CN112789505B - 用于生产大麻素和其它异戊二烯化的化合物的生物合成平台 - Google Patents

用于生产大麻素和其它异戊二烯化的化合物的生物合成平台 Download PDF

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CN112789505B
CN112789505B CN201980063307.7A CN201980063307A CN112789505B CN 112789505 B CN112789505 B CN 112789505B CN 201980063307 A CN201980063307 A CN 201980063307A CN 112789505 B CN112789505 B CN 112789505B
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J·U·鲍伊
M·瓦莱瑞
T·P·科尔曼
N·伍道尔
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Abstract

提供了可用于异戊二烯化的酶和用于在无细胞系统中生产大麻素、大麻素前体和其它异戊二烯化的化学物质的重组途径以及催化反应的重组微生物。

Description

用于生产大麻素和其它异戊二烯化的化合物的生物合成平台
相关申请的交叉引用
本申请要求2018年8月1日提交的美国临时申请系列号62/713,348的优先权，其公开内容通过引用整体并入本文。
关于联邦资助的研究的声明
本发明在美国能源部颁布的授予号DE-FC02-02ER63421和国立卫生研究院颁布的授予号GM008496下在政府支持下完成。政府具有本发明的某些权利。
序列表
本申请含有序列表，其已经以ASCII格式电子地提交，且特此通过引用整体并入。于2019年8月1日创建的所述ASCII副本命名为Sequence_ST25.txt，且是287,021字节大小。
技术领域
提供了通过使合适的底物与本公开内容的代谢修饰的微生物或酶促制剂接触来生产大麻素和其它异戊二烯化的化学物质和化合物的方法。
背景
天然化合物的异戊二烯化增加结构多样性，改变生物活性，并增强治疗潜力。异戊二烯化的化合物经常具有低天然丰度或难以分离。一些异戊二烯化的天然产物包括一大类具有经证实的医学特性的生物活性分子。实例包括异戊二烯基-黄酮类化合物、异戊二烯基-芪类化合物和大麻素。
大麻素是一大类具有生物活性的植物衍生的天然产物，它们调节人内源性大麻素系统的大麻素受体(CB1和CB2)。大麻素是有前途的药理学试剂，目前正在进行100多项研究其作为止吐药、抗惊厥药、镇痛药和抗抑郁药的治疗益处的临床试验。进一步，三种大麻素疗法已被FDA批准用于治疗化学疗法引起的恶心、MS痉挛状态和与严重癫痫相关的癫痫发作。
尽管它们具有治疗潜力，但药用级(>99％)大麻素的生产仍然面临重大技术挑战。印度大麻脂(Cannibis)植物如食用性大麻(marijuana)和工业大麻(hemp)产生高水平的四氢大麻酚酸(THCA)和大麻二酚酸(CBDA)，以及多种较低丰度的大麻素。但是，由于污染性大麻素的高度结构相似性以及每种作物的大麻素组成的变化，即使高度表达的大麻素(如CBDA和THCA)的分离也是有挑战性的。当尝试分离稀有大麻素时，这些问题被放大。此外，当前的印度大麻(cannabis)种植实践带来了严重的环境挑战。结果，对开发用于生产大麻素和大麻素类似物的替代方法存在相当大的兴趣。
发明内容
本公开内容提供了一种重组多肽，其包含选自以下的序列：(a)SEQ ID NO:30且具有至少Y288X突变，其中X是A、N、S、V或非天然的氨基酸；(b)SEQ ID NO:30，其具有至少Y288X突变，其中X是A、N、S、V或非天然的氨基酸，和至少一个选自V49Z1、F213Z2、A232S、I234T、V271Z3和/或G286S的其它突变，其中Z1是S、N、T或G，Z2是H、N或G且Z3是N或H；(c)在表1中所示的突变组合中的任一种；(d)(a)、(b)或(c)中的任一种，其包含1-20个保守氨基酸置换且具有NphB异戊二烯基转移酶活性；(e)与SEQ ID NO:30具有至少85％、90％、95％、98％或99％同一性且至少具有在(a)、(b)或(c)中列举的突变的序列；(f)从氨基酸21开始在SEQ ID NO:1-28或29中列举的序列；和(g)与SEQ ID NO:1-28或29中的任一个具有至少99％同一性的任何序列，其中(a)-(g)中的任一项的多肽执行异戊二烯化反应。在一个实施方案中，所述异戊二烯化反应包括从GPP和油橄榄醇酯(Olivetolate)产生CBGA，或从GPP和2,4-二羟基-6-丙基苯甲酸(divarinic acid)产生CBGVA，或从2,4-二羟基苯甲酸或其在C6位置具有化学基团的衍生物(参见，例如，式I)产生CBGXA。
式I
其中‘X’可以是卤素、羟基、氰基、硝基、酯、烷氧基、氨基、巯基、亚磺酰基、磺酰基、亚磺基、磺基、硫氰酰基、异硫氰酰基、硫醛、二羟硼基、硼酸酯、磷酸酯、醛、羧基、羧酰氨基、叠氮基、氰酰、异氰酰、任选地被取代的(C1-C10)烷基、任选地被取代的(C2-C10)烯基、任选地被取代的(C2-C10)炔基、任选地被取代的(C1-C10)杂烷基、任选地被取代的(C2-C10)杂烯基、任选地被取代的(C2-C10)杂炔基、任选地被取代的(C3-C10)环烷基、任选地被取代的芳基和任选地被取代的杂环。在一个实施方案中，X是被取代的或未被取代的含有2-10个碳的烷基。
本公开内容也提供了包含多肽的重组途径，所述多肽具有选自以下的序列：(a)SEQ ID NO:30且具有至少Y288X突变，其中X是A、N、S、V或非天然的氨基酸；(b)SEQ ID NO:30，其具有至少Y288X突变，其中X是A、N、S、V或非天然的氨基酸，和至少一个选自V49Z1、F213Z2、A232S、I234T、V271Z3和/或G286S的其它突变，其中Z1是S、N、T或G，Z2是H、N或G且Z3是N或H；(c)在表1中所示的突变组合中的任一种；(d)(i)、(ii)或(iii)中的任一种，其包含1-20个保守氨基酸置换且具有NphB活性；(e)与SEQ ID NO:30具有至少85％、90％、95％、98％或99％同一性且至少具有在(i)、(ii)或(iii)中列举的突变的序列；(f)从氨基酸21开始在SEQ ID NO:1-28或29中列举的序列；和(g)与SEQ ID NO:1-28或29中的任一个具有至少99％同一性的任何序列，和多种将葡萄糖转化为香叶基焦磷酸酯的酶；和(h)与SEQ ID NO:1-28或29中的任一个具有至少99％同一性的任何序列，和多种将(异)戊二烯醇转化为香叶基焦磷酸酯的酶。在另一个实施方案中，所述方法还包括丙酮酸脱氢酶旁路酶途径，其包含丙酮酸氧化酶和乙酰基磷酸转移酶。在另一个或其它实施方案中，所述途径包含再循环NADH/NAD和NADPH/NADP的“净化阀”。在前述任一个的另一个或其它实施方案中，所述途径包含以下酶:(i)己糖激酶(Hex)；(ii)葡萄糖-6-磷酸异构酶(Pgi)；(iii)磷酸果糖激酶(Pfk)；(iv)果糖-1,6-二磷酸醛缩酶(Fba)；(v)磷酸丙糖异构酶(Tpi)；(vi)Gald-3-P脱氢酶(Gap)；(vii)突变体Gald-3-P脱氢酶(mGap)；(viii)NADH氧化酶(Nox)；(ix)磷酸甘油酸激酶(Pgk)；(x)磷酸甘油酸酯变位酶(2,3BPG依赖性的)(dPgm)；(xi)烯醇化酶(eno)；(xii)丙酮酸激酶(FBP依赖性的)；(xiii)丙酮酸氧化酶(PyOx)；(xiv)乙酰基磷酸转移酶(PTA)；(xv)乙酰辅酶A乙酰基转移酶(PhaA)；(xvi)HMG-CoA合酶(HMGS)；(xvii)HMG-CoA还原酶(HMGR)；(xviii)甲羟戊酸激酶(MVK)；(xix)磷酸甲羟戊酸激酶(PMVK)；(xx)二磷酸甲羟戊酸脱羧酶(MDC)；(xxi)异戊烯基二磷酸异构酶(IDI)；(xxii)香叶基-PP合酶(GPPS)；和；(xxiii)突变体芳族异戊二烯基转移酶。在任一个前述实施方案的另一个实施方案中，除了磷酸甲羟戊酸脱羧酶(PMDC)和异戊烯基-磷酸激酶(IPK)以外，所述途径包含上面的酶(i)至(xviii)和(xxii)至(xxiii)。在另一个或其它实施方案中，所述途径包含4-步骤途径以使用ATP和一个或多个再循环ADP/ATP的步骤将异戊二烯醇或戊二烯醇转化为GPP。在前述任一个的另一个或其它实施方案中，所述途径包含(a)(异)戊二烯醇激酶(PRK)；(b)异戊烯基磷酸激酶(IPK)；(c)异戊烯基二磷酸异构酶(IDI)；和(d)香叶基焦磷酸酯合酶(GPPS)。在再一个或其它实施方案中，给所述途径补充ATP和油橄榄醇酯(或2,4-二羟基苯甲酸或其衍生物)且所述途径产生大麻素前体。在另一个实施方案中，所述途径进一步包含大麻二酚酸合酶。在再一个或其它实施方案中，所述途径产生大麻二酚酸。
本公开内容也提供了一种生产异戊二烯化的化合物的方法，所述方法包括在有重组多肽存在下使底物与具有以下通用结构的异戊二烯基接触：
所述重组多肽具有选自以下的序列：(a)SEQ ID NO:30且具有至少Y288X突变，其中X是A、N、S、V或非天然的氨基酸；(b)SEQ ID NO:30，其具有至少Y288X突变，其中X是A、N、S、V或非天然的氨基酸，和至少一个选自V49Z1、F213Z2、A232S、I234T、V271Z3和/或G286S的其它突变，其中Z1是S、N、T或G，Z2是H、N或G且Z3是N或H；(c)在表1中所示的突变组合中的任一种；(d)(i)、(ii)或(iii)中的任一种，其包含1-20个保守氨基酸置换且具有NphB活性；(e)与SEQ ID NO:30具有至少85％、90％、95％、98％或99％同一性且至少具有在(i)、(ii)或(iii)中列举的突变的序列；(f)从氨基酸21开始在SEQ ID NO:1-28或29中列举的序列；和(g)与SEQ ID NO:1-28或29中的任一个具有至少99％同一性的任何序列，其中所述异戊二烯基添加至所述底物。
在附图和以下描述中阐述了本公开内容的一个或多个实施方案的细节。从说明书和附图以及从权利要求书会明白其它特征、目的和优点。
附图简要说明
并入本说明书中并构成本说明书的一部分的附图说明了本公开内容的一个或多个实施方案，并且与详细描述一起用于解释本发明的原理和实施方式。
图1A-B描绘了本公开内容的示例性生物合成途径。(A)用于产生异戊二烯基天然产物的生物化学合成平台.首先，通过经修饰以调节NADPH水平的糖酵解途径将葡萄糖分解为丙酮酸(12个酶促步骤)。然后，PDH或PDH旁路将丙酮酸转化为乙酰辅酶A。乙酰辅酶A通过甲羟戊酸途径转化为GPP(8个酶促步骤)。通过改变芳族异戊二烯基转移酶(aPT)和芳族底物，可以使用相同的中心途径产生多种异戊二烯基-类黄酮和异戊二烯基-芪类化合物。开发了异戊二烯基转移酶NphB的变体(dNphB)以生产CBGA或CBGVA。通过大麻二酚酸合酶(CBDAS)，CBGA转化为大麻二酚酸(CBDA)且CBGVA转化为次大麻二酚酸(CBDVA)。通过使用不同的大麻素合酶(THCAS和CBCAS)可能生产其它大麻素。(B)描绘了(A)的途径的更详细视图。葡萄糖通过糖酵解分解为丙酮酸(深蓝色)。以深蓝色描绘的净化阀允许碳通量继续通过糖酵解途径，而不会积累过量的NADPH。丙酮酸通过以浅蓝色描绘的PDH旁路转化为乙酰辅酶A。乙酰辅酶A通过甲羟戊酸途径(水)积累在高能磷酸酯分子中以产生GPP。然后，来自甲羟戊酸途径的GPP被用于对芳族聚酮化合物进行异戊二烯化。此处显示了油橄榄醇酯的异戊二烯化以产生CBGA；但是，油橄榄醇酯可以用多种底物(芳族和非芳族)代替以生成各种异戊二烯化的产物。最后，使用CBDAS将CBGA转化为CBDA。自发脱羧完成向CBDA的生物合成途径。CBDA的生产完成了以绿色显示的大麻素模块。
图2A-D显示了用于异戊二烯化芳族聚酮化合物的PDH旁路的发展。(A)在各种芳族聚酮化合物和2％乙醇(媒介物)的存在下测量丙酮酸脱氢酶(Ec PDH)的活性(n＝3)。(B)使用PDH(PDH系统-灰色迹线)和PDH旁路系统(蓝色迹线)在不同浓度的1,6DHN下通过完整途径实现的最终滴度的对比。误差条代表样品之间的标准偏差(n＝3)。(C)使用WT NphB用PDH旁路系统随时间产生的5-异戊二烯基-1,6-DHN蓝色迹线和CBGA绿色迹线的量。误差条代表样品之间的标准偏差(n＝3)。(D)使用NphB、AtaPT或NovQ异戊二烯基转移酶将各种芳族底物添加至所述途径(生物学重复，n＝3)。结果是各种C5和C10异戊二烯基-天然产物(*指示未确定滴度)。
图3A-C显示了NphB的工程改造以改善CBGA生产。(A)WT NphB的活性位点中的油橄榄醇酯的模型。在设计过程期间，允许残基A288、G286和A232和I234、V271和V49发生变化。残基A288、G286和A232对关于OA的活性具有最大影响，并且是在聚焦文库中靶向的位置。(B)活性测定的结果，以确定以油橄榄醇酯为底物的NphB突变体的近似活性。倍数提高是用GPP(2.5mM)、油橄榄醇酯(5mM)、MgCl2(5mM)和1mg/mL WT NphB和突变体进行三次重复反应的平均值。(C)与CBGA标准品相比使用M23和WT NphB的完整途径反应产物的GC-MS色谱图。M23突变体显著提高了对于正确产物的特异性。
图4A-C显示了用于生产各种大麻素的无细胞异戊二烯化系统的评价。(A)随着时间的流逝，大麻素前体的无细胞酶促生产(从葡萄糖)。使用M23的CBGA生产显示为浅绿色迹线，且WT NphB显示为深绿色迹线。使用M31的CBGVA生产显示为浅蓝色迹线。用于WT、M23和M31的NphB的浓度固定在0.5mg/mL(n＝3)。(B)使用壬烷流CBGA捕获系统，获得更高的CBGA滴度(1.2g/L)。使用蠕动泵交换壬烷层，该蠕动泵使壬烷沿箭头所示方向循环。该系统能够将CBGA稀释进数毫升的壬烷和缓冲液中，这减少反应中CBGA的量。(C)使用CBDAS随时间推移生产大麻素。CBDA生产显示为深紫色迹线，且CBDVA生产显示为浅紫色迹线。
图5A-C显示了MatB和MdcA(转移酶)路径的途径示意图。(A)这是MatB路径的示意图。丙二酰基辅酶A生产是ATP依赖性的，但是在其它方面与途径无关。所述途径的滴度为12mg/L。(B)这是MdcA转移酶路径的示意图。丙二酰基辅酶A生产不再是ATP依赖性的，而是与丙酮酸氧化路径和甲羟戊酸路径有关。该系统的滴度为42mg/L。(C)显示了在(A)和(B)中示出的途径的聚酮化合物模块中的示例性步骤的额外细节。
图6显示了(异)戊二烯醇至GPP路径的途径示意图。可以使用ATP和必要的激酶将异戊二烯醇或戊二烯醇转化成香叶基焦磷酸酯。
图7显示了可用于从乙酰辅酶A(或甲羟戊酸)产生IPP/DMAPP的各种规范的(真核)和非规范的(古细菌I和II)甲羟戊酸途径。
具体实施方式
如在本文中和在所附权利要求书中所使用的，单数形式“一个/种(a)”、“一个/种(an)”和“该”包括复数指示物，除非上下文另外清楚地指明。因此，例如，对“一种多核苷酸”的提及包括多种这样的多核苷酸，且对“该酶”的提及包括对一种或多种酶的提及，诸如此类。
除非另外定义，在本文中使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些类似或等同的方法和材料可以用于所公开的方法和组合物的实践中，但是本文描述了示例性的方法、装置和材料。
并且，除非另外说明，“或”的使用是指“和/或”。类似地，“包含(comprise)”、“包含(comprises)”、“包含(comprising)”、“包括(include)”、“包括(includes)”和“包括(including)”是可互换的，并不旨在进行限制。
还应当理解，在各个实施方案的描述使用术语“包含”的情况下，本领域技术人员会理解，在一些具体情况下，可以可替换地使用语言“基本上由……组成”或“由……组成”描述一个实施方案。
在上面以及贯穿全文所讨论的任何出版物仅仅出于其在本申请的申请日之前公开的目的而提供。本文中的任何内容均不应解释为承认：发明人由于在先公开而无权领先于这样的公开。
异戊二烯化(也被称作异戊二烯基化或脂质化)是将疏水性分子添加至蛋白或化学化合物。通常假定异戊二烯基(3-甲基丁-2-烯-1-基)有助于向细胞膜附着，类似于脂质锚，如GPI锚。已显示异戊二烯基对于通过专门的异戊二烯基-结合结构域的蛋白-蛋白结合而言是重要的。
异戊二烯化的天然产物是具有经证实的医学特性的一大类生物活性分子。实例包括异戊二烯基-黄酮类化合物、异戊二烯基-芪类化合物和大麻素。由于污染性分子的结构相似性以及作物之间的可变组成，难以分离植物来源的异戊二烯化合物。当尝试分离低丰度化合物时，这些挑战会进一步加剧。已经开发了许多化学合成来解决与制备异戊二烯化的天然产物有关的挑战，但是由于复杂程度和低产率，它们对于药物制造而言通常是不切实际的。
对于异戊二烯化的天然产物，微生物生产是自然提取的有用替代方案，但是伴随着许多挑战，诸如需要将碳通量从中心代谢和产物毒性转移，仅举几个例子。例如，异戊二烯基-天然产物如异戊二烯基-柚皮素、异戊二烯基-白藜芦醇和大麻二酚酸(CBDA)源自脂肪酸、类异戊二烯和聚酮化合物生物合成的代谢途径的组合。因此，高水平的生产需要有效地重新设计长的、必不可少的和高度调节的途径。尽管存在挑战，但许多研究组已经工程改造微生物来产生未异戊二烯化的聚酮化合物如柚皮素、白藜芦醇和油橄榄醇酯，但在相对低的水平(分别为110、391和80mg/L)。获得异戊二烯化的产物甚至更有挑战性，因为香叶基-焦磷酸酯(GPP)是必需的代谢产物，其在中等浓度对细胞有毒，从而为高水平微生物生产造成重要障碍。
大麻素特别显示出巨大的治疗潜力，正在进行超过100项作为止吐药、抗惊厥药、抗抑郁药和镇痛药的临床试验。尽管如此，尽管异戊二烯基-天然产物具有治疗潜力，但由于缺乏有成本效益的生产方法，其研究和应用受到限制。
基于植物的大麻素生产的两个主要替代方案是有机合成和在代谢工程改造的宿主(例如植物、酵母或细菌)中的生产。已经阐明了用于生产一些大麻素(诸如THCA和CBDA)的总合成，但它们通常不适用于药物制造。另外，合成方法不是模块化的，需要对每种大麻素进行独特合成。模块方案可以通过使用天然的生物合成途径来实现。
三种主要的大麻素(THCA、CBDA和cannibichromene或CBCA)衍生自单一前体CBGA。此外，从CBGVA衍生出三种低丰度大麻素(图1A)。因此，在异源宿主中制备CBGA和CBGVA的能力将为生产一系列大麻素打开大门。不幸的是，工程改造微生物以生产CBGA和CBGVA已被证明极具挑战性。
大麻素衍生自脂肪酸、聚酮化合物和萜烯生物合成途径的组合，所述途径生成关键的结构单元香叶基焦磷酸酯(GPP)和油橄榄醇酸(OA)(图1A)。高水平CBGA生物合成需要重新设计长的、必不可少的和高度调节的途径。此外，GPP对细胞有毒，对在微生物中的高水平生产造成明显障碍。尽管Gagne等人(Proc.Natl.Acad.Sci.,109:12811,2012)工程改造了一个途径来在酵母中生产OA，但是滴度是非常低的(0.5mg L-1)，从而提示，在所述途径上的中间体的高水平生产并不容易。在一项单独的研究中，Zirpel等人在含有混杂的异戊二烯基转移酶(NphB)和THCA合酶并补充了GPP和油橄榄醇酸(OA)的酵母裂解物中生产了THCA(J.Biotechnol.,259:204-212,2017)。但是，仍然没有公开的报告表明从低成本原料在经工程改造的活细胞中生产大麻素。
在合成生物化学中，使用酶的混合物无细胞地进行复杂的生物化学转化，与传统的代谢工程改造相比具有潜在的优点，包括：在途径设计中更高的灵活性水平；对组分优化的更大控制；更快的设计-建造-测试周期；和没有中间体或产物的细胞毒性。本公开内容提供了用于生产大麻素的无细胞系统。
本公开内容提供了酶变体和包含此类变体的途径，其用于化合物的异戊二烯化，包括大麻素的产生。另外，本文所述的生物合成途径使用“净化阀”来调节NAD(P)H水平。这样的“净化阀”已经证实从葡萄糖高水平生产单萜，表明可以无细胞产生大量GPP(参见，国际专利公开WO2017/015429，其公开内容通过引用并入本文)。这些净化阀用于升级和多样化原始系统，以生产复杂天然产物诸如大麻素。图1A、1B、5A和5B中概述了合成生物化学方法。在一个实施方案中，本公开内容提供了一种使用衍生自葡萄糖的GPP用于异戊二烯化的无细胞系统(参见，图1A、1B、5A、5B和7)。在另一个实施方案中，本公开内容提供了一种使用衍生自(异)戊二烯醇或戊二烯醇的GPP进行异戊二烯化的无细胞系统(参见，图6)。图6的途径可以与任何ATP产生系统偶联以产生反应所需的ATP。例如，所述途径可以与肌酸激酶ATP产生系统；乙酸激酶系统；糖酵解系统以及其它系统偶联。图6的酶(核酸编码序列和多肽)提供在SEQ ID NO:54-65中(例如，PRK酶提供在SEQ ID NO:54-57中；IPK酶提供在SEQ IDNO:58-61中；IDI酶提供在SEQ ID NO:62-63中；且FPPS酶提供在SEQ ID NO:64-65中)。
NphB是一种芳族异戊二烯基转移酶，其催化10-碳香叶基与芳族底物的连接。NphB表现出富集底物选择性和产物区域选择性。从链霉菌属(Streptomyces)鉴定出的NphB催化10-碳香叶基向许多小的有机芳族底物的添加。NphB具有一个宽敞且溶剂可接近的结合口袋，两种底物分子香叶基二磷酸酯(GPP)和1,6-二羟基萘(1,6-DHN)可以结合在该结合口袋中。除了Mg2+以外，GPP通过其带负电荷的二磷酸酯部分与几个氨基酸侧链(包括Lys119、Thr171、Arg228、Tyr216和Lys284)之间的相互作用而稳定。NphB的活性需要Mg2+辅因子。来自链霉菌属的NphB具有如SEQ ID NO:30中所示的序列。
NovQ(登记号AAF67510,通过引用并入本文)是异戊二烯基转移酶的CloQ/NphB类的一个成员。novQ基因可以从雪白链霉菌(Streptomyces niveus)克隆，其产生氨基香豆素抗生素新生霉素。重组NovQ可在大肠杆菌中表达并纯化至同质。纯化的酶是40-kDa的可溶性单体蛋白，其独立于二价阳离子催化二甲基烯丙基向4-羟基苯基丙酮酸酯(4-HPP)的转移，以生成3-二甲基烯丙基-4-HPP，即新生霉素的中间体。除了4-HPP的异戊二烯化外，NovQ还催化各种苯丙素(phenylpropanoids)、类黄酮和二羟基萘的基于碳-碳的和基于碳-氧的异戊二烯化。尽管其催化混杂，但NovQ催化的异戊二烯化以区域特异性方式发生。NovQ是第一种报告的异戊二烯基转移酶，其能够催化二甲基烯丙基向苯丙素(诸如对香豆酸和咖啡酸)和类黄酮的B环的转移。NovQ可以充当用于合成异戊二烯化的苯丙素和异戊二烯化的类黄酮的有用生物催化剂。
最近被发现和表征的土曲霉(Aspergillus terreus)芳族异戊二烯基转移酶(AtaPT；登记号AMB20850,通过引用并入本文)负责各种芳族化合物的异戊二烯化。重组AtaPT可以在大肠杆菌中过表达并纯化。在有不同的异戊二烯基二磷酸酯存在下，土曲霉芳族异戊二烯基转移酶(AtaPT)主要催化酰基间苯三酚的C-单异戊二烯化。
油橄榄醇酸(OA)是野生型NphB的相对较差的底物。所以，通过使用更优选的NphB底物1,6二羟基萘(1,6DHN)来测试无细胞系统对共底物异戊二烯化的能力。当从2.5mM 1,6DHN和500mM葡萄糖开始时，获得约400mg/L(1.3mM)的异戊二烯化的产物。但是，当起始1,6DHN浓度从2.5mM增加到5mM时，最终滴度降低2倍，这表明1,6DHN抑制一种或多种酶。酶测定揭示，大肠杆菌丙酮酸脱氢酶(EcPDH)不仅被1,6DHN抑制，而且还被几种其它芳族聚酮化合物抑制(图2B)。在1mM的1,6DHN、油橄榄醇或白藜芦醇，PDH的活性降低了2倍(图2B)。因此，设计了通过实施PDH旁路来消除PDH的实验(参见图1A和2B)。在PDH旁路中，使用丙酮酸氧化酶(PyOx)和乙酰基磷酸转移酶(PTA)将丙酮酸转化为乙酰辅酶A，从而消除PDH(图1A)。如在图2A中所示，当从5mM 1,6DHN开始时，新系统消除了在较高1,6DHN浓度下所见的抑制，并使5-异戊二烯基-1,6DHN的滴度比PDH系统高4倍(图2B)。图2C显示了利用PDH旁路从5mM1,6DHN开始的5-异戊二烯基-1,6DHN生物合成的时程。在最初的24小时内转化了大约50％的1,6DHN，最终达到705±12mg/L的最终滴度。
NphB对芳族聚酮化合物的异戊二烯化被认为是通过碳阳离子中间体进行，其中第一步是将二磷酸酯与GPP解离，以在GPP的C1碳上生成碳阳离子，其随后攻击附近的亲核体。为了提高异戊二烯基转移的区域特异性，使用与1,6DHN、Mg2+和不可水解的GPP类似物(香叶基S-硫羟二磷酸酯)形成复合物的NphB的晶体结构作为起始点(PDBID 1ZB6；蛋白数据库参照1ZB6)，将OA模型化进NphB的活性位点。对于设计，使用1,6DHN作为引导物将OA放入结合口袋中，将期望的异戊二烯化位点(OA的C3碳)定位在新生的香叶基C1碳阳离子以上(图3A)。选择的距离是基于1,6DHN的C5碳与GPP的C1碳的距离。然后使用ROSETTA软件改变与OA接触的残基，以优化NphB用于结合OA的活性位点。与GPP接触或可能提供催化功能的侧链保持不变。结果是建议的NphB变体的整体。
为了减少要进行实验测试的变体的数目，使用评分系统将可能对OA结合具有最显著影响的变化进行排序。挑选一组代表性的变体(表1)，并在其它突变的背景下将每个残基系统化地变回野生型侧链，并在能量评分中评价变化(表2)。Y288替代对能量评分的影响最大，因此在每个实验评价的构建体中使用了Y288A或Y288N突变。突变的频率、多个突变可能如何协同工作、以及用于进一步塑造NphB文库的计算能量评分都被考虑在内。考虑到这些因素，如表1所示，生成了一个文库，该文库包含29种构建体，范围从单点突变体到每个构建体最多6个突变(也参见SEQ ID NO:1-29；注意SEQ ID NO:1-29包括来自表达构建体的六组氨酸前导序列，即对生物活性不是必需的氨基酸1-20)。
表1：提供了相对于野生型(即，SEQ ID NO:30的多肽)的示例性突变和倍数改善。NphB文库构建体和突变(参考SEQ ID NO:30的氨基酸位置)。
表2:NphB突变体的动力学参数
b2,4-二羟基-6-丙基苯甲酸的动力学参数
本文描述了用于产生和分离本公开内容的经修饰的NphB多肽的重组方法。除了重组生产外，通过使用固相技术的直接肽合成也可以产生多肽(例如，Stewart等人(1969)Solid-Phase Peptide Synthesis(WH Freeman Co,San Francisco)；和Merrifield(1963)J.Am.Chem.Soc.85:2149-2154；它们中的每一篇通过引用并入)。可以使用手工技术或通过自动化进行肽合成。可以实现自动化的合成，例如，根据制造商提供的说明，使用AppliedBiosystems 431A肽合成仪(Perkin Elmer,Foster City,Calif.)。
获得了粗纯化的NphB突变体，并使用对于野生型NphB而言饱和的浓度的GPP和OA进行用于CBGA生产的初始筛选。鉴定出六种与WT NphB相比具有>10倍的表观活性增加的构建体(M1、M2、M3、M6、M10和M15)和4种与WT NphB相比具有2-10倍表观改善的构建体(M5、M7、M12和M20)，而其余的构建体具有与WT NphB相似的活性。将来自初始筛选的顶部命中(M1、M3、M10和M15)纯化并更仔细地表征(图3B)。从初始筛选可以明显看出以下观察结果：(1)Y288A(M1)和Y288N(M2)本身显著增强了活性，如通过计算所预测的；(2)Y288N在任何构建体中的存在降低了纯化产率，表明Y288N可能是不稳定的突变，使Y288A成为更期望的突变；(3)G286S在Y288N(M10)背景中的添加似乎比Y288N(M2)进一步提高了活性，从而提示G286S可能是另一种有利的突变；(4)Y288A/F213N/A232S(M15)与Y288A(M1)相比活性略有提高，即使F213N在Y288A/F213N(M5)构建体中具有中性或有害作用，表明A232S也可能是有利的突变。
从这些初步观察结果，设计了一个聚焦文库，其包括各种组合的变体Y288A、GS86S和A232S。添加了与Y288V的其它组合，其原理是它可能提高稳定性，同时仍然减小Y288侧链的尺寸。在一小时的终点测定中，第二个文库中除一个构建体外的所有构建体均表现出比WT NphB高至少100倍的活性。来自第一轮的最佳突变体与来自第二轮的最佳突变体的对比显示在图3B中。显然，来自第一轮的有益突变的组合改善了CBGA生产。另外，与Y288N相比，Y288A和Y288V构建体改善了NphB的表达，而没有牺牲活性。
进一步表征了来自初始筛选的最佳两种突变体以及来自聚焦文库的最佳三种构建体。动力学参数总结在表2中。尽管所有突变体具有相对不大的对Km的影响，但观察到kcat值的显著改善。M23(SEQ ID NO:23的NphB)特别地将kcat提高了750倍，从0.0021±0.00008min-1提高至1.58±0.05min-1。与野生型酶相比，M23和M31的催化效率(kcat/Km)提高了1000倍以上。尽管M31具有比M23更高的kcat/Km，但使用M23而不是M31，因为M23具有更高的kcat，并且合成生物化学系统通常在饱和OA条件下运行。
设计的突变体M23不仅显示出急剧提高的对OA的异戊二烯化的催化效率，而且还是非常特异性的，仅产生正确的CBGA产物。WT NphB产生CBGA，但是主要产物是异戊二烯化的异构体(图3C)。相反，设计的突变体M23几乎只能制成CBGA。总体而言，设计的酶是比非特异性的异戊二烯化野生型酶远远更有效的CBGA合酶。
本公开内容因此提供了突变体NphB变体，其包含：(i)SEQ ID NO:30且具有至少Y288X突变，其中X是A、N、S、V或非天然的氨基酸；(ii)SEQ ID NO:30，其具有至少Y288X突变，其中X是A、N、S、V或非天然的氨基酸，和至少一个选自V49Z1、F213Z2、A232S、I234T、V271Z3和/或G286S的其它突变，其中Z1是S、N、T或G，Z2是H、N或G且Z3是N或H；(iii)在表1中所示的突变组合中的任一种；(iv)(i)、(ii)或(iii)中的任一个，其包含1-20(例如，2、5、10、15或20；或1-20之间的任何值)个保守氨基酸置换且具有NphB活性；(v)与SEQ ID NO:1-29或30具有至少85％、90％、95％、98％或99％同一性且至少具有在(i)、(ii)或(iii)中列举的突变的序列；(vi)从氨基酸21开始包含在SEQ ID NO:1-28或29中列举的序列中的任一个的NphB突变；或(vii)与SEQ ID NO:1-28或29中的任一个具有至少99％同一性且具有NphB活性的任何序列。“NphB活性”是指酶使底物异戊二烯化、和更具体地从OA产生CBGA的能力。
本文中使用的非天然的氨基酸表示在自然界中不存在的氨基酸，诸如N-甲基氨基酸(例如，N-甲基L-丙氨酸、N-甲基L-缬氨酸等)或α-甲基氨基酸、β-同型氨基酸、同型氨基酸和D-氨基酸。在一个特定实施方案中，可用于本公开内容中的非天然氨基酸包括小的疏水的非天然氨基酸(例如，N-甲基L-丙氨酸、N-甲基L-缬氨酸等)。
另外，本公开内容提供了编码前述NphB变体中的任一个的多核苷酸。因为遗传密码的简并性，实际的编码序列可以变化，同时仍然到达为NphB突变体和变体列举的多肽。在SEQ ID NO:66、67和68中提供了示例性的多核苷酸序列(分别对应于SEQ ID NO:23、29和69的多肽序列)。再次显而易见的是，遗传密码的简并性将允许与SEQ ID NO:66、67和68的同一性百分比的广泛变化，同时仍编码SEQ ID NO:23、29和69的多肽。
