser Phe lie Ser Glu Thr Ser Phe Gly
Ser Leu Lys Val His lie Glu Leu Val Thr Thr Leu Ser Gln Leu Lys Gln Ala Phe
Tyr Arg Phe Phe 10
Lys Leu Ser Glu 25
Arg lie Asn Lys 40
Trp Asp Glu Thr 55
Ser Leu Arg Tyr 70
Arg Leu Glu lie 85
Leu Leu Tyr Ser 100
Thr Phe Asn Leu 115
Leu lie Asp Thr
130 Gly 145 Pro 160 lie 175 Gln 190 Gln 205 Thr 220 Gly 235 Ala 250 Met 265 lie
Val Val Ser lie Leu Leu Phe lie Arg Phe Thr Ala Glu Ala Lys Gln lie Leu Glu Ala lie Pro Ser lie lie Met Leu lie Arg Pro
lie Gln 15
Arg Glu
30 Pro Met 45 Phe Glu 60 Leu Val 75 Asp Gly
90 Lys Gly 105 His Gln 120 Pro Tyr 135 Thr Leu 150 Ser Asp 165 Leu lie 180 Thr Pro 195 Gly Leu 210 Pro Phe 225 Leu Lys 240 Trp Gly 255 Leu lie 270 Val Gln
54Arg Leu Ala Arg Lys
Glu Leu Pro Ser Phe His Phe Asn Ala Leu Leu Gly
Thr Leu Leu Ser Ala Asn
Leu Leu
lie Gly Phe Phe Lys
Asp Asp Lys lie
Arg Lys Tyr Glu Arg
Val Cys Ser Phe Leu Tyr Thr lie
Arg Arg Phe Ala Ala
Met Leu
Leu Ser Glu
His Pro Ser Ser
Gln lie Gln Thr
Val Thr Ala Ala
275 Asn 290 Lys 305 Asn 320 His 335 Ala 350 Gln 365 Lys 380 Leu 395 Glu 410 Tyr 425 lie 440 Glu 455 Leu 470 Gly 485 Lys 500 Asn 515
lie Lys Ala Met Asn Tyr lie lie
Gln Phe Asn Lys Lys Asp Ser Leu Val
Val Tyr Val Glu Leu His lie Gly Phe Tyr Asn Asp Glu Val Ala Met Cys Ala Asp lie Pro Gln Ser Leu Ser Asp Tyr
Pro Met Pro Asn Lys Gln lie Thr Glu Ser Gly Arg Asp lie Pro Ser
280 Asp 295 Thr 310 Thr 325 Asp 340 Leu 355 Thr 370 Thr 385 Lys 400 Arg 415 Phe 430 Gln 445 lie 460 His 475 Glu 490 Asn 505 Asp 520
Lys Lys Gln Lys Glu Leu Arg Lys Val Gln Arg Gln Glu Leu Thr Gln Ala Thr Gln lie Leu lie Leu Gly Leu Gly His Leu Met Leu Glu Gln Phe Gly Gly Glu Ala Pro Leu Glu
His Thr Ser Met Gln Asn
Arg Val Tyr Ala Tyr Lys
Ala Leu Ser Phe Gln Asp Asp Lys Tyr Lys Asp
285 Tyr 300 lie 315 Asn 330 lie 345 Gln 360 Asp 375 Ala 390 His 405 Glu 420 Arg 435 Pro 450 Gly 465 Asn 480 Ala 495 Val 510
权利要求
一种EPA合成基因簇，其具有SEQ ID NO 1所示的核苷酸序列，该EPA合成基因簇包含编码以下蛋白的基因，具体为金属β 内酰胺酶基因，位于第1 1998碱基，编码金属β 内酰胺酶；PPTase基因，位于2062 2835碱基，编码磷酸泛酰巯基乙胺转移酶；TonB依赖受体基因，位于3912 6818碱基，编码TonB依赖受体蛋白；Fis调控因子基因，位于7251 8117碱基，编码Fis转录调控因子；PfaA基因，位于第8090 16279碱基，编码omega 3多不饱和脂肪酸合成酶；PfaB基因，位于第16279 8525碱基，编码omega 3多不饱和脂肪酸合成酶；β 酮脂酰合成酶基因，位于第18525 24395碱基，编码β 酮脂酰合成酶；PfaD基因，位于第24588 26216碱基，编码omega 3多不饱和脂肪酸合成酶；GGDEF基因，位于第27322 28893碱基。
2.—种含EPA合成基因簇的基因工程菌，其特征在于该基因工程菌在中国典型培养 物保藏中心的保藏号为CCTCC M 209092。
3.一种获得权利要求3所述的含EPA合成基因簇的基因工程菌的方法，包括1)菌株培养采用2216E培养基，10 20°C常压下培养WP3菌株；cosmid文库的克隆 子于35 40°C在常压下培养，所用培养基为LB培养基加上终浓度为50 200ug/ml的氨 苄青霉素；2)WP3基因组cosmid文库的构建先按照标准的提总DNA的方法提取制备大分子量 的基因组DNA，并溶于一定量的TE缓冲液中。接着，将总DNA随机用枪头进行机械打断至 30-45kb，然后用脉冲电泳进行分离，并用电洗脱回收30-45kb的DNA片段；将回收到的DNA 进行末端补平反应以获得平末端的DNA片段，接着把补平后的DNA片段与pTOB载体进行连 接。连接后，产物用MaXPlaXTMX噬菌体包装蛋白进行包装，接着将包装产物去侵染大肠杆 菌EPI100，并用含氨苄青霉素抗性的LB平板进行筛选，完成WP3基因组cosmid文库的构 建；2) Cosmid文库的筛选选用两对引物对cosmid文库进行PCR筛选，以获得包含EPA合 成基因簇的克隆子；其中，第一对引物的扩增区域位于EPA基因簇的第一个开放性阅读框， 第二对引物的扩增区域位于第八个开放性阅读框；用分别位于ORFl和0RF8的两对引物分 别对文库进行PCR筛选，有扩增产物的为携带WP3 EPA合成的基因簇的目的菌株。
4.一种生产EPA的方法，其特征在于使用权利要求3所述的含EPA合成基因簇的基 因工程菌。
全文摘要
本发明涉及希瓦氏菌EPA合成基因簇及含该基因簇的基因工程菌。本发明获得了包含希瓦氏菌shewanella piezotolerans WP3的EPA合成基因簇的大肠杆菌基因工程菌并确定了基因簇负责EPA在WP3中的合成，该EPA合成基因簇包含有一个磷酸泛酰巯基乙胺转移酶(PPTase)基因和pfaA，B，C，D基因，基因转录量受到PfaA上游Fis型转录调控因子的调控。低温和高压条件能导致其提高EPA的合成。
文档编号C12N15/55GK101942469SQ20091011220
公开日2011年1月12日 申请日期2009年7月7日 优先权日2009年7月7日
发明者姜丽晶, 彭华煜, 王峰, 王风平, 肖湘 申请人:国家海洋局第三海洋研究所