本公开内容还提供了包含本公开内容的任何NphB变体酶的重组宿主细胞和无细胞系统。在一些实施方案中，使用所述重组细胞和无细胞系统进行异戊二烯化过程。
本公开内容的一个目的是，从葡萄糖或戊二烯醇和/或异戊二烯醇生产前体GPP，然后可以将其用于用本公开内容的突变体NphB对添加的OA进行异戊二烯化，从而产生CBGA。
本公开内容因此提供了一种无细胞系统，其包括多个将葡萄糖转化为香叶基焦磷酸酯的酶促步骤，其中所述途径包括净化阀和PDH旁路酶促过程。
如图1B中所描绘，本公开内容的一种途径包括使用己糖激酶将葡萄糖转化为葡萄糖-6-磷酸。己糖激酶(EC 2.7.1.1)是将己糖(六碳糖)磷酸化从而形成己糖磷酸酯的酶。己糖激酶具有将无机磷酸酯基团从ATP转移至底物的能力。已经克隆并表达了来自各种生物的许多己糖激酶蛋白。在一些实施方案中，己糖激酶包含来自酿酒酵母(Saccharomycescerevisiae，Sc)的在UniProtKB登录号P04806中所示的序列(通过引用并入本文)以及与其具有至少60％、70％、80％、85％、90％、95％、98％、99％同一性且具有己糖激酶活性的序列。
然后葡萄糖-6-磷酸被磷酸葡萄糖异构酶(Pgi)(EC 5.3.1.9)转化为果糖-6-磷酸。因此，除上述内容外，术语“磷酸葡萄糖异构酶”或“Pgi”表示能够催化从葡萄糖-6-磷酸形成果糖-6-磷酸的蛋白，并且它们与SEQ ID NO:31具有至少约40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更大序列同一性，或至少约50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或更大的序列相似性，如通过NCBI BLAST使用默认参数所计算的，并且其中所述酶具有磷酸葡萄糖异构酶活性。
在另一个或其它实施方案中，本文提供的系统或重组微生物包括磷酸果糖激酶(Pfk,多磷酸酯依赖性的Pfk或其同系物或变体)的表达。该表达可以与代谢途径中的其它酶组合。Pfk可以衍生自嗜热脂肪土芽孢杆菌(G.stearothermophilus)(SEQ ID NO:32)。在另一个实施方案中，可以使用Pfk的经工程改造的变体，只要它具有磷酸果糖激酶活性且可以将果糖-6-磷酸转化为果糖-1,6-二磷酸。这样的经工程改造的变体可以通过定位诱变、定向进化等获得。因此，在本公开内容中包括与SEQ ID NO:32中所示的序列具有至少85-99％同一性并且具有磷酸果糖激酶活性的多肽(参见，例如，SEQ ID NO:33-34)。
除前述内容外，术语“果糖1,6二磷酸醛缩酶”或“Fba”表示能够催化从果糖1,6-二磷酸形成二羟基丙酮磷酸和甘油醛-3-磷酸的蛋白，并且其与SEQ ID NO:35具有至少约40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更大序列同一性、或至少约50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或更大序列相似性，如通过NCBI BLAST使用默认参数所计算的。另外的同系物包括：与SEQ ID NO:35具有26％同一性的细长聚球蓝细菌(Synechococcus elongatus)PCC6301YP_170823.1；与SEQ ID NO:35具有80％同一性的黑美人弧菌(Vibrionigripulchritudo)ATCC 27043ZP_08732298.1；与SEQ ID NO:35具有76％同一性的白色甲基微菌(Methylomicrobium album)BG8 ZP_09865128.1；与SEQ ID NO:35具有25％同一性的荧光假单胞菌(Pseudomonas fluorescens)Pf0-1 YP_350990.1；和与SEQ ID NO:35具有24％同一性的Methylobacterium nodulans ORS2060YP_002502325.1。因此，本公开内容包括与SEQ ID NO:35具有26％至100％同一性的多肽的用途，其中所述多肽具有二磷酸醛缩酶活性。与前述登录号相关的序列通过引用并入本文。
除前述内容外，术语“磷酸丙糖异构酶”或“Tpi”表示能够催化从二羟基丙酮磷酸(DHAP)形成甘油醛-3-磷酸的蛋白，并且其与SEQ ID NO:36具有至少约40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更大序列同一性、或至少约50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或更大序列相似性，如通过NCBI BLAST使用默认参数所计算的。另外的同系物包括:与SEQ ID NO:36具有45％同一性的褐家鼠(Rattus norvegicus)AAA42278.1；与SEQ ID NO:36具有45％同一性的智人(Homo sapiens)AAH17917.1；与SEQ ID NO:36具有40％同一性的枯草芽孢杆菌(Bacillus subtilis)BEST7613 NP_391272.1；与SEQ ID NO:36具有40％同一性的细长聚球蓝细菌(Synechococcus elongatus)PCC 6301YP_171000.1；和与SEQ ID NO:36具有98％同一性的肠道沙门氏菌肠道亚种Typhi株血清变型(Salmonella entericasubsp.enterica serovar Typhi str.)AG3 ZP_06540375.1。因此，本公开内容包括与SEQID NO:36具有40％至100％同一性且具有磷酸丙糖异构酶活性的多肽的用途。与前述登录号相关的序列通过引用并入本文。
在所述途径的另一个步骤中，可以将甘油醛-3-磷酸转化为1,3-二磷酸甘油酸酯。该酶促步骤可以包括“净化阀系统”(如在本文别处所讨论的)。例如，甘油醛-3-磷酸脱氢酶(Gap,Tdh)将甘油醛-3-磷酸转化为1,3-二磷酸-甘油酸酯。在一个实施方案中，使用用NAD+作为辅因子的野生型Gap(参见，例如，SEQ ID NO:37)或包含P191D突变的突变体Gap(相对于SEQ ID NO:37的序列，和如在SEQ ID NO:38中所示)。在另一个实施方案中，使用突变体Gap(mGap；例如，具有D34A/L35R/T35K突变；相对于SEQ ID NO:37的序列和如在SEQ ID NO:39中所示)，其使用NADP+作为辅因子。在另一个实施方案中，使用Gap和mGap的组合(GapM6)。当使用优先使用NAD+的野生型gap或P118D突变体gap时，包含产生水的NADH氧化酶(NoxE)的分子净化阀可用于再循环(“净化”)NADH，所述NADH氧化酶特异性地氧化NADH、但不氧化NADPH。
除前述内容外，术语“NADH氧化酶”或“NoxE”表示能够将NADH氧化成NAD+的蛋白，并且其与SEQ ID NO:18具有至少约40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更大序列同一性、或至少约50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或更大序列相似性，如通过NCBI BLAST使用默认参数所计算的。
所述途径通过使用磷酸甘油酸激酶(EC 2.7.2.3)(PGK；例如，如在SEQ ID NO:40，或与其具有至少80％同一性的其同系物或变体中提供的)可以将1,3-二磷酸甘油酸酯进一步转化成3-磷酸甘油酸酯，所述磷酸甘油酸激酶催化磷酸酯基团从1,3-二磷酸甘油酸酯(1,3-BPG)至ADP的可逆转移，从而产生3-磷酸甘油酸酯(3-PG)和ATP。使用例如GTPase或其它酶或其同系物或变体，用于ATP的分子净化阀可以呈现用于再循环ADP。
3-磷酸甘油酸酯然后可以被磷酸甘油酸酯变位酶(pgm；例如，如在SEQ ID NO:41，或与其具有至少80％同一性的其同系物或变体中提供的)转化成2-磷酸甘油酸酯。
烯醇化酶(eno；例如，如在SEQ ID NO:42，或与其具有至少80％同一性的其同系物或变体中提供的)然后可以将2-磷酸甘油酸酯转化成磷酸烯醇丙酮酸(PEP)。
丙酮酸激酶(pyk；例如，如在SEQ ID NO:43、44和45，或与SEQ ID NO:43、44或45中的任一个具有至少80％同一性的其同系物或变体中提供的)将PEP转化成丙酮酸。
如上所提及，丙酮酸脱氢酶(PDH)被所述途径的产物抑制。因此，可以使用PDH旁路将丙酮酸转化为乙酰辅酶A。PDH旁路包括两个酶促步骤：(i)由丙酮酸氧化酶(例如，来自绿色气球菌(Aerococcus viridans)的PyOx；EC 1.2.3.3；参见SEQ ID NO:46)催化的丙酮酸→乙酰基磷酸酯；和(ii)由乙酰基磷酸转移酶(又名磷酸乙酰基转移酶)(例如，来自嗜热脂肪土芽孢杆菌的PTA)催化的乙酰基磷酸酯→乙酰辅酶A。
如本文中使用的，在本公开内容的组合物和方法中使用的PyOx包括与SEQ ID NO:46具有至少85％、90％、95％、98％、99％同一性并且具有丙酮酸氧化酶活性的序列。
磷酸乙酰基转移酶(EC 2.3.1.8)是一种催化乙酰辅酶A+磷酸酯至辅酶A+乙酰基磷酸酯的化学反应的酶，且反之亦然。磷酸乙酰基转移酶由pta在大肠杆菌中编码。PTA参与乙酸向乙酰辅酶A的转化。具体而言，PTA催化乙酰辅酶A向乙酰基磷酸酯的转化。PTA同系物和变体是已知的。在NCBI上可得到大约1075种细菌磷酸乙酰基转移酶。例如，这样的同系物和变体包括：磷酸乙酰基转移酶Pta(猫立克次氏体(Rickettsia felis)URRWXCal2)gi|67004021|gb|AAY60947.1|(67004021)；磷酸乙酰基转移酶(蚜虫巴克纳氏菌(Buchneraaphidicola)Cc(Cinara cedri)株)gi|116256910|gb|ABJ90592.1|(116256910)；pta(蚜虫巴克纳氏菌Cc(Cinara cedri)株)gi|116515056|ref|YP_802685.1|(116515056)；pta(Glossina brevipalpis的Wigglesworthia glossinidia内共生体)gi|25166135|dbj|BAC24326.1|(25166135)；Pta(多杀巴斯德氏菌多杀亚种Pm70株(Pasteurella multocidasubsp.multocida str.Pm70))gi|12720993|gb|AAK02789.1|(12720993)；Pta(深红红螺菌(Rhodospirillum rubrum))gi|25989720|gb|AAN75024.1|(25989720)；pta(威氏李斯特菌6b血清变型SLCC5334株(Listeria welshimeri serovar 6b str.SLCC5334))gi|116742418|emb|CAK21542.1|(116742418)；Pta(鸟分枝杆菌副结核亚种K-10(Mycobacterium avium subsp.paratuberculosis K-10))gi|41398816|gb|AAS06435.1|(41398816)；磷酸乙酰基转移酶(pta)(布氏疏螺旋体(Borrelia burgdorferi)B31)gi|15594934|ref|NP_212723.1|(15594934)；磷酸乙酰基转移酶(pta)(布氏疏螺旋体B31)gi|2688508|gb|AAB91518.1|(2688508)；磷酸乙酰基转移酶(pta)(流感嗜血菌(Haemophilusinfluenzae)Rd KW20)gi|1574131|gb|AAC22857.1|(1574131)；磷酸乙酰基转移酶Pta(Rickettsia bellii RML369-C)gi|91206026|ref|YP_538381.1|(91206026)；磷酸乙酰基转移酶Pta(Rickettsia bellii RML369-C)gi|91206025|ref|YP_538380.1|(91206025)；磷酸乙酰基转移酶pta(结核分枝杆菌(Mycobacterium tuberculosis)F11)gi|148720131|gb|ABR04756.1|(148720131)；磷酸乙酰基转移酶pta(结核分枝杆菌Haarlem株)gi|134148886|gb|EBA40931.1|(134148886)；磷酸乙酰基转移酶pta(结核分枝杆菌C)gi|124599819|gb|EAY58829.1|(124599819)；磷酸乙酰基转移酶Pta(Rickettsia belliiRML369-C)gi|91069570|gb|ABE05292.1|(91069570)；磷酸乙酰基转移酶Pta(Rickettsiabellii RML369-C)gi|91069569|gb|ABE05291.1|(91069569)；磷酸乙酰基转移酶(pta)(苍白密螺旋体苍白亚种Nichols株(Treponema pallidum subsp.pallidum str.Nichols))gi|15639088|ref|NP_218534.1|(15639088)；和磷酸乙酰基转移酶(pta)(苍白密螺旋体苍白亚种Nichols株)gi|3322356|gb|AAC65090.1|(3322356)，与登录号相关的每个序列通过引用整体并入本文。
再次转向图1B，所述途径包括乙酰辅酶A向乙酰乙酰辅酶A的转化。乙酰辅酶A向乙酰乙酰辅酶A的转化由乙酰辅酶A乙酰基转移酶(例如，PhaA)执行。许多乙酰辅酶A乙酰基转移酶是本领域已知的。例如，来自富养产碱菌(R.eutropha)的乙酰辅酶A乙酰基转移酶。在另一个实施方案中，所述乙酰辅酶A乙酰基转移酶具有与SEQ ID NO:47具有至少85％、90％、95％、98％、99％或100％同一性的氨基酸序列。
乙酰乙酰辅酶A和乙酰辅酶A可以被具有A110G突变的酶HMG-CoA合酶(参见，例如，SEQ ID NO:48)或与其具有85％-99％序列同一性的其同系物或变体转化为HMG-CoA。
然后通过NADPH和HMG-CoA还原酶(参见，例如，SEQ ID NO:49)或与其具有85％-99％序列同一性的其同系物或变体的作用，将HMG-CoA还原成甲羟戊酸。
甲羟戊酸然后通过ATP和甲羟戊酸激酶(MVK)的作用而磷酸化以产生甲羟戊酸-5-磷酸和ADP。甲羟戊酸激酶是本领域已知的且包括与SEQ ID NO:50的序列具有至少85-100％(例如，85％、90％、95％、98％、99％)同一性且具有甲羟戊酸激酶活性的序列。
甲羟戊酸-5-磷酸通过ATP和磷酸甲羟戊酸激酶(PMVK)的作用而进一步磷酸化以产生甲羟戊酸-5-二磷酸和ADP。磷酸甲羟戊酸激酶是本领域已知的且包括与SEQ ID NO:51的序列具有至少85-100％(例如，85％、90％、95％、98％、99％)同一性且具有磷酸甲羟戊酸激酶活性的序列。
甲羟戊酸-5-二磷酸通过ATP和二磷酸甲羟戊酸脱羧酶(MDC)的作用而脱羧以产生ADP、CO2和异戊基焦磷酸酯。二磷酸甲羟戊酸脱羧酶是本领域已知的且包括与SEQ ID NO:52的序列具有至少85-100％(例如，85％、90％、95％、98％、99％)同一性且具有二磷酸甲羟戊酸激酶活性的序列。
可以使用各种其它甲羟戊酸途径(参见，例如，图7)。
然后，在相对于SEQ ID NO:53具有S82F突变的法呢基-PP合酶存在下，从DMAPP和异戊基焦磷酸酯的组合形成香叶基焦磷酸酯(GPP)。在一个实施方案中，法呢基-二磷酸合酶具有与具有S82F突变的SEQ ID NO:53具有至少95％、98％、99％或100％同一性的序列，且其能够从DMAPP和异戊基焦磷酸酯形成香叶基焦磷酸酯。
GPP然后可以用作许多途径的底物，所述途径导致产生异戊二烯基-黄酮类化合物、香叶基-flavonoics、异戊二烯基-芪类化合物、香叶基-芪类化合物、CBGA、CBGVA、CBDA、CBDVA、CBGVA、CBCVA、THCA和THCVA(参见，例如，FIG.1A)。
例如，如上所述，使用NphB突变体(例如，M23突变体)，使用包括PDH旁路的完全合成生物化学系统测试了从葡萄糖和OA直接产生CBGA的能力(参见，图1A和图1B)。在系统中使用M23的初始生产力为67mg L-1hr-1，其中最终滴度为744±34mg L-1的CBGA。这比使用WTNphB的CBGA生产快100倍，并达到高21倍的滴度。应当指出，使用突变体NphB酶，在24小时内达到最大滴度，并且生产停止，但是使用野生型酶，系统连续运行长达4天，这表明所述酶和辅因子在更长的时间段内保持活性和活力。注意到一旦产生了约500mg L-1CBGA，反应就变得浑浊。收集沉淀物，并通过SDS-PAGE分析来鉴定沉淀物中的酶混合物，表明溶液中高水平的CBGA造成酶沉淀。开发了一种更有效的系统以在反应过程中移取产物。
尽管在反应中使用壬烷覆盖物来提取CBGA，但是CBGA比壬烷更易溶于水，这限制了可以用简单覆盖物提取的CBGA的量。因此，设计了一种流动系统，该系统将从壬烷层捕获CBGA并将其捕集在单独的水蓄池中(图4b)。通过实施该流动系统，将较低浓度的CBGA维持在反应容器中以减轻酶沉淀。该流动系统确实将最终滴度提高到1.2g/L。
然后进行实验以通过用2,4-二羟基-6-丙基苯甲酸(DA)代替系统中的OA来产生许多稀有大麻素的前体CBGVA(参见，例如，图1B)。首先测试设计的酶以确定它们是否对DA底物有活性。测试了两种最佳突变体M23和M31以及WT NphB的产生CBGVA的能力。表2中显示的动力学数据表明，M31远远更优，其催化效率比M23高15倍，并且比WT NphB高650倍。因此，进一步的努力利用M31从葡萄糖和2,4-二羟基-6-丙基苯甲酸生产CBGVA。如在图4A中所示，CBGVA以约107mg L-1hr-1的最大生产力生产，并达到1.74±0.09g L-1的最终滴度，将92％的添加的2,4-二羟基-6-丙基苯甲酸转化成CBGVA。CBGVA的生产不需要壬烷流动系统，因为CBGVA在沉淀酶中的功效较小。
为了证明该方法最终可以用于制备其它大麻素，使用CBDA合酶将CBGA转化为CBDA，并将CBGVA转化为CBDVA。对于CBDA，壬烷覆盖物含有大量的CBGA，因此通过简单地将壬烷覆盖物转移至包含CBDA合酶的溶液，CBGA就以14.4±0.8mg L-1小时-1mg总蛋白-1的恒定速率转化为CBDA保持4天。
由于CBGVA在壬烷中的有限溶解度，因此将CBGVA提取并添加到含有CBDA合酶的反应中。使用GC-MS，CBDA合酶的产物实际上是CBDVA。
本公开内容因此提供了一种用于生产GPP的无细胞系统。进一步，本公开内容提供了一种无细胞方法，其用于如下生产一系列纯的大麻素和其它异戊二烯化的天然产物：使用与突变体NphB组合的GPP途径，或使用本公开内容的突变体NphB的底物。该方法的成功使用了本公开内容的经工程改造的异戊二烯基转移酶(例如，如上所述的NphB突变体)，该酶是有活性的，高特异性的，且消除了对天然跨膜异戊二烯基转移酶的需要。本文提供的生物化学合成平台的模块性和灵活性具有基于生物的方法的益处，但消除了令人满意的生命系统的复杂性。例如，GPP毒性不影响设计过程。此外，OA不被酵母吸收，因此外源添加OA的方法在细胞中不一定是可能的。实际上，无细胞系统的灵活性可以极大地促进进一步优化、另外途径酶以及试剂和辅因子修饰所需的设计-构建-测试循环。
转向图1的总途径，本公开内容提供了由酶催化的许多步骤以将“底物”转化成产物。在一些情况下，一个步骤可以利用辅因子，但是一些步骤不使用辅因子(例如，NAD(P)H,ATP/ADP等)。表3提供了所使用的酶、生物和反应量以及登录号的列表(与此类登录号相关的序列通过引用并入本文)。
表3：在酶促平台中使用的酶
如上所述，通过本文和上面描述的突变体NphB多肽的活性，进行GPP对油橄榄醇酯的异戊二烯化。
本公开内容提供了生产异戊二烯化的化合物的体外方法，且此外，提供了用于生产大麻素和大麻素前体(例如，CBGA、CBGVA或CBGXA，其中‘X’表示任何化学基团)的体外方法。在本公开内容的一个实施方案中，无细胞制剂可以通过例如三种方法制备。在一个实施方案中，如本文所述，购买所述途径的酶并在合适的缓冲液中混合，并添加合适的底物，并在适合生产异戊二烯化的化合物或大麻素或大麻素前体(视情况而定)的条件下温育。在一些实施方案中，所述酶可以结合至支持物或在噬菌体展示或其它表面表达系统中表达，并且例如，固定在与代谢途径的循环中的点相对应的流体途径中。
图5A-B将途径描绘为各种“模块”(例如，糖酵解模块、甲羟戊酸/类异戊二烯模块、大麻素模块、聚酮化合物模块)。例如，类异戊二烯模块通过甲羟戊酸途径从乙酰辅酶A产生类异戊二烯香叶基焦磷酸酯(GPP)。芳族聚酮化合物模块利用III型聚酮化合物合酶(PKS)将己酰基辅酶A和丙二酰基辅酶A(源自乙酰辅酶A)转化成油橄榄醇酸(OA)。大麻素模块使用来自类异戊二烯模块和聚酮化合物模块的产物以得到大麻萜酚酸，其然后通过大麻素合酶转化为最终的大麻素。
在另一个实施方案中，在表达所述酶的条件下，将编码所述途径的一种或多种酶的一种或多种多核苷酸克隆到一种或多种微生物中。随后将细胞裂解，并将含有一种或多种衍生自细胞的酶的裂解制剂与合适的缓冲液和底物(如果必要的话，以及所述途径的一种或多种其它酶)组合，以生成异戊二烯化的化合物或大麻素或大麻素前体。可替换地，可以从裂解制剂中分离酶，且然后在适当的缓冲液中重组。在又一个实施方案中，使用购买的酶和表达的酶的组合在适当的缓冲液中提供途径。在一个实施方案中，克隆并表达所述途径的热稳定的多肽/酶。在一个实施方案中，所述途径的酶衍生自嗜热微生物。然后将微生物裂解，将制剂加热至一定温度，在该温度，所述途径的热稳定多肽是有活性的，且其它多肽(不感兴趣的多肽)被变性并变为无活性的。因此，该制剂包括微生物中所有酶的子集，且包括有活性的热稳定酶。然后该制剂可用于实现所述途径以生产异戊二烯化的化合物或大麻素或大麻素前体。
例如，为了构建一个体外系统，所有的酶可从市场上获得或通过亲和色谱法纯化，测试活性，并且在适当地选择的反应缓冲液中混合在一起。
还设想了体内系统，其在被工程改造进微生物中的生物合成途径中使用前述酶的全部或部分来获得重组微生物。
本公开内容还提供了包含代谢工程改造的生物合成途径的重组生物，所述途径包含用于生产异戊二烯化的化合物的突变体nphB，并且本公开内容可以进一步包括一种或多种表达用于生产大麻素的酶的其它生物(例如，一组表达部分途径的微生物和第二组表达所述途径的另一部分或最终部分等的微生物等的共培养物)。
在一个实施方案中，本公开内容提供了一种重组微生物，其包含与亲本微生物相比升高的至少一种靶酶的表达，或编码在亲本生物中未发现的酶。在另一个或其它实施方案中，所述微生物包含至少一种编码酶的基因的减少、破坏或敲除，所述酶与产生期望的代谢物所必需的代谢物竞争或其产生不希望的产物。重组微生物表达一种酶，该酶产生至少一种参与生物合成途径的代谢物，所述生物合成途径用于生产例如异戊二烯化的化合物或大麻素或大麻素前体。一般而言，重组微生物包含至少一种重组代谢途径，其包含靶酶，并且可以进一步包括在竞争性生物合成途径中的酶的活性或表达的降低。所述途径起作用以在例如异戊二烯化的化合物或大麻素或大麻素前体的生产中修饰底物或代谢中间体。所述靶酶由衍生自合适的生物学来源的多核苷酸编码并由其表达。在一些实施方案中，所述多核苷酸包含衍生自细菌或酵母来源并重组工程改造进本公开内容的微生物中的基因。在另一个实施方案中，编码期望的靶酶的多核苷酸天然存在于生物体中，但是被重组工程改造以与天然表达水平相比过表达。
术语“微生物”包括来自古细菌域、细菌域和真核生物域的原核和真核微生物物种，所述真核生物域包括酵母和丝状真菌、原生动物、藻类或高级原生生物。术语“微生物细胞”和“微生物”与术语微生物互换使用。
术语“原核生物”是本领域公知的，并且表示不包含细胞核或其它细胞器的细胞。通常将原核生物分类为细菌和古细菌两个域之一。古细菌域和细菌域的生物之间的决定性差异是基于16S核糖体RNA中的核苷酸碱基序列的根本差异。
“细菌”或“真细菌界”表示原核生物域。细菌包括如下至少11个不同的组：(1)革兰氏阳性(gram+)细菌，其中存在两个主要小类：(1)高G+C组(放线菌属(Actinomycetes)、分枝杆菌属(Mycobacteria)、微球菌属(Micrococcus)等)；(2)低G+C组(芽孢杆菌属(Bacillus)、梭状芽胞杆菌属(Clostridia)、乳杆菌属(Lactobacillus)、葡萄球菌属(Staphylococci)、链球菌属(Streptococci)、支原体属(Mycoplasmas))；(2)变形菌(Proteobacteria)，例如，紫色光合的+非光合的革兰氏阴性细菌(包括大多数“常见的”革兰氏阴性细菌)；(3)蓝细菌(Cyanobacteria)，例如氧化性光能利用菌；(4)螺旋体属(Spirochetes)和有关的物种；(5)浮霉状菌属(Planctomyces)；(6)拟杆菌属(Bacteroides)，黄杆菌属(Flavobacteria)；(7)衣原体属(Chlamydia)；(8)绿硫细菌；(9)绿色非硫细菌(也称作厌氧性光能利用菌)；(10)耐辐射微球菌(Radioresistantmicrococci)和有关的物种；和(11)栖热袍菌属(Thermotoga)和嗜热栖热腔菌(Thermosipho thermophiles)。
“革兰氏阴性细菌”包括球菌、非肠道杆菌和肠道杆菌。革兰氏阴性细菌的属包括例如奈瑟球菌属(Neisseria)、螺菌属(Spirillum)、巴斯德氏菌属(Pasteurella)、布鲁杆菌属(Brucella)、耶尔森氏菌属(Yersinia)、弗朗西丝氏菌属(Francisella)、嗜血菌属(Haemophilus)、博德特氏菌属(Bordetella)、埃希氏菌属(Escherichia)、沙门氏菌属(Salmonella)、志贺氏菌属(Shigella)、克雷伯氏菌属(Klebsiella)、变形菌属(Proteus)、弧菌属(Vibrio)、假单胞菌属(Pseudomonas)、拟杆菌属(Bacteroides)、醋杆菌属(Acetobacter)、气杆菌属(Aerobacter)、土壤杆菌属(Agrobacterium)、固氮菌属(Azotobacter)、螺旋状菌属(Spirilla)、沙雷氏菌属(Serratia)、弧菌属(Vibrio)、根瘤菌属(Rhizobium)、衣原体属(Chlamydia)、立克次氏体属(Rickettsia)、密螺旋体属(Treponema)和梭杆菌属(Fusobacterium)。
“革兰氏阳性细菌”包括球菌、不形成孢子的杆菌和形成孢子的杆菌。革兰氏阳性细菌的属包括例如放线菌属(Actinomyces)、芽孢杆菌属(Bacillus)、梭菌属(Clostridium)、棒杆菌属(Corynebacterium)、丹毒丝菌属(Erysipelothrix)、乳杆菌属(Lactobacillus)、李斯特菌属(Listeria)、分枝杆菌属(Mycobacterium)、粘球菌属(Myxococcus)、诺卡氏菌属(Nocardia)、葡萄球菌属(Staphylococcus)、链球菌属(Streptococcus)和链霉菌属(Streptomyces)。
如本文中使用的，酶的“活性”是其催化产生代谢物的反应(即，“发挥功能”)的能力的量度，并且可以表示为产生反应的代谢物的速率。例如，酶活性可以表示为每单位时间或每单位酶产生的代谢物的量(例如，浓度或重量)，或以亲和力或解离常数表示。
术语“生物合成途径”也被称作“代谢途径”，表示一组用于将一种化学物质转化(转变)成另一种的合成代谢或分解代谢生物化学反应(参见，例如，图1A-B)。如果基因产物平行地或串联地作用于相同的底物、产生相同的产物、或作用于或产生相同的底物和代谢物终产物之间的代谢中间体(即，代谢物)，则所述基因产物属于相同的“代谢途径”。本公开内容提供了具有用于生产期望的产物或中间体的代谢工程改造的途径的重组微生物。
因此，如下生产代谢“工程改造的”或“修饰的”微生物：将遗传物质引入选择的宿主或亲本微生物中，从而修饰或改变该微生物的细胞生理学和生物化学。通过遗传物质的引入，亲本微生物获得新的特性，例如产生新的或更大量的细胞内代谢物或表达通常不表达的多肽的能力。在一个示例性实施方案中，遗传物质向亲本微生物中的引入导致新的或经修饰的能力以使用丙酮酸氧化酶和乙酰基磷酸转移酶通过PDH旁路产生乙酰基磷酸酯和/或乙酰辅酶A。引入亲本微生物中的遗传物质含有编码一种或多种酶的基因或部分基因，所述酶参与用于生产异戊二烯化的化合物或大麻素或大麻素前体的生物合成途径，且所述遗传物质也可以包括用于表达这些基因和/或调节这些基因的表达的其它元件，例如启动子序列。
除了将遗传物质引入宿主或亲本微生物中以外或作为替代方案，经工程改造的或修饰的微生物还可以包括基因或多核苷酸的破坏、缺失或敲除，以改变微生物的细胞生理学和生物化学。通过基因或多核苷酸的减少、破坏或敲除，微生物获得新的或改良的特性(例如，产生新的或更大量的细胞内代谢物，改善代谢物沿期望的途径的通量，和/或减少不希望的副产物的产生的能力)，或从无细胞制剂消除酶，所述酶可能与从裂解制剂产生的生物合成途径竞争。
“酶”是指通常全部或大部分由构成蛋白或多肽的氨基酸组成的任何物质，其或多或少地特异性地催化或促进一种或多种化学或生物化学反应。
在本文中可互换地使用的术语“蛋白”或“多肽”包含一条或多条称为氨基酸的化学结构单元的链，所述氨基酸通过称为肽键的化学键连接在一起。蛋白或多肽可以作为酶发挥功能。
本文中使用的术语“代谢工程改造的”或“代谢工程改造”涉及在微生物中的合理途径设计，以及生物合成基因、与操纵子相关的基因和这样的多核苷酸的控制元件的组装，以生产期望的代谢物，诸如乙酰基磷酸酯和/或乙酰辅酶A、高级醇或其它化学物质。“代谢工程改造的”可以进一步包括通过使用基因工程改造和适当的培养条件(包括减少、破坏或敲除与通向期望途径的中间体竞争的竞争性代谢途径)调节和优化转录、翻译、蛋白稳定性和蛋白功能来优化代谢通量。生物合成基因可以对于宿主微生物而言是异源的，这是由于对于宿主而言是外来的，或者由于诱变、重组和/或与内源宿主细胞中的异源表达控制序列结合被修饰。在一个实施方案中，在多核苷酸对宿主生物而言是异源的情况下，可以对多核苷酸进行密码子优化。
“代谢物”表示由代谢产生的任何物质，或对于特定代谢过程而言必需的或参与特定代谢过程的物质，所述特定代谢过程产生期望的代谢物、化学物质、醇或酮。代谢物可以是作为代谢的起始材料(例如，葡萄糖等)、中间体(例如，乙酰辅酶A)或终产物(例如，CBDA)的有机化合物。代谢物可以用于构建更复杂的分子，或者它们可以分解成更简单的分子。中间代谢物可以从其它代谢物合成，可能用于制备更复杂的物质，或分解成更简单的化合物，这经常伴随化学能的释放。
“突变”是指产生突变蛋白、酶、多核苷酸、基因或细胞的任何过程或机制。这包括其中蛋白、酶、多核苷酸或基因序列被改变的任何突变，以及由这样的突变引起的细胞中的任何可检测的变化。通常，通过点突变、单个或多个核苷酸残基的缺失或插入，在多核苷酸或基因序列中发生突变。突变包括在基因的蛋白编码区内产生的多核苷酸改变，以及在蛋白编码序列之外的区域(例如，但不限于，调节序列或启动子序列)中的改变。基因中的突变可以是“沉默的”，即，不反映在表达后的氨基酸改变中，从而导致基因的“序列保守”变体。当一种氨基酸对应于超过一种密码子时，通常出现这种情况。产生蛋白的不同一级序列的突变可以被称作突变蛋白或蛋白变体。
“天然的”或“野生型”蛋白、酶、多核苷酸、基因或细胞是指在自然界中存在的蛋白、酶、多核苷酸、基因或细胞。
“亲本微生物”表示用于产生重组微生物的细胞。在一个实施方案中，术语“亲本微生物”描述了在自然界中存在的细胞，即未经遗传修饰的“野生型”细胞。术语“亲本微生物”进一步描述用作用于进一步工程改造的“亲本”的细胞。在该后一个实施方案中，细胞可以已经被遗传工程改造，但是用作进一步遗传工程改造的来源。
例如，可以对野生型微生物进行遗传修饰以表达或过表达第一靶酶诸如己糖激酶。该微生物可以在被修饰以表达或过表达第二靶酶(例如，果糖-1,6-二磷酸醛缩酶)的微生物的产生中充当亲本微生物。该微生物又可以被修饰以表达或过表达例如NADH氧化酶和Gald-3-磷酸脱氢酶(及其突变体)，其可以进一步被修饰以表达或过表达第三靶酶，例如，磷酸甘油酸激酶等。本文中使用的“表达”或“过表达”表示期望的基因产物的表型表达。在一个实施方案中，可以工程改造生物体中天然存在的基因，使得其与异源启动子或调节结构域连接，其中调节结构域引起该基因的表达，从而相对于野生型生物体修饰其正常表达。可替换地，可以工程改造生物体以除去或减少对该基因的阻抑功能，从而修饰其表达。在又一个实施方案中，将包含与期望的表达控制/调节元件可操作地连接的基因序列的盒工程改造到微生物中。
因此，亲本微生物作为连续遗传修饰事件的参考细胞发挥功能。每个修饰事件可以通过将一种或多种核酸分子引入参考细胞来完成。所述引入促进一种或多种靶酶的表达或过表达或一种或多种靶酶的减少或消除。应当理解，术语“促进”涵盖通过遗传修饰(例如亲本微生物中的启动子序列的遗传修饰)来激活编码靶酶的内源多核苷酸。还应当理解，术语“促进”涵盖将编码靶酶的外源多核苷酸引入亲本微生物中。
将编码可用于产生代谢物的酶(包括其同系物、变体、片段、相关融合蛋白或功能等同物)的多核苷酸用在重组核酸分子中，其指导这样的多肽在适当的宿主细胞(诸如细菌或酵母细胞)中表达。本文提供的序列和登录号为本领域技术人员提供了使用容易得到的软件和基础生物学知识来获得和得到本公开内容的各种酶的编码序列的能力。
本文所附的序列表提供了可用于本文所述方法中的示例性多肽。应当理解，不改变多肽分子活性的序列的添加，诸如非功能性或非编码序列(例如，多HIS标签)的添加，是基本分子的保守变异。
应当理解，本文描述的多核苷酸包括“基因”，并且上述的核酸分子包括“载体”或“质粒”。
术语“多核苷酸”、“核酸”或“重组核酸”表示多核苷酸诸如脱氧核糖核酸(DNA)，且在适当的情况下，表示核糖核酸(RNA)。
关于基因或多核苷酸的术语“表达”表示基因或多核苷酸的转录，和适当的话，得到的mRNA转录物向蛋白或多肽的翻译。因此，如从上下文将显而易见，蛋白或多肽的表达源自开放读码框的转录和翻译。
本领域技术人员会认识到，由于遗传密码的简并性质，可以使用其核苷酸序列不同的多种密码子来编码给定的氨基酸。本文提及编码上述生物合成酶或多肽的特定多核苷酸或基因序列仅用于举例说明本公开内容的一个实施方案，并且本公开内容包括任何序列的多核苷酸，所述序列编码包含在本公开内容的方法中使用的酶的多肽和蛋白的相同氨基酸序列的多肽。以类似的方式，多肽通常可以在其氨基酸序列中耐受一个或多个氨基酸置换、缺失和插入，而不损失或显著损失期望的活性。本公开内容包括具有替代氨基酸序列的此类多肽，并且由本文所示的DNA序列编码的氨基酸序列仅举例说明本公开内容的示例性实施方案。
如本文其它地方更详细描述的，本公开内容提供了重组DNA表达载体或质粒形式的多核苷酸，其编码一种或多种靶酶。通常，此类载体可以在宿主微生物的细胞质中复制，或整合到宿主微生物的染色体DNA中。在任一种情况下，载体可以是稳定载体(即，即使仅采用选择压力，载体经多次细胞分裂后仍存在)或瞬时载体(即，随着细胞分裂次数的增加，载体逐渐被宿主微生物遗失)。本公开内容提供了分离形式(即，非纯的，但在制备物中以自然界中未发现的丰度和/或浓度存在)以及纯化形式(即，基本上不含有污染物质，或基本上不含有与相应DNA一起存在于自然界中的物质)的DNA分子。
使用cDNA、mRNA或可替换地基因组DNA作为模板和适当的寡核苷酸引物，根据标准PCR扩增技术和下面实施例部分中描述的那些程序，可以扩增本公开内容的多核苷酸。可以将如此扩增的核酸克隆到适当的载体中并通过DNA序列分析进行表征。此外，通过标准合成技术，例如使用自动化的DNA合成仪，可以制备对应于核苷酸序列的寡核苷酸。
本公开内容提供了本申请所附序列表中的许多多肽序列，其可以用于使用遗传密码的简并性或使用公众可得到的数据库搜索编码序列来设计、合成和/或分离多核苷酸序列。
还理解，可以如下产生分离的编码与本文所述酶同源的多肽的多核苷酸分子：向编码特定多肽的核苷酸序列中引入一个或多个核苷酸置换、添加或缺失，使得将一个或多个氨基酸置换、添加或缺失引入编码的蛋白中。通过标准技术，诸如定位诱变和PCR介导的诱变，可以将突变引入多核苷酸中。与那些可能需要进行非保守氨基酸置换的位置相反，在一些位置，优选进行保守氨基酸置换。
如本领域技术人员会理解的，修饰编码序列以增强其在特定宿主中的表达可以是有利的。遗传密码是冗余的，具有64种可能密码子，但大多数生物体通常使用这些密码子的一部分。在物种中最常利用的密码子被称为最佳密码子，而那些不被经常利用的密码子被分类为稀有或利用率低的密码子。密码子可被置换以反映宿主的优选密码子选择，该过程有时被称为“密码子优化”或“控制物种密码子偏爱”。
可以制备含有被特定原核或真核宿主偏爱的密码子的优化编码序列(也参见,Murray等人(1989)Nucl.Acids Res.17:477-508)，例如，与由非优化序列产生的转录物相比，提高翻译速率或产生具有期望特性(例如更长的半衰期)的重组RNA转录物。翻译终止密码子也可以被修饰以反映宿主偏好。例如，酿酒酵母和哺乳动物的典型终止密码子分别是UAA和UGA。单子叶植物的典型终止密码子是UGA，而昆虫和大肠杆菌通常使用UAA作为终止密码子(Dalphin等人(1996)Nucl.Acids Res.24:216-218)。用于优化用于在植物中表达的核苷酸序列的方法提供在例如美国专利号6,015,891以及其中引用的参考文献中。
术语“底物”或“合适的底物”表示通过酶的作用转化成或意图转化成另一种化合物的任何物质或化合物。该术语不仅包括单一化合物，而且包括化合物的组合，诸如溶液、混合物和含有至少一种底物的其它物质或其衍生物。此外，术语“底物”不仅包括提供起始材料的化合物，而且包括用在如本文所述的与代谢工程改造的微生物有关的途径中的中间体和终产物代谢物。
“转化”表示将载体引入宿主细胞中的过程。转化(或转导或转染)可以通过许多方式中的任何一种来实现，所述方式包括电穿孔、显微注射、生物弹道技术(或颗粒轰击介导的递送)或土壤杆菌介导的转化。
“载体”通常表示可以在生物体、细胞或细胞组分之间传播和/或转移的多核苷酸。载体包括病毒、细菌噬菌体、原病毒(pro-viruses)、质粒、噬菌粒、转座子和人工染色体诸如YAC(酵母人工染色体)、BAC(细菌人工染色体)以及PLAC(植物人工染色体)等，它们是“附加体”，即自主复制或可以整合到宿主细胞的染色体中。载体也可以是裸RNA多核苷酸、裸DNA多核苷酸、在同一链内由DNA和RNA组成的多核苷酸、聚赖氨酸缀合的DNA或RNA、肽缀合的DNA或RNA、脂质体缀合的DNA等，它们在自然界中不是附加型的(episomal)，或者其可以是包含一种或多种上述多核苷酸构建体的生物体，诸如土壤杆菌或细菌。
表达载体的各种组分可以广泛变化，取决于载体的预期用途和意图使载体在其中复制或驱动表达的宿主细胞。适于在大肠杆菌、酵母、链霉菌和其它常用细胞中表达基因和维持载体的表达载体组分是广泛已知的和商购可得的。例如，用于包含在本公开内容的表达载体中的合适启动子包括在真核或原核宿主微生物中发挥功能的那些启动子。启动子可以包含调节序列，其允许调节与宿主微生物的生长相关的表达或者响应于化学或物理刺激而使基因的表达开启或关闭。对于大肠杆菌和某些其它细菌宿主细胞，可以使用来源于生物合成酶、赋予抗生素抗性的酶和噬菌体蛋白的基因的启动子，且包括例如半乳糖启动子、乳糖(lac)启动子、麦芽糖启动子、色氨酸(trp)启动子、β-内酰胺酶(bla)启动子、细菌噬菌体λPL启动子和T5启动子。此外，还可以使用合成启动子，诸如tac启动子(美国专利号4,551,433，其通过引用整体并入本文)。对于大肠杆菌表达载体，包括大肠杆菌复制起点(诸如来自pUC、p1P、p1和pBR)是有用的。
因此，重组表达载体含有至少一种表达系统，该表达系统又由与启动子可操作地连接的基因编码序列的至少一部分和任选的终止序列组成，所述终止序列起作用以实现编码序列在相容的宿主细胞中的表达。通过用本公开内容的重组DNA表达载体转化来修饰宿主细胞，以包含表达系统序列作为染色体外元件或整合到染色体中。
另外，且如上所述，本文提供的微生物和方法包括可用于产生代谢物的酶的同系物。关于第一家族或物种的原始酶或基因使用的术语“同系物”表示第二家族或物种的不同酶或基因，其通过功能、结构或基因组分析被确定为对应于第一家族或物种的原始酶或基因的第二家族或物种的酶或基因。最经常地，同系物将具有功能、结构或基因组相似性。使用遗传探针和PCR可以容易地克隆酶或基因的同系物的技术是已知的。使用功能测定和/或通过基因的基因组作图，可以确认克隆的序列作为同系物的身份。
如果编码蛋白的核酸序列具有与编码第二蛋白的核酸序列相似的序列，则蛋白与第二蛋白具有“同源性”或是“同源的”。可替换地，如果两种蛋白具有“相似的”氨基酸序列，则蛋白与第二蛋白具有同源性。(因此，术语“同源蛋白”定义为表示两种蛋白具有相似的氨基酸序列)。
如本文中使用的，当氨基酸序列具有至少约30％、40％、50％60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性时，两种蛋白(或蛋白的区域)是基本上同源的。为了确定两个氨基酸序列或两个核酸序列的同一性百分比，将所述序列比对以达到最佳对比目的(例如，可以在第一和第二氨基酸或核酸序列中的一个或两个中引入缺口用以最佳比对，并且为了对比目的可以忽略不同源的序列)。在一个实施方案中，出于对比目的而比对的参考序列的长度为所述参考序列的长度的至少30％，通常至少40％，更通常至少50％，且甚至更通常至少60％，甚至更通常至少70％、80％、90％、100％。然后对比相应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置被与第二序列中相应位置相同的氨基酸残基或核苷酸占据时，则所述分子在该位置是相同的(本文中使用的氨基酸或核酸“同一性”等同于氨基酸或核酸“同源性”)。两个序列之间的同一性百分比是所述序列共有的相同位置的数目的函数，考虑缺口的数目和每个缺口的长度，所述缺口为了两个序列的最佳比对而需要引入。
当“同源的”用于指蛋白或肽时，应当认识到，不相同的残基位置经常由于保守氨基酸置换而不同。“保守氨基酸置换”是这样的置换：其中一个氨基酸残基被置换为另一个含有具有类似化学特性(例如，电荷或疏水性)的侧链(R基团)的氨基酸残基。一般而言，保守氨基酸置换不会实质上改变蛋白的功能特性。在两个或更多个氨基酸序列彼此差别在于保守置换的情况下，可以上调序列同一性百分比或同源性程度以校正置换的保守性质。进行这种调节的方法是本领域技术人员众所周知的(参见，例如，Pearson等人,1994,特此通过引用并入本文)。
在一些情况下，可以使用“同工酶”，其实现相同的功能转化/反应，但其结构如此不同以至于它们通常被确定为不是“同源的”。
“保守氨基酸置换”是其中氨基酸残基被具有相似侧链的氨基酸残基替换的置换。具有类似侧链的氨基酸残基家族已经在本领域中定义。这些家族包括具有碱性侧链的氨基酸(例如，赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如，天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-支化侧链的氨基酸(例如，苏氨酸、缬氨酸、异亮氨酸)和具有芳族侧链的氨基酸(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。以下六组各自含有对于彼此为保守置换的氨基酸：1)丝氨酸(S)、苏氨酸(T)；2)天冬氨酸(D)、谷氨酸(E)；3)天冬酰胺(N)、谷氨酰胺(Q)；4)精氨酸(R)、赖氨酸(K)；5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、丙氨酸(A)、缬氨酸(V)和6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
通常使用序列分析软件来测量多肽的序列同源性，其也可以被称作序列同一性百分比。参见，例如，威斯康辛州大学生物技术中心(University of WisconsinBiotechnology Center,910University Avenue,Madison,Wis.53705)遗传计算机组(Genetics Computer Group，GCG)的序列分析软件包。蛋白分析软件使用为各种置换、缺失和其它修饰(包括保守氨基酸置换)指定的同源性的量度来匹配相似的序列。例如，GCG含有程序诸如“Gap”和“Bestfit”，其可以与默认参数一起用来确定密切相关的多肽(诸如来自不同生物物种的同源多肽)之间或野生型蛋白和其突变蛋白之间的序列同源性或序列同一性。参见，例如，GCG 6.1版。
将分子序列与含有来自不同生物体的大量序列的数据库进行对比而使用的典型算法是计算机程序BLAST(Altschul,1990；Gish,1993；Madden,1996；Altschul,1997；Zhang,1997)，特别是blastp或tblastn(Altschul,1997)。用于BLASTp的典型参数是：期望值：10(默认)；过滤器：seg(默认)；开放缺口罚分(Cost to extend a gap)：11(默认)；延伸缺口罚分(Cost to extend agap)：1(默认)；最大比对：100(默认)；字长：11(默认)；描述数目(No.of descriptions)：100(默认)；罚分矩阵：BLOWSUM62。
当搜索含有来自大量不同生物体的序列的数据库时，通常对比氨基酸序列。可以通过本领域已知的除BLASTp之外的算法来测量使用氨基酸序列的数据库搜索。例如，可以使用FASTA(GCG 6.1版中的一个程序)对比多肽序列。FASTA提供了查询序列和搜索序列之间的最佳重叠区域的比对和序列同一性百分比(Pearson,1990,特此通过引用并入本文)。例如，使用FASTA及其默认参数(字长2和PAM250评分矩阵)可以确定氨基酸序列之间的序列同一性百分比，如在GCG 6.1版中所提供，特此通过引用并入本文。
本公开内容提供了可用于产生用在体外系统中的重组微生物和蛋白的各种基因、同系物和变体的登录号和序列。应当理解，本文描述的同系物和变体是示例性的和非限制性的。使用各种数据库，包括例如可在万维网上访问的国家生物技术信息中心(NCBI)，本领域技术人员可获得另外的同系物、变体和序列。
利用本文描述的序列和登录号来鉴定可用于或替代本文使用的任何多肽的同系物和同工酶完全是在本领域的技术水平内。实际上，本文提供的任一种序列的BLAST搜索将鉴定多个相关的同系物。
适合于培养和维持本文提供的重组微生物的培养条件是已知的(参见，例如，Freshney的“Culture of Animal Cells--A Manual of Basic Technique”,Wiley-Liss,N.Y.(1994),第三版)。熟练的技术人员将认识到，可以改变这样的条件以适应每种微生物的需求。
应当理解，可以修饰一系列微生物以包括适合于生产异戊二烯化的化合物或大麻素或大麻素前体的重组代谢途径的全部或部分。还理解，多种微生物可以充当编码适用于本文提供的重组微生物中的靶酶的遗传物质的“来源”。
如以前讨论的，描述本文中可用的分子生物学技术(包括载体、启动子的使用和许多其它相关主题)的通用教材包括Berger和Kimmel,Guide to Molecular CloningTechniques,Methods in Enzymology第152卷,(Academic Press,Inc.,San Diego,Calif.)(“Berger”)；Sambrook等人,Molecular Cloning--ALaboratory Manual,第2版,第1-3卷,Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y.,1989(“Sambrook”)和Current Protocols in Molecular Biology,F.M.Ausubel等人,编,CurrentProtocols,a joint venture between Greene Publishing Associates,Inc.and JohnWiley&Sons,Inc.,(1999年增刊)(“Ausubel”)，它们中的每一篇通过引用整体并入本文。
足以指导技术人员进行体外扩增方法(包括聚合酶链式反应(PCR)、连接酶链式反应(LCR)、Qβ-复制酶扩增和其它RNA聚合酶介导的技术(例如，NASBA)(例如，用于生产本公开内容的同源核酸))的方案的例子参见Berger,Sambrook,和Ausubel,以及Mullis等人(1987)美国专利号4,683,202；Innis等人,编(1990)PCR Protocols:A Guide to Methodsand Applications(Academic Press Inc.San Diego,Calif.)(“Innis”)；Arnheim和Levinson(1990年10月1日)C&EN 36-47；The Journal Of NIH Research(1991)3:81-94；Kwoh等人(1989)Proc.Natl.Acad.Sci.USA 86:1173；Guatelli等人(1990)Proc.Nat'l.Acad.Sci.USA 87:1874；Lomell等人(1989)J.Clin.Chem 35:1826；Landegren等人(1988)Science 241:1077-1080；Van Brunt(1990)Biotechnology 8:291-294；Wu和Wallace(1989)Gene 4:560；Barringer等人(1990)Gene 89:117；以及Sooknanan和Malek(1995)Biotechnology 13:563-564。
用于克隆体外扩增的核酸的改进方法描述于Wallace等人,美国专利号5,426,039中。
通过PCR扩增大核酸的改进方法总结在Cheng等人(1994)Nature 369:684-685和其中引用的参考文献中，其中产生了高达40kb的PCR扩增子。技术人员将明白，使用逆转录酶和聚合酶，基本上任何RNA都可被转化为适合于限制性消化、PCR扩增和测序的双链DNA。参见，例如，Ausubel,Sambrook和Berger,都出处同上。
在以下实施例中举例说明了本发明，这些实施例以举例说明的方式提供且无意进行限制。
实施例
化学物质和试剂.酵母己糖激酶和谷氨酸棒杆菌(Corynebacterium glutamicum)过氧化氢酶购自Sigma Aldrich。绿色气球菌丙酮酸氧化酶购自A.G.Scientific。所有的辅因子和试剂都购自Sigma Aldrich或Thermo Fisher Scientific，例外是购自Santa CruzBiotechnology的油橄榄醇酸和购自Toronto Research Chemicals的2,4-二羟基-6-丙基苯甲酸。
酶的克隆和纯化.从IDT DNA购买NphB基因作为基因块，并使用Gibson Assembly方法克隆到pET 28(+)载体中。从基因组DNA或质粒扩增剩余的酶，并使用相同的Gibsonassembly方法克隆到pET28(+)中。将所有质粒转化到BL21(DE3)Gold中，并在含50μg/mL卡那霉素的LB培养基中表达酶。给1L培养物接种2mL在相同培养基中的饱和培养物，并在37℃生长至0.5-0.8的OD600。用1mM IPTG诱导培养物，并在18℃表达16小时。通过以2,500x g离心收获细胞，并重新悬浮于约20mL裂解缓冲液:50mM Tris[pH 8.0],150mM NaCl,和10mM咪唑。使用Emulsiflex仪器裂解细胞。通过以20,000x g离心将裂解物澄清，并将上清液在4℃分批结合1mL NiNTA树脂30分钟。将该树脂转移至重力流动柱。将树脂用10柱体积的洗涤缓冲液(50mM Tris[pH 8.0],150mM NaCl,和10mM咪唑)洗涤。然后用2柱体积的洗脱缓冲液(50mM Tris[pH 8.0],150mM NaCl,250mM咪唑和30％(v/v)甘油)洗脱蛋白。使用液氮将酶在洗脱缓冲液中快速冷冻，并将酶储备液保存在-80℃。
PDH无细胞反应.PDH反应以两部分组装。首先，将辅因子和底物在一个试管中组合，并在另一个试管中组合酶。通过以200μL的终体积混合辅因子和酶来引发反应。最终的底物和辅因子浓度如下：500mM葡萄糖,1mM1,6果糖二磷酸,4mM ATP,0.5mM 2,3二磷酸甘油酸酯,0.5mM NAD+,1.5mM CoA,1.5mM NADP+,0.5mM TPP,6mM MgCl2,10mM KCl,50mM Tris[pH 8.0]和20mM磷酸盐缓冲液[pH 8.0],5mM谷胱甘肽和0.5-5mM 1,6DHN。在24小时淬灭反应。
PDH活性测定.在几种芳族聚酮化合物存在下测定PDH的活性。媒介物对照是1％乙醇，并且将活性与没有芳族聚酮化合物的测定进行比较。最终反应体积为200μL，且含有2mMNAD+、2mM CoA、1mM TPP、5mM MgCl2、5mM KCl、50mM Tris pH 8.0和5μL 1.25mg/mL PDH。在96孔板中设置反应。加入芳族聚酮化合物至1mM的终浓度，并加入乙醇对照至1％(v/v)的终浓度。将板在室温温育10分钟，并用10μL 100mM丙酮酸引发反应。使用M200波谱仪监测在340nm的吸光度10分钟。由于芳族分子在340nm处具有背景吸光度，因此使用反应混合物和芳族分子给反应建立空白，但不用丙酮酸引发反应，而是添加水。使用线性拟合的初始斜率确定初始速率。使用Beer定律和6.22x 103M-1cm-1的消光系数，计算每单位时间产生的NADH的量。一式三份进行反应，并计算平均值和标准误差。
PyOx/PTA无细胞反应.PyOx/PTA反应以两部分组装。首先，将辅因子和底物在一个试管中组合，并在另一个试管中组合酶。在200μL反应中的最终辅因子和底物浓度如下：500mM葡萄糖,1mM 1,6果糖二磷酸,4mM ATP,0.5mM 2,3二磷酸甘油酸酯,0.5mM NAD+,1.5mM CoA,3mM NADP+,0.5mM TPP,6mM MgCl2,10mM KCl,50mM Tris pH 8.0和50mM磷酸盐缓冲液[pH 8.0]。在表3中详细列出了添加到每个反应中的酶的量。混合辅因子和酶以引发反应，并在顶部添加500μL壬烷覆盖物。将反应物在室温在凝胶振荡器上轻轻振荡温育。
对于1,6DHN/5-p-1,6DHN:当芳族底物是变化的组分时，将0.5-5mM芳族底物加入反应中，并在24小时淬灭反应。当时间是变化的组分时，加入5mM 1,6DHN，并在约12、24、48和72小时淬灭单独的反应。
对于油橄榄醇酯/CBGA:大麻素途径的优化显示，用较少的葡萄糖可达到相同的滴度，因此葡萄糖浓度降至150mM。此外，将NADP+浓度增加到6mM并将ATP浓度减少到1mM导致更高的CBGA滴度。油橄榄醇酯浓度设定为5mM。添加到反应中的NphB的量是可变的。在图2c中显示的数据利用1.5mg/mL NphB，并在约4、8、14、24、48、72和96小时淬灭反应。在图4a中显示的数据是使用0.5mg/mL WT NphB和M23获得，并且在约6、9、12、24、48、72和96小时淬灭反应。
对于2,4-二羟基-6-丙基苯甲酸/CBGVA:条件与上述一般方法非常相似，不同之处在于使用了150mM葡萄糖、1mM ATP和6mM NADP+，并在约6、9、12、24和48小时淬灭反应。此外，异戊二烯基转移酶的终浓度为1mg/mL，并且我们用芹菜配基、黄豆苷元、染料木黄酮、柚皮素和白藜芦醇测试了AtaPT、NovQ和NphB。我们还用油橄榄醇、油橄榄醇酯和1,6DHN测试了NphB。在24小时淬灭反应。
淬灭反应.为了淬灭反应，将水层和有机层转移至1.5mL微量离心管。用200μL乙酸乙酯洗涤反应小瓶，然后将其与微量离心管中的反应物合并。将样品涡旋5-10秒，且然后以13,000rpm离心3分钟。除去有机层，并将剩余的水层用200μL乙酸乙酯再萃取两次。对于每个样品，将有机萃取物合并，且然后使用真空离心机蒸发。将样品重新溶解在甲醇中用于HPLC分析。
对于油橄榄醇酯/CBGA:由于观察到蛋白沉淀，在存在0.12g尿素(固体)的情况下提取了图4a中所示的CBGA反应物，以促进CBGA的提取。这对于图2c中的WT NphB CBGA数据来说是不必要的，因为蛋白不沉淀。
产物的定量.使用Thermo Ultimate 3000HPLC在C18柱(4.6×100mm)上通过反相色谱法分级分离反应物。将柱隔室温度设定为40℃，并且流速为1mL/min。使用水+0.1％TFA(溶剂A)和乙腈+0.1％ TFA(溶剂B)作为流动相，用梯度洗脱分离化合物。将溶剂B保持在20％持续第1分钟。然后在4分钟内将溶剂B增加到95％ B，且然后将95％ B保持3分钟。然后将柱重新平衡至20％ B持续3分钟，总运行时间为11分钟。
使用从由Sigma Aldrich购买的分析标准品所衍生出的外部校正曲线定量大麻素(CBGA、CBDA和CBDVA)。5-p-1,6-DHN和CBGVA核磁共振(NMR)样品用于生成外部校正曲线，因为没有可靠标准品可用。将已知浓度的标准品溶解在水中，且然后使用上面详述的方法提取。
在没有可靠标准品的情况下定量异戊二烯基产物.由于缺乏异戊二烯基产物异戊二烯基-芹菜配基、异戊二烯基-黄豆苷元、异戊二烯基-柚皮素、异戊二烯基-染料木黄酮、异戊二烯基-白藜芦醇和异戊二烯基-橄榄醇的可靠标准品，因此基于底物消耗来定量异戊二烯基产物。为了产生标准曲线，将每种芳族底物的系列稀释液进行反应混合，但是为了防止产物形成，异戊二烯基-转移酶被省去。与标准曲线相比，使用液相色谱法-质谱法定量被反应消耗的底物的量。
在由MassLynx 4.1软件(Waters Corporation,Milford,MA)控制的Waters LCT-Premier XE飞行时间仪器上进行电喷射电离飞行时间测量。该仪器配备有以电喷雾模式操作的多模式电离源。将亮氨酸脑啡肽(Sigma Chemical,L9133)的溶液用于Lock-Spray以获得准确的质量测量值。在Waters Acquity UPLC系统上使用直接环注射注入样品。使用Acquity BEH C18 1.7μm柱(50×2.1mm)在Waters Acquity UPLC系统上分离样品，并用30-95％溶剂B的梯度历时10min(溶剂A：水，溶剂B：乙腈，二者含有0.2％甲酸(vol/vol))洗脱。从300-2000Da的质量记录质谱图。
NMR光谱法.使用NMR光谱法鉴定异戊二烯基产物，并定量5-p-1,6-DHN。
对于1,6DHN/5-p-1,6DHN:使用PyOx/PTA无细胞系统生产异戊二烯基-DHN。合并200μL反应物，并用等量壬烷萃取3次，且然后蒸发壬烷。将反应的产物悬浮于500μL氘代甲醇(CD3OD)中，以2mM 1,3,5-三甲氧基苯(TMB)作为内部标准品。在AV400 Bruker NMR谱仪上收集波谱。参考内部TMB标准品测定样品中异戊二烯化的化合物的量。将来自6.05ppm的TMB(3H,s)的质子信号与对应于7.27ppm的5-p-1,6-DHN(1H,d)的芳族质子进行比较。
对于2,4-二羟基-6-丙基苯甲酸/CBGVA:还使用NMR来鉴定以2,4-二羟基-6-丙基苯甲酸作为芳族底物的酶促系统的产物。如上详述建立PyOx/PTA系统，并将在24小时淬灭反应。如上详述提取反应，并在HPLC上分析。在6.7分钟时出现一个新的主峰，其预计为异戊二烯化的2,4-二羟基-6-丙基苯甲酸。纯化HPLC峰，除去溶剂，并将纯组分重新溶解在600μLCD3OD中。将使用AV500 Bruker NMR谱仪收集的质子谱图与Shoyama等人发表的CBGVA的质子谱图进行比较，以确认CBGVA是主要产物。基于Shoyama等人的论文，Bohlman等人的论文，结论是，2,4-二羟基-6-丙基苯甲酸的异戊二烯化发生在2,4-二羟基-6-丙基苯甲酸的C3碳处。
修饰NphB的结合口袋以接受油橄榄醇酯的Rosetta设计.将油橄榄醇酯放在NphB的活性位点中在六个不同的起始位置，所述位置表示为表4中的油橄榄醇酯P1-6。对每个油橄榄醇酯位置运行ROSETTA 5次，总共30次设计。表4列出了在每种设计中预测的突变。对于每个油橄榄醇酯位置，选择一组共有突变(即，最常选择的残基)以进一步评价：共有组A至F(表4)。然后评价每个ROSSETTA建议的突变的相对重要性。对于每个共有组，将突变设置回WT残基，一次一个，并使用ROSETTA计算能量评分的变化(参见表5)。引起最大能量变化的那些突变被认为是最重要的突变体，以包括在用于实验测试的文库中。
表4
表5
为了建模油橄榄醇酸，使用来自5B09晶体结构的油橄榄醇酯的4MX.sdf3-D结构，并使用Open Babel 2.3.1在假定pH 7的情况下将氢原子添加至该结构。使用生物化学文库(BCL)分子为油橄榄醇酸生成了旋转异构体文库：使用PDB文库生成了ConformerGenerator 3.5。最后，在使用Rosetta 3.7版本中的脚本main/source/python/public/molfile_to_params.py生成由Rosetta读出的参数文件之前，将芳族键手动注释到文件中。使用来自1ZB6晶体结构的GST.sdf文件，在没有旋转异构体文库的情况下生成了香叶基s-巯基二磷酸酯(GST)的参数文件。然后将油橄榄醇酸分子手工放置进NphB与GST和DHN的共晶体结构(1ZB6)中，其中使用pymol脱除DHN和结晶水。将油橄榄醇酸放置在活性位点的6个不同位置，其中油橄榄醇酯芳族环的平面平行于GST烷基尾巴，且期望的异戊二烯化位点与最终的碳阳离子相距3.7埃，这反映了DHN在1ZB6晶体结构中的位置。在Rosetta设计过程中，允许残基49、162、213、224、232、233、234、271、286和288是任何氨基酸，而其它侧链则保持在固定位置，且主链固定。设计的残基与油橄榄醇酯直接接触，而不与GST直接接触。来自Rosetta 3.7版本的固定主链脚本main/source/bin/fixbb.static.linuxgccrelease用所有可能的旋转异构体(-ex4)运行，使用输入侧链(-use_input_sc)、设计后最小化的侧链(minimize_sidechains)、线性记忆节点交互图(-linmem_ig 10)以及有和没有配体加权得分函数(-score:weights ligand)。从相同的起点，使用-nstruct输入将每个设计运行5次。从Rosetta建议的一组突变中，选择发生频率最高且对Rosetta评分函数贡献最大的突变，从而创建了22个突变体的文库用于实验测试。
初始NphB突变体文库筛选.为了筛选初始文库，进行了小规模表达和纯化。给25mLLB培养基接种25μL带有NphB表达质粒的BL21 DE3 Gold的饱和培养物。将培养物在37℃温育直至OD600达到0.4-0.6。通过添加1mM IPTG来诱导NphB构建体的表达，然后在18℃温育18小时。通过以2500xg离心收获细胞。将沉淀重新悬浮于500μL裂解缓冲液:50mM[Tris pH8.0],150mM NaCl,和5mM咪唑，并通过声处理裂解。通过在4℃以20,000x g离心10分钟来澄清细胞裂解物，并将上清液在4℃与50μL NiNTA树脂一起温育。使用96-孔旋转柱板纯化NphB构建体。将上清液/树脂上样至柱，并以500x g离心2分钟。然后加入500μL裂解缓冲液，并将板再次以500xg离心1分钟。使用200μL洗脱缓冲液(50mM Tris[pH 8.0],150mM NaCl,250mM咪唑和30％(v/v)甘油)洗脱蛋白。
在以下条件下测定酶：2.5mM香叶基焦磷酸酯,5mM油橄榄醇酯,5mM MgCl2,50mMTris pH 8.0,约0.1mg/mL NphB突变体，最终体积为100μL。首先使用洗脱缓冲液将所有酶稀释至0.5mg/mL，使得在每个反应中咪唑的终浓度相同。将反应在室温温育12小时，然后用100μL乙酸乙酯萃取3次。为每个反应合并有机萃取物，并使用真空离心机除去溶剂。将样品重新溶解在100μL甲醇中，并进行HPLC分析。
聚焦NphB突变体文库筛选.对于聚焦文库，进行如上所述的1L规模的NphB构建体表达和纯化。在以下条件下测定酶：2.5mM GPP,5mM油橄榄醇酯,5mM MgCl2,50mM Tris pH8.0和约1mg/mL NphB酶，最终体积为100μL。将反应在室温温育1小时。在80μL乙腈中淬灭40μL每个反应。将样品以13,000rpm离心5分钟，以除去沉淀的蛋白。如上所述，使用HPLC分析上清液。
酶动力学参数.在以下条件下设置反应：50mM Tris[pH 8.0],2.5mM GPP,5mMMgCl2,约27μM酶,和在0.1mM至6mM变化的油橄榄醇酯或2,4-二羟基-6-丙基苯甲酸，最终体积为200μL。以下面详述的时间间隔，在80μl乙腈+0.1％ TFA中淬灭40μL反应。将反应物以13,000-16,060x g离心5分钟以使蛋白沉淀，并使用上文详述的HPLC方法分析上清液。绘制初始速率相对于底物浓度的图，并与Michaelis-Menten方程拟合以确定动力学参数kcat和KM(OriginPro)。一式三份地执行每条Michaelis-Menten曲线。报道了动力学参数的平均值和标准偏差。
对于油橄榄醇酯/CBGA:对于WT、M1、M10和M30，时程为3、6、9和12分钟。对于突变体25，在1、2、4和8分钟淬灭反应，且对于M31，在1、2、4和6分钟淬灭反应。
对于2,4-二羟基-6-丙基苯甲酸/CBGVA:对于M31，时程为0.5、1、1.5和2分钟。对于M23，时程为5、10、15和20分钟，且对于WT NphB，时程为8、16、24和32分钟。突变体的酶浓度为约27μM，且WT NphB的浓度为约35μM。
来自WT NphB和M23的异构体概况的GC-MS表征.将样品溶解在200μL乙酸乙酯中。使用Agilent Model 7693自动采样器、7890B气相色谱仪和7250Q-TOF质量选择性检测器(以电子电离模式)进行GC-MS测量。样品注射以分流模式进行，其中入口温度设置为280℃。在尺寸为30m x250μm x 0.25μm的Agilent HP5-MS柱上进行分离。以恒定流模式，将超高纯等级He(Airgas)用作载气，流量设置为1.1mL/min。将初始恒温箱温度设置为120℃持续1分钟，然后以20℃/min的速度升至300℃的最终温度并保持4分钟。使用了3.0分钟的溶剂延迟。El能量设置为15eV。MSD设置为扫描50-500m/z范围。使用Mass Hunter采集和定性分析软件(Agilent)进行数据收集和分析。
由于GC入口的温度升高，CBGA经历了自发的脱羧，如Radwan等人所述，导致在316m/z的M+离子。对应于CBGA标准品的316m/z离子的保留时间为10.48分钟。
用于从溶液中萃取CBGA的壬烷-流动系统.如上所详述建立了PyOx/PTA反应。将500μL壬烷覆盖物添加到在2ml玻璃小瓶中的反应物中，该玻璃小瓶覆盖有2层可呼吸的细胞培养膜。将两个针插入15mL falcon管中的约750μL标记和3.5mL标记处。将管道连接器的路厄锁连接到针头，并将Viton管道连接到路厄锁的另一端。将针通过路厄锁连接器连接到管道的另一端，并穿过网状覆盖物插入，所以它们仅接触壬烷层且不接触反应物。将2mLTris缓冲液[pH 8.5]添加到15mL锥形管中，并添加6mL壬烷。使用蠕动泵将壬烷泵送通过系统，使得壬烷从反应顶部流过缓冲溶液。将壬烷泵入蓄池，分离到15mL锥形管的顶层。将来自15mL锥形管的顶部的壬烷泵入反应小瓶的顶部。这基本上稀释了整个系统中的CBGA，从而驱动CBGA扩散到壬烷层中并扩散出反应。
克隆CBDAS.从针对巴斯德毕赤酵母(Pichia pastoris)优化的IDT密码子订购了CBDAS的基因块。通过从蛋白序列的第28个残基(NPREN…)到蛋白末端进行PCR扩增，除去信号序列，其突出端与pPICZα载体相容。使用Gibson克隆方法将PCR产物克隆到用EcoRI和XbaI消化的pPICZα载体中。将组装反应的产物转化进BL21 Gold(DE3)细胞，分离具有正确序列的克隆。将该质粒用PmeI消化2小时，且然后使用Qiagen PCR纯化方案纯化。使用电穿孔将质粒转化到巴斯德毕赤酵母X33中。在电穿孔以后，立即将细胞在1mL冷的1M山梨醇和1mL YPD培养基中在没有摇动下温育2小时。将细胞铺板在具有500μg/mL zeocin的YPDS平板上。关于CBDAS基因在AOX1启动子和终止子之间的存在，使用PCR筛选菌落。为了筛选，将菌落重新悬浮于15μL无菌水中，并将5μL重新悬浮的菌落转移进含0.2％SDS的PCR管中。将样品在99℃加热10分钟，且然后将1μL用作PCR的模板。关于CBDAS的表达，筛选具有阳性菌落PCR命中的六个菌落。
CBDAS表达测试.使六个菌落在30℃生长过夜以获得饱和培养物。将过夜培养物用于接种在BMGY培养基中的25mL培养物，并生长至约2的OD。通过以2,000x g离心10分钟来收获细胞。将细胞沉淀物重新悬浮在90mL BMMY培养基中，并在30℃温育5天。每天，取出1mL培养物用于SDS-PAGE分析，并添加500μL甲醇。在第3天，针对CBDAS活性筛选培养物。测定条件如下：100μL 200mM柠檬酸盐缓冲液,100μM CBGA,5mM MgCl2,5mM KCl,1mM FAD和50μL表达培养基，最终体积为200μL。将反应物在室温温育过夜，且然后用200μL乙酸乙酯萃取3次。为每个样品合并乙酸乙酯萃取液，并用真空离心机除去。将样品重新悬浮于200μL甲醇中，并通过HPLC分析。所有克隆均产生活性CBDAS。
收集来自三个克隆(总共约300mL)的培养物以获得CBDAS活性。通过在4℃以约3,000x g离心20分钟，使细胞沉淀。然后使上清液穿过0.22μm过滤器。浓缩培养基，并使用来自Millipore的50,000MWCO蛋白浓缩器将缓冲液更换为100mM柠檬酸盐缓冲液pH 5.0。使用Bradford测定法确定培养基浓缩物中的总蛋白为0.4mg/mL，总产率为约5mg/L总蛋白。
CBDVA和CBDA的生产.为了将前体CBGA和CBGVA分别转化为CBDA和CBGVA，用CBDAS合酶建立了二级反应。
对于CBGA/CBDA:如上所详述建立了PyOx/PTA酶促系统以生产CBGA。24小时后，将200μL来自CBGA反应的壬烷覆盖物转移至CBDAS反应容器。在水层中：50mM Hepes[pH 7.0],5mM MgCl2,5mM KCl,25μMFAD,0.1mg/mL CBDAS浓缩物。将反应物在30℃在轻轻摇动下温育。在12、24、48、72和96小时淬灭反应。
对于CBGVA/CBDVA:将HPLC纯化的CBGVA转化为CBDVA。最终反应体积为200μL，含有50mM Hepes[pH 7.0]、5mM MgCl2、5mM KCl、25μMFAD和0.1mg/mL(总蛋白)的CBDAS浓缩物。添加200μL壬烷覆盖物，并在30℃在轻轻摇动下温育反应。在约24、48、72和96小时淬灭反应。
MatB活性测定.在有OA和DA存在下使用偶联酶促测定来确定来自R.palustris的丙二酰基辅酶A合成酶(MatB)(参见，例如，SEQ ID NO:82-83)的活性。反应条件为:2.5mM丙二酸,2mM ATP,1mM CoA,2.5mM磷酸烯醇丙酮酸(PEP),1mM NADH,5mM MgCl2,10mM KCl,0.35mg/mL ADK,0.75μg/mL MatB,1.6单位的PK和2.5单位的LDH,和50mM Tris[pH 8.0]。通过忽略底物(丙二酸)来控制背景ATP酶活性，并将1％乙醇、250μM或5mM OA或5mM DA添加到其余反应中。通过使用M2 SpectraMax监测由于NADH消耗引起的在340nm的吸光度的降低，确定MatB的活性。为确保MatB在5mM OA或DA是限制性的，将MatB加倍至1.5μg/mL。反应速率增加了一倍，表明MatB是系统中的限制性组分。将在5mM OA和5mM DA的NADH消耗率针对1％乙醇对照归一化。
AAE3活性测定.在有OA和DA存在下，使用与上述相似的偶联酶促测定来确定酰基活化酶3(AAE3)(参见，例如，SEQ ID NO:70-71和同系物-SEQ ID NO:72-75)的活性。条件与MatB测定相同，具有以下修改：添加2.5mM己酸代替丙二酸，并添加15μg/mL AAE3代替MatB。为了确保AAE3是限制性的，在有5mM OA或DA存在下使AAE3加倍。反应速率加倍，表明AAE3是限制性的。
ADK活性测定.在有OA和DA存在下，使用偶联酶促测定来确定腺苷酸激酶(ADK)(参见，例如，SEQ ID NO:)的活性。条件与MatB测定相同，具有以下修改：添加2mM AMP代替丙二酸，不添加CoA，并添加0.001mg/mL ADK。为了确保ADK在5mM OA和DA下是限制性试剂，将ADK的量加倍。速率的2倍增加表明ADK是限制因素。
CPK活性测定.在有OA或DA存在下使用偶联酶促测定来确定肌酸激酶(CPK)的活性。反应条件为:5mM磷酸肌酸,2mM ADP,5mM葡萄糖,2mM NADP+,5mM MgCl2,5mM KCl,0.3mg/mL Zwf,0.1mg/mL Sc Hex和0.08单位CPK。阳性对照反应含有1％乙醇，并将5mM OA或DA添加到其余反应中。监测NADPH在340nm处的吸光度。为了确保CPK是限制性的，在5mM OA和5mMDA将其加倍。得到的速率加倍，其表明，即使在高OA和DA，CPK也是限制性的。
OLS活性测定.通过设置以下条件来测定油橄榄醇合酶(OLS)(参见，例如，SEQ IDNO:76-77)：200μM丙二酰基辅酶A,100μM己酰基辅酶A,0.65mg/mL OAS,在50mM柠檬酸盐缓冲液pH 5.5或50mM Tris缓冲液pH 8.0中。通过加入OAS来引发反应，且然后在30分钟通过向50μL反应物中添加150μL甲醇来淬灭反应。将样品以约16,000x g离心2分钟以沉淀蛋白。使用HPLC分析上清液。
对于抑制实验，将条件改变为：1mM丙二酰基辅酶A,400μM己酰基辅酶A，在50mM柠檬酸盐缓冲液pH 5.5中，最终体积为200μL。将1％乙醇、250μM OA或1mM DA添加到反应中，且然后通过添加0.65mg/mL OLS来引发反应。将50μL等分试样在2、4、6和8分钟在150μL甲醇中淬灭。将反应物短暂涡旋并以16,000x g离心2分钟以沉淀蛋白。通过HPLC分析上清液。将HTAL、PDAL和油橄榄醇的原始峰面积相加，并相对于时间作图以确定速率。将补充了OA的反应和补充了DA的反应的速率针对乙醇对照归一化。
OLS/OAC活性测定.为了产生OA，使用与上面指定的相同的OLS条件，但是将油橄榄醇酸环化酶(OAC)(参见，例如，SEQ ID NO:78-79)以0.6mg/mL加入到反应中。将反应淬灭，并以与OLS测定相同的方式进行分析。将乙酰基磷酸酯和BSA分别以5mM-40mM AcP和10-30mg/mL BSA终浓度添加到测定中。
完整途径设置.在该研究中使用的酶和终浓度(mg/mL)可以在表6(对于MatB路径)和表7(对于MdcA路径)中找到。对于MatB路径，以以下浓度添加辅因子：150mM葡萄糖,1mM果糖二磷酸,2mM ATP,0.25mM NAD+,3mM NADP+,2mM CoA,0.25mM 2,3-二磷酸甘油酸酯,6mMMgCl2,10mM KCl,0.5mM硫胺素焦磷酸盐,50mM磷酸盐pH 8.0,5mM己酸,15mM丙二酸,5mM磷酸肌酸,和50mM Tris,pH 8.0。通过添加表6中列出的酶来引发反应。将反应在室温温育过夜，并将反应淬灭并用200μL乙酸乙酯萃取3次。使用真空离心机除去乙酸乙酯。将样品溶解在200μL甲醇中，并使用HPLC分析。
表6:在完整大麻素MatB途径中使用的酶以及最终酶浓度
表7:在完整大麻素MdcA途径中使用的酶以及最终酶浓度
MdcA路径的酶可以在表7中找到。在上面指定的相同辅因子条件下设置MdcA反应，但有以下变化：3mM ATP,0.25mM AMP,25mM磷酸肌酸且无Tris缓冲液。
在图5A-B中提供了MatB和MdcA途径的途径。
已经描述了本发明的某些实施方案。应该理解，在不脱离本发明的精神和范围的情况下可以做出各种修改。其它实施方案在下述权利要求的范围内。
序列表
<110> 加利福尼亚大学董事会
<120> 用于生产大麻素和其它异戊二烯化的化合物的生物合成平台
<130> 00011-089WO1
<140> 尚未指定
<141> 2019-08-01
<150> US 62/713,348
<151> 2018-08-01
<160> 83
<170> PatentIn 3.5版
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20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Gly Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Asn Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Asn Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 12
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M12
<400> 12
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Asn Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Thr Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Ala Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 13
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M13
<400> 13
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Asn Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Asn Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Ser Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 14
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M14
<400> 14
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Gly Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Thr Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Asn Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 15
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M15
<400> 15
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Asn Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ser Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Ala Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 16
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M16
<400> 16
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Asn Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ser Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Asn Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 17
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M17
<400> 17
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Thr Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Gly Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Asn Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 18
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M18
<400> 18
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Ser Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Asn Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Asn Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 19
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M19
<400> 19
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Ser Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Asn Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Asn Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Asn Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 20
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M20
<400> 20
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Thr Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Gly Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu His Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Asn Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 21
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M21
<400> 21
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Ser Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Asn Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ser Val Thr Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Asn Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Asn Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 22
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M22
<400> 22
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Thr Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Gly Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Thr Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu His Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Gly Ala Asn Tyr His Ile Thr Asp Val Gln Arg Gly Ile Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 23
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M23
<400> 23
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Ser Ala Ala Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 24
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M24
<400> 24
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ser Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Ser Ala Ala Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 25
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M25
<400> 25
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser His Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ser Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Ser Ala Ala Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 26
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M27
<400> 26
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Ser Ala Val Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 27
<211> 327
<212> PRT
<213> 人工序列
<220>
<223> NphB M28
<400> 27
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ser Val Ile Ser Asn
245 250 255
Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe
260 265 270
His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg
275 280 285
Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys
290 295 300
Leu Ser Ala Val Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys
305 310 315 320
Ala Phe Asp Ser Leu Glu Asp
325
<210> 28
<211> 328
<212> PRT
<213> 人工序列
<220>
<223> NphB M30
<400> 28
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ser Ala Val Ile Ser
245 250 255
Asn Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys
260 265 270
Phe His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys
275 280 285
Arg Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr
290 295 300
Lys Leu Gly Ala Ala Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu
305 310 315 320
Lys Ala Phe Asp Ser Leu Glu Asp
325
<210> 29
<211> 328
<212> PRT
<213> 人工序列
<220>
<223> NphB M31
<400> 29
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala
20 25 30
Ala Met Glu Glu Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp
35 40 45
Lys Ile Tyr Pro Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly
50 55 60
Gly Ser Val Val Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu
65 70 75 80
Leu Asp Phe Ser Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala
85 90 95
Thr Val Val Glu Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp
100 105 110
Asp Leu Leu Ala Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala
115 120 125
Ile Asp Gly Glu Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe
130 135 140
Pro Thr Asp Asn Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser
145 150 155 160
Met Pro Pro Ala Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly
165 170 175
Leu Asp Lys Val Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val
180 185 190
Asn Leu Tyr Phe Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser
195 200 205
Val Leu Ala Leu Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu
210 215 220
Gly Leu Lys Phe Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn
225 230 235 240
Trp Glu Thr Gly Lys Ile Asp Arg Leu Cys Phe Ser Ala Val Ile Ser
245 250 255
Asn Asp Pro Thr Leu Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys
260 265 270
Phe His Asn Tyr Ala Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys
275 280 285
Arg Thr Leu Val Tyr Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr
290 295 300
Lys Leu Gly Ala Val Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu
305 310 315 320
Lys Ala Phe Asp Ser Leu Glu Asp
325
<210> 30
<211> 307
<212> PRT
<213> 链霉菌属
<400> 30
Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu
1 5 10 15
Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro
20 25 30
Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val
35 40 45
Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser
50 55 60
Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu
65 70 75 80
Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala
85 90 95
Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu
100 105 110
Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn
115 120 125
Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala
130 135 140
Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val
145 150 155 160
Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe
165 170 175
Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu
180 185 190
Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe
195 200 205
Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly
210 215 220
Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu
225 230 235 240
Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala
245 250 255
Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr
260 265 270
Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Tyr
275 280 285
Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser
290 295 300
Leu Glu Asp
305
<210> 31
<211> 449
<212> PRT
<213> 嗜热脱氮土壤芽孢杆菌
<400> 31
Met Thr His Ile Arg Phe Asp Tyr Ser Lys Ala Leu Ala Phe Phe Gly
1 5 10 15
Glu His Glu Leu Thr Tyr Leu Arg Asp Ala Val Lys Val Ala His His
20 25 30
Ser Leu His Glu Lys Thr Gly Val Gly Asn Asp Phe Leu Gly Trp Leu
35 40 45
Asp Trp Pro Val Asn Tyr Asp Lys Glu Glu Phe Ala Arg Ile Lys Gln
50 55 60
Ala Ala Lys Lys Ile Gln Ser Asp Ser Asp Val Leu Leu Val Ile Gly
65 70 75 80
Ile Gly Gly Ser Tyr Leu Gly Ala Arg Ala Ala Ile Glu Met Leu His
85 90 95
His Ser Phe Tyr Asn Ala Leu Pro Lys Glu Lys Arg Ser Thr Pro Gln
100 105 110
Ile Ile Phe Val Gly Asn Asn Ile Ser Ser Thr Tyr Met Lys Asp Val
115 120 125
Ile Asp Phe Leu Glu Gly Lys Asp Phe Ser Ile Asn Val Ile Ser Lys
130 135 140
Ser Gly Thr Thr Thr Glu Pro Ala Ile Ala Phe Arg Ile Phe Arg Lys
145 150 155 160
Leu Leu Glu Asp Lys Tyr Gly Lys Glu Glu Ala Arg Arg Arg Ile Tyr
165 170 175
Ala Thr Thr Asp Arg Ala Arg Gly Ala Leu Arg Thr Leu Ala Asp Glu
180 185 190
Glu Gly Tyr Glu Thr Phe Val Ile Pro Asp Asp Ile Gly Gly Arg Tyr
195 200 205
Ser Val Leu Thr Ala Val Gly Leu Leu Pro Ile Ala Ala Ser Gly Ala
210 215 220
Asp Ile Asp Ala Met Met Glu Gly Ala Ala Lys Ala Arg Glu Asp Phe
225 230 235 240
Ser Arg Ser Glu Leu Glu Glu Asn Ala Ala Tyr Gln Tyr Ala Ala Ile
245 250 255
Arg Asn Ile Leu Tyr Asn Lys Gly Lys Thr Ile Glu Leu Leu Val Asn
260 265 270
Tyr Glu Pro Ala Leu His Tyr Phe Ala Glu Trp Trp Lys Gln Leu Phe
275 280 285
Gly Glu Ser Glu Gly Lys Asp Gln Lys Gly Ile Tyr Pro Ala Ser Ala
290 295 300
Asp Phe Ser Thr Asp Leu His Ser Leu Gly Gln Tyr Ile Gln Glu Gly
305 310 315 320
Arg Arg Asp Leu Phe Glu Thr Val Leu Lys Leu Glu Glu Pro Arg His
325 330 335
Glu Leu Val Ile Glu Ala Glu Glu Ser Asp Leu Asp Gly Leu Asn Tyr
340 345 350
Leu Ala Gly Gln Thr Val Asp Phe Val Asn Thr Lys Ala Phe Glu Gly
355 360 365
Thr Leu Leu Ala His Thr Asp Gly Gly Val Pro Asn Leu Val Val Thr
370 375 380
Leu Pro Lys Leu Asp Glu Tyr Thr Phe Gly Tyr Leu Val Tyr Phe Phe
385 390 395 400
Glu Lys Ala Cys Ala Met Ser Gly Tyr Leu Leu Gly Val Asn Pro Phe
405 410 415
Asp Gln Pro Gly Val Glu Ala Tyr Lys Lys Asn Met Phe Ala Leu Leu
420 425 430
Gly Lys Pro Gly Tyr Glu Glu Leu Lys Asp Glu Leu Glu Lys Arg Leu
435 440 445
Lys
<210> 32
<211> 319
<212> PRT
<213> 嗜热脂肪土芽孢杆菌
<400> 32
Met Lys Arg Ile Gly Val Leu Thr Ser Gly Gly Asp Ser Pro Gly Met
1 5 10 15
Asn Ala Ala Ile Arg Ser Val Val Arg Lys Ala Ile Tyr His Gly Val
20 25 30
Glu Val Tyr Gly Val Tyr His Gly Tyr Ala Gly Leu Ile Ala Gly Asn
35 40 45
Ile Lys Lys Leu Glu Val Gly Asp Val Gly Asp Ile Ile His Arg Gly
50 55 60
Gly Thr Ile Leu Tyr Thr Ala Arg Cys Pro Glu Phe Lys Thr Glu Glu
65 70 75 80
Gly Gln Lys Lys Gly Ile Glu Gln Leu Lys Lys His Gly Ile Glu Gly
85 90 95
Leu Val Val Ile Gly Gly Asp Gly Ser Tyr Gln Gly Ala Lys Lys Leu
100 105 110
Thr Glu His Gly Phe Pro Cys Val Gly Val Pro Gly Thr Ile Asp Asn
115 120 125
Asp Ile Pro Gly Thr Asp Phe Thr Ile Gly Phe Asp Thr Ala Leu Asn
130 135 140
Thr Val Ile Asp Ala Ile Asp Lys Ile Arg Asp Thr Ala Thr Ser His
145 150 155 160
Glu Arg Thr Tyr Val Ile Glu Val Met Gly Arg His Ala Gly Asp Ile
165 170 175
Ala Leu Trp Ser Gly Leu Ala Gly Gly Ala Glu Thr Ile Leu Ile Pro
180 185 190
Glu Ala Asp Tyr Asp Met Asn Asp Val Ile Ala Arg Leu Lys Arg Gly
195 200 205
His Glu Arg Gly Lys Lys His Ser Ile Ile Ile Val Ala Glu Gly Val
210 215 220
Gly Ser Gly Val Asp Phe Gly Arg Gln Ile Gln Glu Ala Thr Gly Phe
225 230 235 240
Glu Thr Arg Val Thr Val Leu Gly His Val Gln Arg Gly Gly Ser Pro
245 250 255
Thr Ala Phe Asp Arg Val Leu Ala Ser Arg Leu Gly Ala Arg Ala Val
260 265 270
Glu Leu Leu Leu Glu Gly Lys Gly Gly Arg Cys Val Gly Ile Gln Asn
275 280 285
Asn Gln Leu Val Asp His Asp Ile Ala Glu Ala Leu Ala Asn Lys His
290 295 300
Thr Ile Asp Gln Arg Met Tyr Ala Leu Ser Lys Glu Leu Ser Ile
305 310 315
<210> 33
<211> 309
<212> PRT
<213> 大肠杆菌
<400> 33
Met Val Arg Ile Tyr Thr Leu Thr Leu Ala Pro Ser Leu Asp Ser Ala
1 5 10 15
Thr Ile Thr Pro Gln Ile Tyr Pro Glu Gly Lys Leu Arg Cys Thr Ala
20 25 30
Pro Val Phe Glu Pro Gly Gly Gly Gly Ile Asn Val Ala Arg Ala Ile
35 40 45
Ala His Leu Gly Gly Ser Ala Thr Ala Ile Phe Pro Ala Gly Gly Ala
50 55 60
Thr Gly Glu His Leu Val Ser Leu Leu Ala Asp Glu Asn Val Pro Val
65 70 75 80
Ala Thr Val Glu Ala Lys Asp Trp Thr Arg Gln Asn Leu His Val His
85 90 95
Val Glu Ala Ser Gly Glu Gln Tyr Arg Phe Val Met Pro Gly Ala Ala
100 105 110
Leu Asn Glu Asp Glu Phe Arg Gln Leu Glu Glu Gln Val Leu Glu Ile
115 120 125
Glu Ser Gly Ala Ile Leu Val Ile Ser Gly Ser Leu Pro Pro Gly Val
130 135 140
Lys Leu Glu Lys Leu Thr Gln Leu Ile Ser Ala Ala Gln Lys Gln Gly
145 150 155 160
Ile Arg Cys Ile Val Asp Ser Ser Gly Glu Ala Leu Ser Ala Ala Leu
165 170 175
Ala Ile Gly Asn Ile Glu Leu Val Lys Pro Asn Gln Lys Glu Leu Ser
180 185 190
Ala Leu Val Asn Arg Glu Leu Thr Gln Pro Asp Asp Val Arg Lys Ala
195 200 205
Ala Gln Glu Ile Val Asn Ser Gly Lys Ala Lys Arg Val Val Val Ser
210 215 220
Leu Gly Pro Gln Gly Ala Leu Gly Val Asp Ser Glu Asn Cys Ile Gln
225 230 235 240
Val Val Pro Pro Pro Val Lys Ser Gln Ser Thr Val Gly Ala Gly Asp
245 250 255
Ser Met Val Gly Ala Met Thr Leu Lys Leu Ala Glu Asn Ala Ser Leu
260 265 270
Glu Glu Met Val Arg Phe Gly Val Ala Ala Gly Ser Ala Ala Thr Leu
275 280 285
Asn Gln Gly Thr Arg Leu Cys Ser His Asp Asp Thr Gln Lys Ile Tyr
290 295 300
Ala Tyr Leu Ser Arg
305
<210> 34
<211> 319
<212> PRT
<213> 嗜热脂肪土芽孢杆菌
<400> 34
Met Lys Arg Ile Gly Val Leu Thr Ser Gly Gly Asp Ser Pro Gly Met
1 5 10 15
Asn Ala Ala Ile Arg Ser Val Val Arg Lys Ala Ile Tyr His Gly Val
20 25 30
Glu Val Tyr Gly Val Tyr His Gly Tyr Ala Gly Leu Ile Ala Gly Asn
35 40 45
Ile Lys Lys Leu Glu Val Gly Asp Val Gly Asp Ile Ile His Arg Gly
50 55 60
Gly Thr Ile Leu Tyr Thr Ala Arg Cys Pro Glu Phe Lys Thr Glu Glu
65 70 75 80
Gly Gln Lys Lys Gly Ile Glu Gln Leu Lys Lys His Gly Ile Glu Gly
85 90 95
Leu Val Val Ile Gly Gly Asp Gly Ser Tyr Gln Gly Ala Lys Lys Leu
100 105 110
Thr Glu His Gly Phe Pro Cys Val Gly Val Pro Gly Thr Ile Asp Asn
115 120 125
Asp Ile Pro Gly Thr Asp Phe Thr Ile Gly Phe Asp Thr Ala Leu Asn
130 135 140
Thr Val Ile Asp Ala Ile Asp Lys Ile Arg Asp Thr Ala Thr Ser His
145 150 155 160
Glu Arg Thr Tyr Val Ile Glu Val Met Gly Arg His Ala Gly Asp Ile
165 170 175
Ala Leu Trp Ser Gly Leu Ala Gly Gly Ala Glu Thr Ile Leu Ile Pro
180 185 190
Glu Ala Asp Tyr Asp Met Asn Asp Val Ile Ala Arg Leu Lys Arg Gly
195 200 205
His Glu Ala Gly Lys Lys His Ser Ile Ile Ile Val Ala Glu Gly Val
210 215 220
Gly Ser Gly Val Asp Phe Gly Arg Gln Ile Gln Glu Ala Thr Gly Phe
225 230 235 240
Glu Thr Arg Val Thr Val Leu Gly His Val Gln Arg Gly Gly Ser Pro
245 250 255
Thr Ala Phe Asp Arg Val Leu Ala Ser Arg Leu Gly Ala Arg Ala Val
260 265 270
Glu Leu Leu Leu Glu Gly Lys Gly Gly Arg Cys Val Gly Ile Gln Asn
275 280 285
Asn Gln Leu Val Asp His Asp Ile Ala Glu Ala Leu Ala Asn Lys His
290 295 300
Thr Ile Asp Gln Arg Met Tyr Ala Leu Ser Lys Glu Leu Ser Ile
305 310 315
<210> 35
<211> 359
<212> PRT
<213> 大肠杆菌
<400> 35
Met Ser Lys Ile Phe Asp Phe Val Lys Pro Gly Val Ile Thr Gly Asp
1 5 10 15
Asp Val Gln Lys Val Phe Gln Val Ala Lys Glu Asn Asn Phe Ala Leu
20 25 30
Pro Ala Val Asn Cys Val Gly Thr Asp Ser Ile Asn Ala Val Leu Glu
35 40 45
Thr Ala Ala Lys Val Lys Ala Pro Val Ile Val Gln Phe Ser Asn Gly
50 55 60
Gly Ala Ser Phe Ile Ala Gly Lys Gly Val Lys Ser Asp Val Pro Gln
65 70 75 80
Gly Ala Ala Ile Leu Gly Ala Ile Ser Gly Ala His His Val His Gln
85 90 95
Met Ala Glu His Tyr Gly Val Pro Val Ile Leu His Thr Asp His Cys
100 105 110
Ala Lys Lys Leu Leu Pro Trp Ile Asp Gly Leu Leu Asp Ala Gly Glu
115 120 125
Lys His Phe Ala Ala Thr Gly Lys Pro Leu Phe Ser Ser His Met Ile
130 135 140
Asp Leu Ser Glu Glu Ser Leu Gln Glu Asn Ile Glu Ile Cys Ser Lys
145 150 155 160
Tyr Leu Glu Arg Met Ser Lys Ile Gly Met Thr Leu Glu Ile Glu Leu
165 170 175
Gly Cys Thr Gly Gly Glu Glu Asp Gly Val Asp Asn Ser His Met Asp
180 185 190
Ala Ser Ala Leu Tyr Thr Gln Pro Glu Asp Val Asp Tyr Ala Tyr Thr
195 200 205
Glu Leu Ser Lys Ile Ser Pro Arg Phe Thr Ile Ala Ala Ser Phe Gly
210 215 220
Asn Val His Gly Val Tyr Lys Pro Gly Asn Val Val Leu Thr Pro Thr
225 230 235 240
Ile Leu Arg Asp Ser Gln Glu Tyr Val Ser Lys Lys His Asn Leu Pro
245 250 255
His Asn Ser Leu Asn Phe Val Phe His Gly Gly Ser Gly Ser Thr Ala
260 265 270
Gln Glu Ile Lys Asp Ser Val Ser Tyr Gly Val Val Lys Met Asn Ile
275 280 285
Asp Thr Asp Thr Gln Trp Ala Thr Trp Glu Gly Val Leu Asn Tyr Tyr
290 295 300
Lys Ala Asn Glu Ala Tyr Leu Gln Gly Gln Leu Gly Asn Pro Lys Gly
305 310 315 320
Glu Asp Gln Pro Asn Lys Lys Tyr Tyr Asp Pro Arg Val Trp Leu Arg
325 330 335
Ala Gly Gln Thr Ser Met Ile Ala Arg Leu Glu Lys Ala Phe Gln Glu
340 345 350
Leu Asn Ala Ile Asp Val Leu
355
<210> 36
<211> 255
<212> PRT
<213> 大肠杆菌
<400> 36
Met Arg His Pro Leu Val Met Gly Asn Trp Lys Leu Asn Gly Ser Arg
1 5 10 15
His Met Val His Glu Leu Val Ser Asn Leu Arg Lys Glu Leu Ala Gly
20 25 30
Val Ala Gly Cys Ala Val Ala Ile Ala Pro Pro Glu Met Tyr Ile Asp
35 40 45
Met Ala Lys Arg Glu Ala Glu Gly Ser His Ile Met Leu Gly Ala Gln
50 55 60
Asn Val Asp Leu Asn Leu Ser Gly Ala Phe Thr Gly Glu Thr Ser Ala
65 70 75 80
Ala Met Leu Lys Asp Ile Gly Ala Gln Tyr Ile Ile Ile Gly His Ser
85 90 95
Glu Arg Arg Thr Tyr His Lys Glu Ser Asp Glu Leu Ile Ala Lys Lys
100 105 110
Phe Ala Val Leu Lys Glu Gln Gly Leu Thr Pro Val Leu Cys Ile Gly
115 120 125
Glu Thr Glu Ala Glu Asn Glu Ala Gly Lys Thr Glu Glu Val Cys Ala
130 135 140
Arg Gln Ile Asp Ala Val Leu Lys Thr Gln Gly Ala Ala Ala Phe Glu
145 150 155 160
Gly Ala Val Ile Ala Tyr Glu Pro Val Trp Ala Ile Gly Thr Gly Lys
165 170 175
Ser Ala Thr Pro Ala Gln Ala Gln Ala Val His Lys Phe Ile Arg Asp
180 185 190
His Ile Ala Lys Val Asp Ala Asn Ile Ala Glu Gln Val Ile Ile Gln
195 200 205
Tyr Gly Gly Ser Val Asn Ala Ser Asn Ala Ala Glu Leu Phe Ala Gln
210 215 220
Pro Asp Ile Asp Gly Ala Leu Val Gly Gly Ala Ser Leu Lys Ala Asp
225 230 235 240
Ala Phe Ala Val Ile Val Lys Ala Ala Glu Ala Ala Lys Gln Ala
245 250 255
<210> 37
<211> 335
<212> PRT
<213> 嗜热脂肪土芽孢杆菌
<400> 37
Met Ala Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Asn
1 5 10 15
Val Phe Arg Ala Ala Leu Lys Asn Pro Asp Ile Glu Val Val Ala Val
20 25 30
Asn Asp Leu Thr Asp Ala Asn Thr Leu Ala His Leu Leu Lys Tyr Asp
35 40 45
Ser Val His Gly Arg Leu Asp Ala Glu Val Ser Val Asn Gly Asn Asn
50 55 60
Leu Val Val Asn Gly Lys Glu Ile Ile Val Lys Ala Glu Arg Asp Pro
65 70 75 80
Glu Asn Leu Ala Trp Gly Glu Ile Gly Val Asp Ile Val Val Glu Ser
85 90 95
Thr Gly Arg Phe Thr Lys Arg Glu Asp Ala Ala Lys His Leu Glu Ala
100 105 110
Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ala Lys Asn Glu Asp Ile
115 120 125
Thr Ile Val Met Gly Val Asn Gln Asp Lys Tyr Asp Pro Lys Ala His
130 135 140
His Val Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Phe
145 150 155 160
Ala Lys Val Leu His Glu Gln Phe Gly Ile Val Arg Gly Met Met Thr
165 170 175
Thr Val His Ser Tyr Thr Asn Asp Gln Arg Ile Leu Asp Leu Pro His
180 185 190
Lys Asp Leu Arg Arg Ala Arg Ala Ala Ala Glu Ser Ile Ile Pro Thr
195 200 205
Thr Thr Gly Ala Ala Lys Ala Val Ala Leu Val Leu Pro Glu Leu Lys
210 215 220
Gly Lys Leu Asn Gly Met Ala Met Arg Val Pro Thr Pro Asn Val Ser
225 230 235 240
Val Val Asp Leu Val Ala Glu Leu Glu Lys Glu Val Thr Val Glu Glu
245 250 255
Val Asn Ala Ala Leu Lys Ala Ala Ala Glu Gly Glu Leu Lys Gly Ile
260 265 270
Leu Ala Tyr Ser Glu Glu Pro Leu Val Ser Arg Asp Tyr Asn Gly Ser
275 280 285
Thr Val Ser Ser Thr Ile Asp Ala Leu Ser Thr Met Val Ile Asp Gly
290 295 300
Lys Met Val Lys Val Val Ser Trp Tyr Asp Asn Glu Thr Gly Tyr Ser
305 310 315 320
His Arg Val Val Asp Leu Ala Ala Tyr Ile Ala Ser Lys Gly Leu
325 330 335
<210> 38
<211> 335
<212> PRT
<213> 人工序列
<220>
<223> mGap突变P191D
<400> 38
Met Ala Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Asn
1 5 10 15
Val Phe Arg Ala Ala Leu Lys Asn Pro Asp Ile Glu Val Val Ala Val
20 25 30
Asn Asp Leu Thr Asp Ala Asn Thr Leu Ala His Leu Leu Lys Tyr Asp
35 40 45
Ser Val His Gly Arg Leu Asp Ala Glu Val Ser Val Asn Gly Asn Asn
50 55 60
Leu Val Val Asn Gly Lys Glu Ile Ile Val Lys Ala Glu Arg Asp Pro
65 70 75 80
Glu Asn Leu Ala Trp Gly Glu Ile Gly Val Asp Ile Val Val Glu Ser
85 90 95
Thr Gly Arg Phe Thr Lys Arg Glu Asp Ala Ala Lys His Leu Glu Ala
100 105 110
Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ala Lys Asn Glu Asp Ile
115 120 125
Thr Ile Val Met Gly Val Asn Gln Asp Lys Tyr Asp Pro Lys Ala His
130 135 140
His Val Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Phe
145 150 155 160
Ala Lys Val Leu His Glu Gln Phe Gly Ile Val Arg Gly Met Met Thr
165 170 175
Thr Val His Ser Tyr Thr Asn Asp Gln Arg Ile Leu Asp Leu Asp His
180 185 190
Lys Asp Leu Arg Arg Ala Arg Ala Ala Ala Glu Ser Ile Ile Pro Thr
195 200 205
Thr Thr Gly Ala Ala Lys Ala Val Ala Leu Val Leu Pro Glu Leu Lys
210 215 220
Gly Lys Leu Asn Gly Met Ala Met Arg Val Pro Thr Pro Asn Val Ser
225 230 235 240
Val Val Asp Leu Val Ala Glu Leu Glu Lys Glu Val Thr Val Glu Glu
245 250 255
Val Asn Ala Ala Leu Lys Ala Ala Ala Glu Gly Glu Leu Lys Gly Ile
260 265 270
Leu Ala Tyr Ser Glu Glu Pro Leu Val Ser Arg Asp Tyr Asn Gly Ser
275 280 285
Thr Val Ser Ser Thr Ile Asp Ala Leu Ser Thr Met Val Ile Asp Gly
290 295 300
Lys Met Val Lys Val Val Ser Trp Tyr Asp Asn Glu Thr Gly Tyr Ser
305 310 315 320
His Arg Val Val Asp Leu Ala Ala Tyr Ile Ala Ser Lys Gly Leu
325 330 335
<210> 39
<211> 335
<212> PRT
<213> 人工序列
<220>
<223> mGap突变D34A/L35R/T36K
<400> 39
Met Ala Val Lys Val Gly Ile Asn Gly Phe Gly Arg Ile Gly Arg Asn
1 5 10 15
Val Phe Arg Ala Ala Leu Lys Asn Pro Asp Ile Glu Val Val Ala Val
20 25 30
Asn Ala Arg Lys Asp Ala Asn Thr Leu Ala His Leu Leu Lys Tyr Asp
35 40 45
Ser Val His Gly Arg Leu Asp Ala Glu Val Ser Val Asn Gly Asn Asn
50 55 60
Leu Val Val Asn Gly Lys Glu Ile Ile Val Lys Ala Glu Arg Asp Pro
65 70 75 80
Glu Asn Leu Ala Trp Gly Glu Ile Gly Val Asp Ile Val Val Glu Ser
85 90 95
Thr Gly Arg Phe Thr Lys Arg Glu Asp Ala Ala Lys His Leu Glu Ala
100 105 110
Gly Ala Lys Lys Val Ile Ile Ser Ala Pro Ala Lys Asn Glu Asp Ile
115 120 125
Thr Ile Val Met Gly Val Asn Gln Asp Lys Tyr Asp Pro Lys Ala His
130 135 140
His Val Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Phe
145 150 155 160
Ala Lys Val Leu His Glu Gln Phe Gly Ile Val Arg Gly Met Met Thr
165 170 175
Thr Val His Ser Tyr Thr Asn Asp Gln Arg Ile Leu Asp Leu Pro His
180 185 190
Lys Asp Leu Arg Arg Ala Arg Ala Ala Ala Glu Ser Ile Ile Pro Thr
195 200 205
Thr Thr Gly Ala Ala Lys Ala Val Ala Leu Val Leu Pro Glu Leu Lys
210 215 220
Gly Lys Leu Asn Gly Met Ala Met Arg Val Pro Thr Pro Asn Val Ser
225 230 235 240
Val Val Asp Leu Val Ala Glu Leu Glu Lys Glu Val Thr Val Glu Glu
245 250 255
Val Asn Ala Ala Leu Lys Ala Ala Ala Glu Gly Glu Leu Lys Gly Ile
260 265 270
Leu Ala Tyr Ser Glu Glu Pro Leu Val Ser Arg Asp Tyr Asn Gly Ser
275 280 285
Thr Val Ser Ser Thr Ile Asp Ala Leu Ser Thr Met Val Ile Asp Gly
290 295 300
Lys Met Val Lys Val Val Ser Trp Tyr Asp Asn Glu Thr Gly Tyr Ser
305 310 315 320
His Arg Val Val Asp Leu Ala Ala Tyr Ile Ala Ser Lys Gly Leu
325 330 335
<210> 40
<211> 394
<212> PRT
<213> 嗜热脂肪土芽孢杆菌
<400> 40
Met Asn Lys Lys Thr Ile Arg Asp Val Asp Val Arg Gly Lys Arg Val
1 5 10 15
Phe Cys Arg Val Asp Phe Asn Val Pro Met Glu Gln Gly Ala Ile Thr
20 25 30
Asp Asp Thr Arg Ile Arg Ala Ala Leu Pro Thr Ile Arg Tyr Leu Ile
35 40 45
Glu His Gly Ala Lys Val Ile Leu Ala Ser His Leu Gly Arg Pro Lys
50 55 60
Gly Lys Val Val Glu Glu Leu Arg Leu Asp Ala Val Ala Lys Arg Leu
65 70 75 80
Gly Glu Leu Leu Glu Arg Pro Val Ala Lys Thr Asn Glu Ala Val Gly
85 90 95
Asp Glu Val Lys Ala Ala Val Asp Arg Leu Asn Glu Gly Asp Val Leu
100 105 110
Leu Leu Glu Asn Val Arg Phe Tyr Pro Gly Glu Glu Lys Asn Asp Pro
115 120 125
Glu Leu Ala Lys Ala Phe Ala Glu Leu Ala Asp Leu Tyr Val Asn Asp
130 135 140
Ala Phe Gly Ala Ala His Arg Ala His Ala Ser Thr Glu Gly Ile Ala
145 150 155 160
His Tyr Leu Pro Ala Val Ala Gly Phe Leu Met Glu Lys Glu Leu Glu
165 170 175
Val Leu Gly Lys Ala Leu Ser Asn Pro Asp Arg Pro Phe Thr Ala Ile
180 185 190
Ile Gly Gly Ala Lys Val Lys Asp Lys Ile Gly Val Ile Asp Asn Leu
195 200 205
Leu Glu Lys Val Asp Asn Leu Ile Ile Gly Gly Gly Leu Ala Tyr Thr
210 215 220
Phe Val Lys Ala Leu Gly His Asp Val Gly Lys Ser Leu Leu Glu Glu
225 230 235 240
Asp Lys Ile Glu Leu Ala Lys Ser Phe Met Glu Lys Ala Lys Glu Lys
245 250 255
Gly Val Arg Phe Tyr Met Pro Val Asp Val Val Val Ala Asp Arg Phe
260 265 270
Ala Asn Asp Ala Asn Thr Lys Val Val Pro Ile Asp Ala Ile Pro Ala
275 280 285
Asp Trp Ser Ala Leu Asp Ile Gly Pro Lys Thr Arg Glu Leu Tyr Arg
290 295 300
Asp Val Ile Arg Glu Ser Lys Leu Val Val Trp Asn Gly Pro Met Gly
305 310 315 320
Val Phe Glu Met Asp Ala Phe Ala His Gly Thr Lys Ala Ile Ala Glu
325 330 335
Ala Leu Ala Glu Ala Leu Asp Thr Tyr Ser Val Ile Gly Gly Gly Asp
340 345 350
Ser Ala Ala Ala Val Glu Lys Phe Gly Leu Ala Asp Lys Met Asp His
355 360 365
Ile Ser Thr Gly Gly Gly Ala Ser Leu Glu Phe Met Glu Gly Lys Gln
370 375 380
Leu Pro Gly Val Val Ala Leu Glu Asp Lys
385 390
<210> 41
<211> 470
<212> PRT
<213> 嗜热脱氮土芽孢杆菌
<400> 41
Met Ala Lys Gln Gln Ile Gly Val Ile Gly Leu Ala Val Met Gly Lys
1 5 10 15
Asn Leu Ala Leu Asn Ile Glu Ser Arg Gly Tyr Ser Val Ala Val Tyr
20 25 30
Asn Arg Ser Arg Glu Lys Thr Asp Glu Phe Leu Glu Glu Ala Lys Gly
35 40 45
Lys Asn Ile Val Gly Thr Tyr Ser Ile Glu Glu Phe Val Asn Ala Leu
50 55 60
Glu Lys Pro Arg Lys Ile Leu Leu Met Val Lys Ala Gly Ala Pro Thr
65 70 75 80
Asp Ala Thr Ile Glu Gln Leu Lys Pro Tyr Leu Glu Lys Gly Asp Ile
85 90 95
Leu Ile Asp Gly Gly Asn Thr Tyr Phe Lys Asp Thr Gln Arg Arg Asn
100 105 110
Glu Glu Leu Ala Lys Leu Gly Ile His Phe Ile Gly Thr Gly Val Ser
115 120 125
Gly Gly Glu Glu Gly Ala Leu Lys Gly Pro Ser Ile Met Pro Gly Gly
130 135 140
Gln Lys Glu Ala His Glu Leu Val Arg Pro Ile Phe Glu Ala Ile Ala
145 150 155 160
Ala Lys Val Asp Gly Glu Pro Cys Thr Thr Tyr Ile Gly Pro Asp Gly
165 170 175
Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly Asp
180 185 190
Met Gln Leu Ile Ala Glu Ala Tyr Phe Leu Leu Lys His Val Leu Gly
195 200 205
Met Asp Ala Ala Glu Leu His Glu Val Phe Ala Asp Trp Asn Lys Gly
210 215 220
Glu Leu Asn Ser Tyr Leu Ile Glu Ile Thr Ala Asp Ile Phe Thr Lys
225 230 235 240
Ile Asp Asp Glu Thr Gly Lys Pro Leu Val Asp Val Ile Leu Asp Lys
245 250 255
Ala Gly Gln Lys Gly Thr Gly Lys Trp Thr Ser Gln Asn Ala Leu Asp
260 265 270
Leu Gly Val Pro Leu Pro Ile Ile Thr Glu Ser Val Phe Ala Arg Phe
275 280 285
Ile Ser Ala Met Lys Asp Glu Arg Val Lys Ala Ser Lys Leu Leu Ser
290 295 300
Gly Pro Ala Val Lys Pro Phe Glu Gly Asp Arg Asp His Phe Ile Glu
305 310 315 320
Ala Val Arg Arg Ala Leu Tyr Met Ser Lys Ile Cys Ser Tyr Ala Gln
325 330 335
Gly Phe Ala Gln Met Lys Ala Ala Ser Asp Glu Tyr Asn Trp Asn Leu
340 345 350
Arg Tyr Gly Asp Ile Ala Met Ile Phe Arg Gly Gly Cys Ile Ile Arg
355 360 365
Ala Gln Phe Leu Gln Lys Ile Lys Glu Ala Tyr Asp Arg Asp Pro Ala
370 375 380
Leu Pro Asn Leu Leu Leu Asp Pro Tyr Phe Lys Asn Ile Val Glu Ser
385 390 395 400
Tyr Gln Asp Ser Leu Arg Glu Ile Val Ala Thr Ala Ala Met Arg Gly
405 410 415
Ile Pro Val Pro Ala Phe Ala Ser Ala Leu Ala Tyr Tyr Asp Ser Tyr
420 425 430
Arg Asn Glu Val Leu Pro Ala Asn Leu Ile Gln Ala Gln Arg Asp Tyr
435 440 445
Phe Gly Ala His Thr Tyr Glu Arg Val Asp Lys Glu Gly Ile Phe His
450 455 460
Thr Glu Trp Leu Ala Lys
465 470
<210> 42
<211> 437
<212> PRT
<213> 大肠杆菌
<400> 42
Met Ser Lys Ile Val Lys Ile Ile Gly Arg Glu Ile Ile Asp Ser Arg
1 5 10 15
Gly Asn Pro Thr Val Glu Ala Glu Val His Leu Glu Gly Gly Phe Val
20 25 30
Gly Met Ala Ala Ala Pro Ser Gly Ala Ser Thr Gly Ser Arg Glu Ala
35 40 45
Leu Glu Leu Arg Asp Gly Asp Lys Ser Arg Phe Leu Gly Lys Gly Val
50 55 60
Thr Lys Ala Val Ala Ala Val Asn Gly Pro Ile Ala Gln Ala Leu Ile
65 70 75 80
Gly Lys Asp Ala Lys Asp Gln Ala Gly Ile Asp Lys Ile Met Ile Asp
85 90 95
Leu Asp Gly Thr Glu Lys Lys Ser Lys Phe Gly Ala Asn Ala Ile Leu
100 105 110
Ala Val Ser Leu Ala Asn Ala Lys Ala Ala Ala Ala Ala Lys Gly Met
115 120 125
Pro Leu Tyr Glu His Ile Ala Glu Leu Asn Gly Thr Pro Gly Lys Tyr
130 135 140
Ser Met Pro Val Pro Met Met Asn Ile Ile Asn Gly Gly Glu His Ala
145 150 155 160
Asp Asn Asn Val Asp Ile Gln Glu Phe Met Ile Gln Pro Val Gly Ala
165 170 175
Lys Thr Val Lys Glu Ala Ile Arg Met Gly Ser Glu Val Phe His His
180 185 190
Leu Ala Lys Val Leu Lys Ala Lys Gly Met Asn Thr Ala Val Gly Asp
195 200 205
Glu Gly Gly Tyr Ala Pro Asn Leu Gly Ser Asn Asp Glu Ala Leu Ala
210 215 220
Val Ile Ala Glu Ala Val Lys Ala Ala Gly Tyr Glu Leu Gly Lys Asp
225 230 235 240
Ile Thr Leu Ala Met Asp Cys Ala Ala Ser Glu Phe Tyr Lys Asp Gly
245 250 255
Lys Tyr Val Leu Ala Gly Glu Gly Asn Lys Ala Phe Thr Ser Glu Glu
260 265 270
Phe Thr His Phe Leu Glu Glu Leu Thr Lys Gln Tyr Pro Ile Val Ser
275 280 285
Ile Glu Asp Gly Leu Asp Glu Ser Asp Trp Asp Gly Phe Ala Tyr Gln
290 295 300
Thr Lys Val Leu Gly Asp Lys Ile Gln Leu Val Gly Asp Asp Leu Phe
305 310 315 320
Val Thr Asn Thr Lys Ile Leu Lys Glu Gly Ile Glu Lys Gly Ile Ala
325 330 335
Asn Ser Tyr Leu Ile Lys Phe Asn Gln Ile Gly Ser Leu Thr Glu Thr
340 345 350
Leu Ala Ala Ile Lys Met Ala Lys Asp Ala Gly Tyr Thr Ala Val Ile
355 360 365
Ser His Arg Ser Gly Glu Thr Glu Asp Ala Thr Ile Ala Asp Leu Ala
370 375 380
Val Gly Thr Ala Ala Gly Gln Ile Lys Thr Gly Ser Met Ser Arg Ser
385 390 395 400
Asp Arg Val Ala Lys Tyr Asn Gln Leu Ile Arg Ile Glu Glu Ala Leu
405 410 415
Gly Glu Lys Ala Arg Thr Thr Val Val Lys Arg Ser Lys Ala Arg His
420 425 430
Lys Thr Asp Phe Ile
435
<210> 43
<211> 587
<212> PRT
<213> 嗜热脂肪土芽孢杆菌
<400> 43
Met Lys Arg Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Ala Ser Glu
1 5 10 15
Ser Val Asp Lys Leu Val Gln Leu Met Glu Ala Gly Met Asn Val Ala
20 25 30
Arg Leu Asn Phe Ser His Gly Asp His Glu Glu His Gly Arg Arg Ile
35 40 45
Ala Asn Ile Arg Glu Ala Ala Lys Arg Thr Gly Arg Thr Val Ala Ile
50 55 60
Leu Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr His Asn Met Glu Asn
65 70 75 80
Gly Ala Ile Glu Leu Lys Glu Gly Ser Lys Leu Val Ile Ser Met Ser
85 90 95
Glu Val Leu Gly Thr Pro Glu Lys Ile Ser Val Thr Tyr Pro Ser Leu
100 105 110
Ile Asp Asp Val Ser Val Gly Ala Lys Ile Leu Leu Asp Asp Gly Leu
115 120 125
Ile Ser Leu Glu Val Asn Ala Val Asp Lys Gln Ala Gly Glu Ile Val
130 135 140
Thr Thr Val Leu Asn Gly Gly Val Leu Lys Asn Lys Lys Gly Val Asn
145 150 155 160
Val Pro Gly Val Lys Val Asn Leu Pro Gly Ile Thr Glu Lys Asp Arg
165 170 175
Ala Asp Ile Leu Phe Gly Ile Arg Gln Gly Ile Asp Phe Ile Ala Ala
180 185 190
Ser Phe Val Arg Arg Ala Ser Asp Val Leu Glu Ile Arg Glu Leu Leu
195 200 205
Glu Ala His Asp Ala Leu His Ile Gln Ile Ile Ala Lys Ile Glu Asn
210 215 220
Glu Glu Gly Val Ala Asn Ile Asp Glu Ile Leu Glu Ala Ala Asp Gly
225 230 235 240
Leu Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Ala Glu Glu
245 250 255
Val Pro Leu Ile Gln Lys Leu Leu Ile Lys Lys Cys Asn Met Leu Gly
260 265 270
Lys Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Gln Arg Asn
275 280 285
Pro Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala Ile Phe
290 295 300
Asp Gly Thr Asp Ala Val Met Leu Ser Gly Glu Thr Ala Ala Gly Gln
305 310 315 320
Tyr Pro Val Glu Ala Val Lys Thr Met His Gln Ile Ala Leu Arg Thr
325 330 335
Glu Gln Ala Leu Glu His Arg Asp Ile Leu Ser Gln Arg Thr Lys Glu
340 345 350
Ser Gln Thr Thr Ile Thr Asp Ala Ile Gly Gln Ser Val Ala His Thr
355 360 365
Ala Leu Asn Leu Asp Val Ala Ala Ile Val Thr Pro Thr Val Ser Gly
370 375 380
Lys Thr Pro Gln Met Val Ala Lys Tyr Arg Pro Lys Ala Pro Ile Ile
385 390 395 400
Ala Val Thr Ser Asn Glu Ala Val Ser Arg Arg Leu Ala Leu Val Trp
405 410 415
Gly Val Tyr Thr Lys Glu Ala Pro His Val Asn Thr Thr Asp Glu Met
420 425 430
Leu Asp Val Ala Val Asp Ala Ala Val Arg Ser Gly Leu Val Lys His
435 440 445
Gly Asp Leu Val Val Ile Thr Ala Gly Val Pro Val Gly Glu Thr Gly
450 455 460
Ser Thr Asn Leu Met Lys Val His Val Ile Ser Asp Leu Leu Ala Lys
465 470 475 480
Gly Gln Gly Ile Gly Arg Lys Ser Ala Phe Gly Lys Ala Val Val Ala
485 490 495
Lys Thr Ala Glu Glu Ala Arg Gln Lys Met Val Asp Gly Gly Ile Leu
500 505 510
Val Thr Val Ser Thr Asp Ala Asp Met Met Pro Ala Ile Glu Lys Ala
515 520 525
Ala Ala Ile Ile Thr Glu Glu Gly Gly Leu Thr Ser His Ala Ala Val
530 535 540
Val Gly Leu Ser Leu Gly Ile Pro Val Ile Val Gly Val Glu Asn Ala
545 550 555 560
Thr Thr Leu Phe Lys Asp Gly Gln Glu Ile Thr Val Asp Gly Gly Phe
565 570 575
Gly Ala Val Tyr Arg Gly His Ala Ser Val Leu
580 585
<210> 44
<211> 475
<212> PRT
<213> 运动发酵单胞菌
<400> 44
Met Thr Glu Gly Leu Phe Pro Arg Gly Arg Lys Val Arg Val Val Ser
1 5 10 15
Thr Leu Gly Pro Ala Ser Ser Thr Ala Glu Gln Ile Arg Asp Arg Phe
20 25 30
Leu Ala Gly Ala Asp Val Phe Arg Ile Asn Met Ser His Gly Thr His
35 40 45
Asp Glu Lys Lys Val Ile Val Asp Asn Ile Arg Ala Leu Glu Lys Glu
50 55 60
Phe Asn Arg Pro Thr Thr Ile Leu Phe Asp Leu Gln Gly Pro Lys Leu
65 70 75 80
Arg Val Gly Asp Phe Lys Glu Gly Lys Val Gln Leu Lys Glu Gly Gln
85 90 95
Thr Phe Thr Phe Asp Gln Asp Pro Thr Leu Gly Asp Glu Thr Arg Val
100 105 110
Asn Leu Pro His Pro Glu Ile Phe Lys Ala Leu Asp Lys Gly His Arg
115 120 125
Leu Leu Leu Asp Asp Gly Lys Ile Val Val Arg Cys Val Glu Ser Ser
130 135 140
Pro Thr Lys Ile Val Thr Arg Val Glu Val Pro Gly Pro Leu Ser Asp
145 150 155 160
His Lys Gly Phe Asn Val Pro Asp Val Val Ile Pro Leu Ala Ala Leu
165 170 175
Thr Pro Lys Asp Arg Lys Asp Leu Asp Phe Ala Leu Lys Glu Lys Ala
180 185 190
Asp Trp Val Ala Leu Ser Phe Val Gln Arg Val Glu Asp Val Ile Glu
195 200 205
Ala Lys Glu Leu Ile Lys Gly Arg Ala Pro Leu Leu Val Lys Leu Glu
210 215 220
Lys Pro Ala Ala Ile Glu Asn Leu Glu Ser Ile Leu Ala Ala Thr Asp
225 230 235 240
Ala Val Met Val Ala Arg Gly Asp Leu Gly Val Glu Cys Leu Pro Glu
245 250 255
Ser Val Pro Pro Thr Gln Lys Arg Ile Val Glu Arg Ser Arg Gln Leu
260 265 270
Gly Lys Pro Val Val Val Ala Thr Ala Met Leu Glu Ser Met Ile Lys
275 280 285
Ala Pro Ala Pro Thr Arg Ala Glu Val Ser Asp Val Ala Asn Ala Ile
290 295 300
Tyr Glu Gly Ala Asp Gly Ile Met Leu Ser Ala Glu Ser Ala Ala Gly
305 310 315 320
Asp Trp Pro His Glu Ala Val Asn Met Met His Arg Ile Ala Ser Tyr
325 330 335
Val Glu Asn Ala Pro Gly Tyr Ile Glu Arg Val Arg Phe Thr Pro Thr
340 345 350
Pro Ala Glu Pro Thr Thr Val Asp Ala Leu Ala Glu Asn Ala Ser Lys
355 360 365
Thr Ala Glu Thr Val Gly Ala Lys Ala Ile Ile Val Phe Thr Glu Thr
370 375 380
Gly Lys Thr Ala Gln Arg Val Ser Arg Ala Arg Pro Val Ala Pro Ile
385 390 395 400
Leu Ser Leu Thr Pro Asp Ala Glu Val Ala Arg Arg Leu Gly Leu Val
405 410 415
Trp Gly Ala Gln Pro Val Gln Val Ser Thr Val Lys Thr Leu Asp Glu
420 425 430
Ala Lys Lys Leu Ala Ala Glu Thr Ala Lys Lys Tyr Gly Phe Ala Lys
435 440 445
Ala Gly Asp Lys Leu Val Val Val Ala Gly Glu Pro Phe Gly Lys Ala
450 455 460
Gly Thr Thr Asn Ile Val Asp Val Ile Glu Ala
465 470 475
<210> 45
<211> 531
<212> PRT
<213> 穴兔
<400> 45
Met Ser Lys Ser His Ser Glu Ala Gly Ser Ala Phe Ile Gln Thr Gln
1 5 10 15
Gln Leu His Ala Ala Met Ala Asp Thr Phe Leu Glu His Met Cys Arg
20 25 30
Leu Asp Ile Asp Ser Ala Pro Ile Thr Ala Arg Asn Thr Gly Ile Ile
35 40 45
Cys Thr Ile Gly Pro Ala Ser Arg Ser Val Glu Thr Leu Lys Glu Met
50 55 60
Ile Lys Ser Gly Met Asn Val Ala Arg Met Asn Phe Ser His Gly Thr
65 70 75 80
His Glu Tyr His Ala Glu Thr Ile Lys Asn Val Arg Thr Ala Thr Glu
85 90 95
Ser Phe Ala Ser Asp Pro Ile Leu Tyr Arg Pro Val Ala Val Ala Leu
100 105 110
Asp Thr Lys Gly Pro Glu Ile Arg Thr Gly Leu Ile Lys Gly Ser Gly
115 120 125
Thr Ala Glu Val Glu Leu Lys Lys Gly Ala Thr Leu Lys Ile Thr Leu
130 135 140
Asp Asn Ala Tyr Met Glu Lys Cys Asp Glu Asn Ile Leu Trp Leu Asp
145 150 155 160
Tyr Lys Asn Ile Cys Lys Val Val Asp Val Gly Ser Lys Val Tyr Val
165 170 175
Asp Asp Gly Leu Ile Ser Leu Gln Val Lys Gln Lys Gly Pro Asp Phe
180 185 190
Leu Val Thr Glu Val Glu Asn Gly Gly Phe Leu Gly Ser Lys Lys Gly
195 200 205
Val Asn Leu Pro Gly Ala Ala Val Asp Leu Pro Ala Val Ser Glu Lys
210 215 220
Asp Ile Gln Asp Leu Lys Phe Gly Val Glu Gln Asp Val Asp Met Val
225 230 235 240
Phe Ala Ser Phe Ile Arg Lys Ala Ala Asp Val His Glu Val Arg Lys
245 250 255
Ile Leu Gly Glu Lys Gly Lys Asn Ile Lys Ile Ile Ser Lys Ile Glu
260 265 270
Asn His Glu Gly Val Arg Arg Phe Asp Glu Ile Leu Glu Ala Ser Asp
275 280 285
Gly Ile Met Val Ala Arg Gly Asp Leu Gly Ile Glu Ile Pro Ala Glu
290 295 300
Lys Val Phe Leu Ala Gln Lys Met Ile Ile Gly Arg Cys Asn Arg Ala
305 310 315 320
Gly Lys Pro Val Ile Cys Ala Thr Gln Met Leu Glu Ser Met Ile Lys
325 330 335
Lys Pro Arg Pro Thr Arg Ala Glu Gly Ser Asp Val Ala Asn Ala Val
340 345 350
Leu Asp Gly Ala Asp Cys Ile Met Leu Ser Gly Glu Thr Ala Lys Gly
355 360 365
Asp Tyr Pro Leu Glu Ala Val Arg Met Gln His Leu Ile Ala Arg Glu
370 375 380
Ala Glu Ala Ala Met Phe His Arg Lys Leu Phe Glu Glu Leu Ala Arg
385 390 395 400
Ala Ser Ser His Ser Thr Asp Leu Met Glu Ala Met Ala Met Gly Ser
405 410 415
Val Glu Ala Ser Tyr Lys Cys Leu Ala Ala Ala Leu Ile Val Leu Thr
420 425 430
Glu Ser Gly Arg Ser Ala His Gln Val Ala Arg Tyr Arg Pro Arg Ala
435 440 445
Pro Ile Ile Ala Val Thr Arg Asn His Gln Thr Ala Arg Gln Ala His
450 455 460
Leu Tyr Arg Gly Ile Phe Pro Val Val Cys Lys Asp Pro Val Gln Glu
465 470 475 480
Ala Trp Ala Glu Asp Val Asp Leu Arg Val Asn Leu Ala Met Asn Val
485 490 495
Gly Lys Ala Arg Gly Phe Phe Lys Lys Gly Asp Val Val Ile Val Leu
500 505 510
Thr Gly Trp Arg Pro Gly Ser Gly Phe Thr Asn Thr Met Arg Val Val
515 520 525
Pro Val Pro
530
<210> 46
<211> 592
<212> PRT
<213> 绿色气球菌
<400> 46
Met Ser Asp Asn Lys Ile Asn Ile Gly Leu Ala Val Met Lys Ile Leu
1 5 10 15
Glu Ser Trp Gly Ala Asp Thr Ile Tyr Gly Ile Pro Ser Gly Thr Leu
20 25 30
Ser Ser Leu Met Asp Ala Met Gly Glu Glu Glu Asn Asn Val Lys Phe
35 40 45
Leu Gln Val Lys His Glu Glu Val Gly Ala Met Ala Ala Val Met Gln
50 55 60
Ser Lys Phe Gly Gly Asn Leu Gly Val Thr Val Gly Ser Gly Gly Pro
65 70 75 80
Gly Ala Ser His Leu Ile Asn Gly Leu Tyr Asp Ala Ala Met Asp Asn
85 90 95
Ile Pro Val Val Ala Ile Leu Gly Ser Arg Pro Gln Arg Glu Leu Asn
100 105 110
Met Asp Ala Phe Gln Glu Leu Asn Gln Asn Pro Met Tyr Asp His Ile
115 120 125
Ala Val Tyr Asn Arg Arg Val Ala Tyr Ala Glu Gln Leu Pro Lys Leu
130 135 140
Val Asp Glu Ala Ala Arg Met Ala Ile Ala Lys Arg Gly Val Ala Val
145 150 155 160
Leu Glu Val Pro Gly Asp Phe Ala Lys Val Glu Ile Asp Asn Asp Gln
165 170 175
Trp Tyr Ser Ser Ala Asn Ser Leu Arg Lys Tyr Glu Pro Ile Ala Pro
180 185 190
Ala Ala Gln Asp Ile Asp Ala Ala Val Glu Leu Leu Asn Asn Ser Lys
195 200 205
Arg Pro Val Ile Tyr Ala Gly Ile Gly Thr Met Gly His Gly Pro Ala
210 215 220
Val Gln Glu Leu Ala Arg Lys Ile Lys Ala Pro Val Ile Thr Thr Gly
225 230 235 240
Lys Asn Phe Glu Thr Phe Glu Trp Asp Phe Glu Ala Leu Thr Gly Ser
245 250 255
Thr Tyr Arg Val Gly Trp Lys Pro Ala Asn Glu Thr Ile Leu Glu Ala
260 265 270
Asp Thr Val Leu Phe Ala Gly Ser Asn Phe Pro Phe Ser Glu Val Glu
275 280 285
Gly Thr Phe Arg Asn Val Asp Asn Phe Ile Gln Ile Asp Ile Asp Pro
290 295 300
Ala Met Leu Gly Lys Arg His His Ala Asp Val Ala Ile Leu Gly Asp
305 310 315 320
Ala Gly Leu Ala Ile Asp Glu Ile Leu Asn Lys Val Asp Ala Val Glu
325 330 335
Glu Ser Ala Trp Trp Thr Ala Asn Leu Lys Asn Ile Ala Asn Trp Arg
340 345 350
Glu Tyr Ile Asn Met Leu Glu Thr Lys Glu Glu Gly Asp Leu Gln Phe
355 360 365
Tyr Gln Val Tyr Asn Ala Ile Asn Asn His Ala Asp Glu Asp Ala Ile
370 375 380
Tyr Ser Ile Asp Val Gly Asn Ser Thr Gln Thr Ser Ile Arg His Leu
385 390 395 400
His Met Thr Pro Lys Asn Met Trp Arg Thr Ser Pro Leu Phe Ala Thr
405 410 415
Met Gly Ile Ala Ile Pro Gly Gly Leu Gly Ala Lys Asn Thr Tyr Pro
420 425 430
Asp Arg Gln Val Trp Asn Ile Ile Gly Asp Gly Ala Phe Ser Met Thr
435 440 445
Tyr Pro Asp Val Val Thr Asn Val Arg Tyr Asn Met Pro Val Ile Asn
450 455 460
Val Val Phe Ser Asn Thr Glu Tyr Ala Phe Ile Lys Asn Lys Tyr Glu
465 470 475 480
Asp Thr Asn Lys Asn Leu Phe Gly Val Asp Phe Thr Asp Val Asp Tyr
485 490 495
Ala Lys Ile Ala Glu Ala Gln Gly Ala Lys Gly Phe Thr Val Ser Arg
500 505 510
Ile Glu Asp Met Asp Arg Val Met Ala Glu Ala Val Ala Ala Asn Lys
515 520 525
Ala Gly His Thr Val Val Ile Asp Cys Lys Ile Thr Gln Asp Arg Pro
530 535 540
Ile Pro Val Glu Thr Leu Lys Leu Asp Ser Lys Leu Tyr Ser Glu Asp
545 550 555 560
Glu Ile Lys Ala Tyr Lys Glu Arg Tyr Glu Ala Ala Asn Leu Val Pro
565 570 575
Phe Arg Glu Tyr Leu Glu Ala Glu Gly Leu Glu Ser Lys Tyr Ile Lys
580 585 590
<210> 47
<211> 393
<212> PRT
<213> 钩虫贪铜菌H16
<400> 47
Met Thr Asp Val Val Ile Val Ser Ala Ala Arg Thr Ala Val Gly Lys
1 5 10 15
Phe Gly Gly Ser Leu Ala Lys Ile Pro Ala Pro Glu Leu Gly Ala Val
20 25 30
Val Ile Lys Ala Ala Leu Glu Arg Ala Gly Val Lys Pro Glu Gln Val
35 40 45
Ser Glu Val Ile Met Gly Gln Val Leu Thr Ala Gly Ser Gly Gln Asn
50 55 60
Pro Ala Arg Gln Ala Ala Ile Lys Ala Gly Leu Pro Ala Met Val Pro
65 70 75 80
Ala Met Thr Ile Asn Lys Val Cys Gly Ser Gly Leu Lys Ala Val Met
85 90 95
Leu Ala Ala Asn Ala Ile Met Ala Gly Asp Ala Glu Ile Val Val Ala
100 105 110
Gly Gly Gln Glu Asn Met Ser Ala Ala Pro His Val Leu Pro Gly Ser
115 120 125
Arg Asp Gly Phe Arg Met Gly Asp Ala Lys Leu Val Asp Thr Met Ile
130 135 140
Val Asp Gly Leu Trp Asp Val Tyr Asn Gln Tyr His Met Gly Ile Thr
145 150 155 160
Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu Ala Gln Asp
165 170 175
Glu Phe Ala Val Gly Ser Gln Asn Lys Ala Glu Ala Ala Gln Lys Ala
180 185 190
Gly Lys Phe Asp Glu Glu Ile Val Pro Val Leu Ile Pro Gln Arg Lys
195 200 205
Gly Asp Pro Val Ala Phe Lys Thr Asp Glu Phe Val Arg Gln Gly Ala
210 215 220
Thr Leu Asp Ser Met Ser Gly Leu Lys Pro Ala Phe Asp Lys Ala Gly
225 230 235 240
Thr Val Thr Ala Ala Asn Ala Ser Gly Leu Asn Asp Gly Ala Ala Ala
245 250 255
Val Val Val Met Ser Ala Ala Lys Ala Lys Glu Leu Gly Leu Thr Pro
260 265 270
Leu Ala Thr Ile Lys Ser Tyr Ala Asn Ala Gly Val Asp Pro Lys Val
275 280 285
Met Gly Met Gly Pro Val Pro Ala Ser Lys Arg Ala Leu Ser Arg Ala
290 295 300
Glu Trp Thr Pro Gln Asp Leu Asp Leu Met Glu Ile Asn Glu Ala Phe
305 310 315 320
Ala Ala Gln Ala Leu Ala Val His Gln Gln Met Gly Trp Asp Thr Ser
325 330 335
Lys Val Asn Val Asn Gly Gly Ala Ile Ala Ile Gly His Pro Ile Gly
340 345 350
Ala Ser Gly Cys Arg Ile Leu Val Thr Leu Leu His Glu Met Lys Arg
355 360 365
Arg Asp Ala Lys Lys Gly Leu Ala Ser Leu Cys Ile Gly Gly Gly Met
370 375 380
Gly Val Ala Leu Ala Val Glu Arg Lys
385 390
<210> 48
<211> 383
<212> PRT
<213> 粪肠球菌
<400> 48
Met Thr Ile Gly Ile Asp Lys Ile Ser Phe Phe Val Pro Pro Tyr Tyr
1 5 10 15
Ile Asp Met Thr Ala Leu Ala Glu Ala Arg Asn Val Asp Pro Gly Lys
20 25 30
Phe His Ile Gly Ile Gly Gln Asp Gln Met Ala Val Asn Pro Ile Ser
35 40 45
Gln Asp Ile Val Thr Phe Ala Ala Asn Ala Ala Glu Ala Ile Leu Thr
50 55 60
Lys Glu Asp Lys Glu Ala Ile Asp Met Val Ile Val Gly Thr Glu Ser
65 70 75 80
Ser Ile Asp Glu Ser Lys Ala Ala Ala Val Val Leu His Arg Leu Met
85 90 95
Gly Ile Gln Pro Phe Ala Arg Ser Phe Glu Ile Lys Glu Ala Cys Tyr
100 105 110
Gly Ala Thr Ala Gly Leu Gln Leu Ala Lys Asn His Val Ala Leu His
115 120 125
Pro Asp Lys Lys Val Leu Val Val Ala Ala Asp Ile Ala Lys Tyr Gly
130 135 140
Leu Asn Ser Gly Gly Glu Pro Thr Gln Gly Ala Gly Ala Val Ala Met
145 150 155 160
Leu Val Ala Ser Glu Pro Arg Ile Leu Ala Leu Lys Glu Asp Asn Val
165 170 175
Met Leu Thr Gln Asp Ile Tyr Asp Phe Trp Arg Pro Thr Gly His Pro
180 185 190
Tyr Pro Met Val Asp Gly Pro Leu Ser Asn Glu Thr Tyr Ile Gln Ser
195 200 205
Phe Ala Gln Val Trp Asp Glu His Lys Lys Arg Thr Gly Leu Asp Phe
210 215 220
Ala Asp Tyr Asp Ala Leu Ala Phe His Ile Pro Tyr Thr Lys Met Gly
225 230 235 240
Lys Lys Ala Leu Leu Ala Lys Ile Ser Asp Gln Thr Glu Ala Glu Gln
245 250 255
Glu Arg Ile Leu Ala Arg Tyr Glu Glu Ser Ile Ile Tyr Ser Arg Arg
260 265 270
Val Gly Asn Leu Tyr Thr Ser Ser Leu Tyr Leu Gly Leu Ile Ser Leu
275 280 285
Leu Glu Asn Ala Thr Thr Leu Thr Ala Gly Asn Gln Ile Gly Leu Phe
290 295 300
Ser Tyr Gly Ser Gly Ala Val Ala Glu Phe Phe Thr Gly Glu Leu Val
305 310 315 320
Ala Gly Tyr Gln Asn His Leu Gln Lys Glu Thr His Leu Ala Leu Leu
325 330 335
Asp Asn Arg Thr Glu Leu Ser Ile Ala Glu Tyr Glu Ala Met Phe Ala
340 345 350
Glu Thr Leu Asp Thr Asp Ile Asp Gln Thr Leu Glu Asp Glu Leu Lys
355 360 365
Tyr Ser Ile Ser Ala Ile Asn Asn Thr Val Arg Ser Tyr Arg Asn
370 375 380
<210> 49
<211> 803
<212> PRT
<213> 粪肠球菌
<400> 49
Met Lys Thr Val Val Ile Ile Asp Ala Leu Arg Thr Pro Ile Gly Lys
1 5 10 15
Tyr Lys Gly Ser Leu Ser Gln Val Ser Ala Val Asp Leu Gly Thr His
20 25 30
Val Thr Thr Gln Leu Leu Lys Arg His Ser Thr Ile Ser Glu Glu Ile
35 40 45
Asp Gln Val Ile Phe Gly Asn Val Leu Gln Ala Gly Asn Gly Gln Asn
50 55 60
Pro Ala Arg Gln Ile Ala Ile Asn Ser Gly Leu Ser His Glu Ile Pro
65 70 75 80
Ala Met Thr Val Asn Glu Val Cys Gly Ser Gly Met Lys Ala Val Ile
85 90 95
Leu Ala Lys Gln Leu Ile Gln Leu Gly Glu Ala Glu Val Leu Ile Ala
100 105 110
Gly Gly Ile Glu Asn Met Ser Gln Ala Pro Lys Leu Gln Arg Phe Asn
115 120 125
Tyr Glu Thr Glu Ser Tyr Asp Ala Pro Phe Ser Ser Met Met Tyr Asp
130 135 140
Gly Leu Thr Asp Ala Phe Ser Gly Gln Ala Met Gly Leu Thr Ala Glu
145 150 155 160
Asn Val Ala Glu Lys Tyr His Val Thr Arg Glu Glu Gln Asp Gln Phe
165 170 175
Ser Val His Ser Gln Leu Lys Ala Ala Gln Ala Gln Ala Glu Gly Ile
180 185 190
Phe Ala Asp Glu Ile Ala Pro Leu Glu Val Ser Gly Thr Leu Val Glu
195 200 205
Lys Asp Glu Gly Ile Arg Pro Asn Ser Ser Val Glu Lys Leu Gly Thr
210 215 220
Leu Lys Thr Val Phe Lys Glu Asp Gly Thr Val Thr Ala Gly Asn Ala
225 230 235 240
Ser Thr Ile Asn Asp Gly Ala Ser Ala Leu Ile Ile Ala Ser Gln Glu
245 250 255
Tyr Ala Glu Ala His Gly Leu Pro Tyr Leu Ala Ile Ile Arg Asp Ser
260 265 270
Val Glu Val Gly Ile Asp Pro Ala Tyr Met Gly Ile Ser Pro Ile Lys
275 280 285
Ala Ile Gln Lys Leu Leu Ala Arg Asn Gln Leu Thr Thr Glu Glu Ile
290 295 300
Asp Leu Tyr Glu Ile Asn Glu Ala Phe Ala Ala Thr Ser Ile Val Val
305 310 315 320
Gln Arg Glu Leu Ala Leu Pro Glu Glu Lys Val Asn Ile Tyr Gly Gly
325 330 335
Gly Ile Ser Leu Gly His Ala Ile Gly Ala Thr Gly Ala Arg Leu Leu
340 345 350
Thr Ser Leu Ser Tyr Gln Leu Asn Gln Lys Glu Lys Lys Tyr Gly Val
355 360 365
Ala Ser Leu Cys Ile Gly Gly Gly Leu Gly Leu Ala Met Leu Leu Glu
370 375 380
Arg Pro Gln Gln Lys Lys Asn Ser Arg Phe Tyr Gln Met Ser Pro Glu
385 390 395 400
Glu Arg Leu Ala Ser Leu Leu Asn Glu Gly Gln Ile Ser Ala Asp Thr
405 410 415
Lys Lys Glu Phe Glu Asn Thr Ala Leu Ser Ser Gln Ile Ala Asn His
420 425 430
Met Ile Glu Asn Gln Ile Ser Glu Thr Glu Val Pro Met Gly Val Gly
435 440 445
Leu His Leu Thr Val Asp Glu Thr Asp Tyr Leu Val Pro Met Ala Thr
450 455 460
Glu Glu Pro Ser Val Ile Ala Ala Leu Ser Asn Gly Ala Lys Ile Ala
465 470 475 480
Gln Gly Phe Lys Thr Val Asn Gln Gln Arg Leu Met Arg Gly Gln Ile
485 490 495
Val Phe Tyr Asp Val Ala Asp Ala Glu Ser Leu Ile Asp Glu Leu Gln
500 505 510
Val Arg Glu Thr Glu Ile Phe Gln Gln Ala Glu Leu Ser Tyr Pro Ser
515 520 525
Ile Val Lys Arg Gly Gly Gly Leu Arg Asp Leu Gln Tyr Arg Ala Phe
530 535 540
Asp Glu Ser Phe Val Ser Val Asp Phe Leu Val Asp Val Lys Asp Ala
545 550 555 560
Met Gly Ala Asn Ile Val Asn Ala Met Leu Glu Gly Val Ala Glu Leu
565 570 575
Phe Arg Glu Trp Phe Ala Glu Gln Lys Ile Leu Phe Ser Ile Leu Ser
580 585 590
Asn Tyr Ala Thr Glu Ser Val Val Thr Met Lys Thr Ala Ile Pro Val
595 600 605
Ser Arg Leu Ser Lys Gly Ser Asn Gly Arg Glu Ile Ala Glu Lys Ile
610 615 620
Val Leu Ala Ser Arg Tyr Ala Ser Leu Asp Pro Tyr Arg Ala Val Thr
625 630 635 640
His Asn Lys Gly Ile Met Asn Gly Ile Glu Ala Val Val Leu Ala Thr
645 650 655
Gly Asn Asp Thr Arg Ala Val Ser Ala Ser Cys His Ala Phe Ala Val
660 665 670
Lys Glu Gly Arg Tyr Gln Gly Leu Thr Ser Trp Thr Leu Asp Gly Glu
675 680 685
Gln Leu Ile Gly Glu Ile Ser Val Pro Leu Ala Leu Ala Thr Val Gly
690 695 700
Gly Ala Thr Lys Val Leu Pro Lys Ser Gln Ala Ala Ala Asp Leu Leu
705 710 715 720
Ala Val Thr Asp Ala Lys Glu Leu Ser Arg Val Val Ala Ala Val Gly
725 730 735
Leu Ala Gln Asn Leu Ala Ala Leu Arg Ala Leu Val Ser Glu Gly Ile
740 745 750
Gln Lys Gly His Met Ala Leu Gln Ala Arg Ser Leu Ala Met Thr Val
755 760 765
Gly Ala Thr Gly Lys Glu Val Glu Ala Val Ala Gln Gln Leu Lys Arg
770 775 780
Gln Lys Thr Met Asn Gln Asp Arg Ala Leu Ala Ile Leu Asn Asp Leu
785 790 795 800
Arg Lys Gln
<210> 50
<211> 301
<212> PRT
<213> 马氏甲烷八叠球菌Go1
<400> 50
Met Val Ser Cys Ser Ala Pro Gly Lys Ile Tyr Leu Phe Gly Glu His
1 5 10 15
Ala Val Val Tyr Gly Glu Thr Ala Ile Ala Cys Ala Val Glu Leu Arg
20 25 30
Thr Arg Val Arg Ala Glu Leu Asn Asp Ser Ile Thr Ile Gln Ser Gln
35 40 45
Ile Gly Arg Thr Gly Leu Asp Phe Glu Lys His Pro Tyr Val Ser Ala
50 55 60
Val Ile Glu Lys Met Arg Lys Ser Ile Pro Ile Asn Gly Val Phe Leu
65 70 75 80
Thr Val Asp Ser Asp Ile Pro Val Gly Ser Gly Leu Gly Ser Ser Ala
85 90 95
Ala Val Thr Ile Ala Ser Ile Gly Ala Leu Asn Glu Leu Phe Gly Phe
100 105 110
Gly Leu Ser Leu Gln Glu Ile Ala Lys Leu Gly His Glu Ile Glu Ile
115 120 125
Lys Val Gln Gly Ala Ala Ser Pro Thr Asp Thr Tyr Val Ser Thr Phe
130 135 140
Gly Gly Val Val Thr Ile Pro Glu Arg Arg Lys Leu Lys Thr Pro Asp
145 150 155 160
Cys Gly Ile Val Ile Gly Asp Thr Gly Val Phe Ser Ser Thr Lys Glu
165 170 175
Leu Val Ala Asn Val Arg Gln Leu Arg Glu Ser Tyr Pro Asp Leu Ile
180 185 190
Glu Pro Leu Met Thr Ser Ile Gly Lys Ile Ser Arg Ile Gly Glu Gln
195 200 205
Leu Val Leu Ser Gly Asp Tyr Ala Ser Ile Gly Arg Leu Met Asn Val
210 215 220
Asn Gln Gly Leu Leu Asp Ala Leu Gly Val Asn Ile Leu Glu Leu Ser
225 230 235 240
Gln Leu Ile Tyr Ser Ala Arg Ala Ala Gly Ala Phe Gly Ala Lys Ile
245 250 255
Thr Gly Ala Gly Gly Gly Gly Cys Met Val Ala Leu Thr Ala Pro Glu
260 265 270
Lys Cys Asn Gln Val Ala Glu Ala Val Ala Gly Ala Gly Gly Lys Val
275 280 285
Thr Ile Thr Lys Pro Thr Glu Gln Gly Leu Lys Val Asp
290 295 300
<210> 51
<211> 335
<212> PRT
<213> 肺炎链球菌
<400> 51
Met Ile Ala Val Lys Thr Cys Gly Lys Leu Tyr Trp Ala Gly Glu Tyr
1 5 10 15
Ala Ile Leu Glu Pro Gly Gln Leu Ala Leu Ile Lys Asp Ile Pro Ile
20 25 30
Tyr Met Arg Ala Glu Ile Ala Phe Ser Asp Ser Tyr Arg Ile Tyr Ser
35 40 45
Asp Met Phe Asp Phe Ala Val Asp Leu Arg Pro Asn Pro Asp Tyr Ser
50 55 60
Leu Ile Gln Glu Thr Ile Ala Leu Met Gly Asp Phe Leu Ala Val Arg
65 70 75 80
Gly Gln Asn Leu Arg Pro Phe Ser Leu Ala Ile Tyr Gly Lys Met Glu
85 90 95
Arg Glu Gly Lys Lys Phe Gly Leu Gly Ser Ser Gly Ser Val Val Val
100 105 110
Leu Val Val Lys Ala Leu Leu Ala Leu Tyr Asn Leu Ser Val Asp Gln
115 120 125
Asn Leu Leu Phe Lys Leu Thr Ser Ala Val Leu Leu Lys Arg Gly Asp
130 135 140
Asn Gly Ser Met Gly Asp Leu Ala Cys Ile Ala Ala Glu Asp Leu Val
145 150 155 160
Leu Tyr Gln Ser Phe Asp Arg Gln Lys Val Ala Ala Trp Leu Glu Glu
165 170 175
Glu Asn Leu Ala Thr Val Leu Glu Arg Asp Trp Gly Phe Ser Ile Ser
180 185 190
Gln Val Lys Pro Thr Leu Glu Cys Asp Phe Leu Val Gly Trp Thr Lys
195 200 205
Glu Val Ala Val Ser Ser His Met Val Gln Gln Ile Lys Gln Asn Ile
210 215 220
Asn Gln Asn Phe Leu Thr Ser Ser Lys Glu Thr Val Val Ser Leu Val
225 230 235 240
Glu Ala Leu Glu Gln Gly Lys Ser Glu Lys Ile Ile Glu Gln Val Glu
245 250 255
Val Ala Ser Lys Leu Leu Glu Gly Leu Ser Thr Asp Ile Tyr Thr Pro
260 265 270
Leu Leu Arg Gln Leu Lys Glu Ala Ser Gln Asp Leu Gln Ala Val Ala
275 280 285
Lys Ser Ser Gly Ala Gly Gly Gly Asp Cys Gly Ile Ala Leu Ser Phe
290 295 300
Asp Ala Gln Ser Thr Lys Thr Leu Lys Asn Arg Trp Ala Asp Leu Gly
305 310 315 320
Ile Glu Leu Leu Tyr Gln Glu Arg Ile Gly His Asp Asp Lys Ser
325 330 335
<210> 52
<211> 344
<212> PRT
<213> 肺炎链球菌
<400> 52
Met Tyr His Ser Leu Gly Asn Gln Phe Asp Thr Arg Thr Arg Thr Ser
1 5 10 15
Arg Lys Ile Arg Arg Glu Arg Ser Cys Ser Asp Met Asp Arg Glu Pro
20 25 30
Val Thr Val Arg Ser Tyr Ala Asn Ile Ala Ile Ile Lys Tyr Trp Gly
35 40 45
Lys Lys Lys Glu Lys Glu Met Val Pro Ala Thr Ser Ser Ile Ser Leu
50 55 60
Thr Leu Glu Asn Met Tyr Thr Glu Thr Thr Leu Ser Pro Leu Pro Ala
65 70 75 80
Asn Val Thr Ala Asp Glu Phe Tyr Ile Asn Gly Gln Leu Gln Asn Glu
85 90 95
Val Glu His Ala Lys Met Ser Lys Ile Ile Asp Arg Tyr Arg Pro Ala
100 105 110
Gly Glu Gly Phe Val Arg Ile Asp Thr Gln Asn Asn Met Pro Thr Ala
115 120 125
Ala Gly Leu Ser Ser Ser Ser Ser Gly Leu Ser Ala Leu Val Lys Ala
130 135 140
Cys Asn Ala Tyr Phe Lys Leu Gly Leu Asp Arg Ser Gln Leu Ala Gln
145 150 155 160
Glu Ala Lys Phe Ala Ser Gly Ser Ser Ser Arg Ser Phe Tyr Gly Pro
165 170 175
Leu Gly Ala Trp Asp Lys Asp Ser Gly Glu Ile Tyr Pro Val Glu Thr
180 185 190
Asp Leu Lys Leu Ala Met Ile Met Leu Val Leu Glu Asp Lys Lys Lys
195 200 205
Pro Ile Ser Ser Arg Asp Gly Met Lys Leu Cys Val Glu Thr Ser Thr
210 215 220
Thr Phe Asp Asp Trp Val Arg Gln Ser Glu Lys Asp Tyr Gln Asp Met
225 230 235 240
Leu Ile Tyr Leu Lys Glu Asn Asp Phe Ala Lys Ile Gly Glu Leu Thr
245 250 255
Glu Lys Asn Ala Leu Ala Met His Ala Thr Thr Lys Thr Ala Ser Pro
260 265 270
Ala Phe Ser Tyr Leu Thr Asp Ala Ser Tyr Glu Ala Met Asp Phe Val
275 280 285
Arg Gln Leu Arg Glu Lys Gly Glu Ala Cys Tyr Phe Thr Met Asp Ala
290 295 300
Gly Pro Asn Val Lys Val Phe Cys Gln Glu Lys Asp Leu Glu His Leu
305 310 315 320
Ser Glu Ile Phe Gly Gln Arg Tyr Arg Leu Ile Val Ser Lys Thr Lys
325 330 335
Asp Leu Ser Gln Asp Asp Cys Cys
340
<210> 53
<211> 297
<212> PRT
<213> 嗜热脂肪土芽孢杆菌
<400> 53
Met Ala Gln Leu Ser Val Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala
1 5 10 15
Val Glu Thr Ala Leu Ser Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala
20 25 30
Lys Leu Lys Lys Ala Met Ala Tyr Ser Leu Glu Ala Gly Gly Lys Arg
35 40 45
Ile Arg Pro Leu Leu Leu Leu Ser Thr Val Arg Ala Leu Gly Lys Asp
50 55 60
Pro Ala Val Gly Leu Pro Val Ala Cys Ala Ile Glu Met Ile His Thr
65 70 75 80
Tyr Ser Leu Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Leu
85 90 95
Arg Arg Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Met Ala
100 105 110
Ile Leu Ala Gly Asp Gly Leu Leu Thr Tyr Ala Phe Gln Leu Ile Thr
115 120 125
Glu Ile Asp Asp Glu Arg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile
130 135 140
Glu Arg Leu Ala Lys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln
145 150 155 160
Ala Ala Asp Met Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu
165 170 175
Glu Tyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val
180 185 190
His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu
195 200 205
Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp
210 215 220
Asp Ile Leu Asp Ile Glu Gly Ala Glu Glu Lys Ile Gly Lys Pro Val
225 230 235 240
Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala Leu Leu Ser
245 250 255
Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile Glu Ala Ala Gln
260 265 270
Arg His Leu Arg Asn Ala Asp Val Asp Gly Ala Ala Leu Ala Tyr Ile
275 280 285
Cys Glu Leu Val Ala Ala Arg Asp His
290 295
<210> 54
<211> 789
<212> DNA
<213> 大肠杆菌
<220>
<221> CDS
<222> (1)..(789)
<400> 54
atg caa gtc gac ctg ctg ggt tca gcg caa tct gcg cac gcg tta cac 48
Met Gln Val Asp Leu Leu Gly Ser Ala Gln Ser Ala His Ala Leu His
1 5 10 15
ctt ttt cac caa cat tcc cct ctt gtg cac tgc atg acc aat gat gtg 96
Leu Phe His Gln His Ser Pro Leu Val His Cys Met Thr Asn Asp Val
20 25 30
gtg caa acc ttt acc gcc aat acc ttg ctg gcg ctc ggt gca tcg cca 144
Val Gln Thr Phe Thr Ala Asn Thr Leu Leu Ala Leu Gly Ala Ser Pro
35 40 45
gcg atg gtt atc gaa acc gaa gag gcc agt cag ttt gcg gct atc gcc 192
Ala Met Val Ile Glu Thr Glu Glu Ala Ser Gln Phe Ala Ala Ile Ala
50 55 60
agt gcc ttg ttg att aac gtt ggc aca ctg acg cag cca cgc gct cag 240
Ser Ala Leu Leu Ile Asn Val Gly Thr Leu Thr Gln Pro Arg Ala Gln
65 70 75 80
gcg atg cgt gct gcc gtt gag caa gca aaa agc tct caa aca ccc tgg 288
Ala Met Arg Ala Ala Val Glu Gln Ala Lys Ser Ser Gln Thr Pro Trp
85 90 95
acg ctt gat cca gta gcg gtg ggt gcg ctc gat tat cgc cgc cat ttt 336
Thr Leu Asp Pro Val Ala Val Gly Ala Leu Asp Tyr Arg Arg His Phe
100 105 110
tgt cat gaa ctt tta tct ttt aaa ccg gca gcg ata cgt ggt aat gct 384
Cys His Glu Leu Leu Ser Phe Lys Pro Ala Ala Ile Arg Gly Asn Ala
115 120 125
tcg gaa atc atg gca tta gct ggc att gct aat ggc gga cgg gga gtg 432
Ser Glu Ile Met Ala Leu Ala Gly Ile Ala Asn Gly Gly Arg Gly Val
130 135 140
gat acc act gac gcc gca gct aac gcg ata ccc gct gca caa aca ctg 480
Asp Thr Thr Asp Ala Ala Ala Asn Ala Ile Pro Ala Ala Gln Thr Leu
145 150 155 160
gca cgg gaa act ggc gca atc gtc gtg gtc act ggc gag atg gat tat 528
Ala Arg Glu Thr Gly Ala Ile Val Val Val Thr Gly Glu Met Asp Tyr
165 170 175
gtt acc gat gga cat cgt atc att ggt att cac ggt ggt gat ccg tta 576
Val Thr Asp Gly His Arg Ile Ile Gly Ile His Gly Gly Asp Pro Leu
180 185 190
atg acc aaa gtg gta gga act ggc tgt gca tta tcg gcg gtt gtc gct 624
Met Thr Lys Val Val Gly Thr Gly Cys Ala Leu Ser Ala Val Val Ala
195 200 205
gcc tgc tgt gcg tta cca ggc gat acg ctg gaa aat gtc gca tct gcc 672
Ala Cys Cys Ala Leu Pro Gly Asp Thr Leu Glu Asn Val Ala Ser Ala
210 215 220
tgt cac tgg atg aaa caa gcc gga gaa cgc gca gtc gcc aga agc gag 720
Cys His Trp Met Lys Gln Ala Gly Glu Arg Ala Val Ala Arg Ser Glu
225 230 235 240
ggg cca ggc agt ttt gtt cca cat ttc ctt gat gcg ctc tgg caa ttg 768
Gly Pro Gly Ser Phe Val Pro His Phe Leu Asp Ala Leu Trp Gln Leu
245 250 255
acg cag gag gtg cag gca tga 789
Thr Gln Glu Val Gln Ala
260
<210> 55
<211> 262
<212> PRT
<213> 大肠杆菌
<400> 55
Met Gln Val Asp Leu Leu Gly Ser Ala Gln Ser Ala His Ala Leu His
1 5 10 15
Leu Phe His Gln His Ser Pro Leu Val His Cys Met Thr Asn Asp Val
20 25 30
Val Gln Thr Phe Thr Ala Asn Thr Leu Leu Ala Leu Gly Ala Ser Pro
35 40 45
Ala Met Val Ile Glu Thr Glu Glu Ala Ser Gln Phe Ala Ala Ile Ala
50 55 60
Ser Ala Leu Leu Ile Asn Val Gly Thr Leu Thr Gln Pro Arg Ala Gln
65 70 75 80
Ala Met Arg Ala Ala Val Glu Gln Ala Lys Ser Ser Gln Thr Pro Trp
85 90 95
Thr Leu Asp Pro Val Ala Val Gly Ala Leu Asp Tyr Arg Arg His Phe
100 105 110
Cys His Glu Leu Leu Ser Phe Lys Pro Ala Ala Ile Arg Gly Asn Ala
115 120 125
Ser Glu Ile Met Ala Leu Ala Gly Ile Ala Asn Gly Gly Arg Gly Val
130 135 140
Asp Thr Thr Asp Ala Ala Ala Asn Ala Ile Pro Ala Ala Gln Thr Leu
145 150 155 160
Ala Arg Glu Thr Gly Ala Ile Val Val Val Thr Gly Glu Met Asp Tyr
165 170 175
Val Thr Asp Gly His Arg Ile Ile Gly Ile His Gly Gly Asp Pro Leu
180 185 190
Met Thr Lys Val Val Gly Thr Gly Cys Ala Leu Ser Ala Val Val Ala
195 200 205
Ala Cys Cys Ala Leu Pro Gly Asp Thr Leu Glu Asn Val Ala Ser Ala
210 215 220
Cys His Trp Met Lys Gln Ala Gly Glu Arg Ala Val Ala Arg Ser Glu
225 230 235 240
Gly Pro Gly Ser Phe Val Pro His Phe Leu Asp Ala Leu Trp Gln Leu
245 250 255
Thr Gln Glu Val Gln Ala
260
<210> 56
<211> 819
<212> DNA
<213> 枯草芽孢杆菌
<220>
<221> CDS
<222> (1)..(819)
<400> 56
atg gat gca caa tca gca gca aaa tgt ctt acg gct gtc cgc cgg cat 48
Met Asp Ala Gln Ser Ala Ala Lys Cys Leu Thr Ala Val Arg Arg His
1 5 10 15
agc cca ctg gtg cat agc ata acc aac aat gtc gta acg aat ttc aca 96
Ser Pro Leu Val His Ser Ile Thr Asn Asn Val Val Thr Asn Phe Thr
20 25 30
gca aac ggc ctg ctc gcg ctc ggc gca tcg ccc gtt atg gcg tac gca 144
Ala Asn Gly Leu Leu Ala Leu Gly Ala Ser Pro Val Met Ala Tyr Ala
35 40 45
aaa gaa gag gtc gcc gat atg gcg aaa att gcg ggt gca ctc gtt tta 192
Lys Glu Glu Val Ala Asp Met Ala Lys Ile Ala Gly Ala Leu Val Leu
50 55 60
aat atc gga aca ctg agc aag gag tca gtc gaa gcg atg atc atc gcg 240
Asn Ile Gly Thr Leu Ser Lys Glu Ser Val Glu Ala Met Ile Ile Ala
65 70 75 80
gga aaa tca gct aat gaa cat ggc gtt ccc gtc att ctt gat cct gtc 288
Gly Lys Ser Ala Asn Glu His Gly Val Pro Val Ile Leu Asp Pro Val
85 90 95
ggt gcc gga gca aca ccg ttc cgc act gaa tcg gca cgt gac atc att 336
Gly Ala Gly Ala Thr Pro Phe Arg Thr Glu Ser Ala Arg Asp Ile Ile
100 105 110
cgt gag gtg cgc ctt gct gca atc aga gga aat gcg gcg gaa att gcc 384
Arg Glu Val Arg Leu Ala Ala Ile Arg Gly Asn Ala Ala Glu Ile Ala
115 120 125
cat acc gtc ggc gtg acc gat tgg ctg atc aaa ggt gtt gat gcg ggt 432
His Thr Val Gly Val Thr Asp Trp Leu Ile Lys Gly Val Asp Ala Gly
130 135 140
gaa ggt gga ggc gac atc atc cgg ctg gct cag cag gcg gca caa aag 480
Glu Gly Gly Gly Asp Ile Ile Arg Leu Ala Gln Gln Ala Ala Gln Lys
145 150 155 160
cta aac acg gtc att gcg ata act ggt gaa gtt gat gtc ata gcc gac 528
Leu Asn Thr Val Ile Ala Ile Thr Gly Glu Val Asp Val Ile Ala Asp
165 170 175
acg tca cat gta tac acc ctt cat aac ggc cac aag ctg ctg aca aaa 576
Thr Ser His Val Tyr Thr Leu His Asn Gly His Lys Leu Leu Thr Lys
180 185 190
gtg aca ggc gcc ggt tgc ctg ctg act tcc gtc gtc ggt gcg ttt tgc 624
Val Thr Gly Ala Gly Cys Leu Leu Thr Ser Val Val Gly Ala Phe Cys
195 200 205
gct gtg gaa gaa aat cca ttg ttt gct gct att gcg gcc att tct tcg 672
Ala Val Glu Glu Asn Pro Leu Phe Ala Ala Ile Ala Ala Ile Ser Ser
210 215 220
tat ggg gtc gcc gct cag ctt gcc gca cag cag acg gct gac aaa ggc 720
Tyr Gly Val Ala Ala Gln Leu Ala Ala Gln Gln Thr Ala Asp Lys Gly
225 230 235 240
cct gga agc ttt cag att gaa ttg ctg aac aag ctt tca act gtt act 768
Pro Gly Ser Phe Gln Ile Glu Leu Leu Asn Lys Leu Ser Thr Val Thr
245 250 255
gaa caa gac gtc caa gaa tgg gcg act ata gaa agg gtg act gtc tca 816
Glu Gln Asp Val Gln Glu Trp Ala Thr Ile Glu Arg Val Thr Val Ser
260 265 270
tga 819
<210> 57
<211> 272
<212> PRT
<213> 枯草芽孢杆菌
<400> 57
Met Asp Ala Gln Ser Ala Ala Lys Cys Leu Thr Ala Val Arg Arg His
1 5 10 15
Ser Pro Leu Val His Ser Ile Thr Asn Asn Val Val Thr Asn Phe Thr
20 25 30
Ala Asn Gly Leu Leu Ala Leu Gly Ala Ser Pro Val Met Ala Tyr Ala
35 40 45
Lys Glu Glu Val Ala Asp Met Ala Lys Ile Ala Gly Ala Leu Val Leu
50 55 60
Asn Ile Gly Thr Leu Ser Lys Glu Ser Val Glu Ala Met Ile Ile Ala
65 70 75 80
Gly Lys Ser Ala Asn Glu His Gly Val Pro Val Ile Leu Asp Pro Val
85 90 95
Gly Ala Gly Ala Thr Pro Phe Arg Thr Glu Ser Ala Arg Asp Ile Ile
100 105 110
Arg Glu Val Arg Leu Ala Ala Ile Arg Gly Asn Ala Ala Glu Ile Ala
115 120 125
His Thr Val Gly Val Thr Asp Trp Leu Ile Lys Gly Val Asp Ala Gly
130 135 140
Glu Gly Gly Gly Asp Ile Ile Arg Leu Ala Gln Gln Ala Ala Gln Lys
145 150 155 160
Leu Asn Thr Val Ile Ala Ile Thr Gly Glu Val Asp Val Ile Ala Asp
165 170 175
Thr Ser His Val Tyr Thr Leu His Asn Gly His Lys Leu Leu Thr Lys
180 185 190
Val Thr Gly Ala Gly Cys Leu Leu Thr Ser Val Val Gly Ala Phe Cys
195 200 205
Ala Val Glu Glu Asn Pro Leu Phe Ala Ala Ile Ala Ala Ile Ser Ser
210 215 220
Tyr Gly Val Ala Ala Gln Leu Ala Ala Gln Gln Thr Ala Asp Lys Gly
225 230 235 240
Pro Gly Ser Phe Gln Ile Glu Leu Leu Asn Lys Leu Ser Thr Val Thr
245 250 255
Glu Gln Asp Val Gln Glu Trp Ala Thr Ile Glu Arg Val Thr Val Ser
260 265 270
<210> 58
<211> 783
<212> DNA
<213> 詹氏甲烷暖球菌
<220>
<221> CDS
<222> (1)..(783)
<400> 58
atg ttg act att ctt aag ttg gga ggg agc att ctg tcc gat aaa aac 48
Met Leu Thr Ile Leu Lys Leu Gly Gly Ser Ile Leu Ser Asp Lys Asn
1 5 10 15
gtt cca tat agc att aag tgg gat aac tta gaa cgt att gct atg gaa 96
Val Pro Tyr Ser Ile Lys Trp Asp Asn Leu Glu Arg Ile Ala Met Glu
20 25 30
atc aaa aac gcg tta gat tat tac aag aac caa aat aaa gaa att aag 144
Ile Lys Asn Ala Leu Asp Tyr Tyr Lys Asn Gln Asn Lys Glu Ile Lys
35 40 45
ctt att ctg gta cat ggc ggc ggg gca ttt ggg cat cca gtg gcc aag 192
Leu Ile Leu Val His Gly Gly Gly Ala Phe Gly His Pro Val Ala Lys
50 55 60
aaa tac ctg aag att gaa gac ggc aaa aaa att ttc atc aac atg gaa 240
Lys Tyr Leu Lys Ile Glu Asp Gly Lys Lys Ile Phe Ile Asn Met Glu
65 70 75 80
aaa gga ttc tgg gag att cag cgt gcg atg cgc cgt ttt aat aac atc 288
Lys Gly Phe Trp Glu Ile Gln Arg Ala Met Arg Arg Phe Asn Asn Ile
85 90 95
atc atc gac acg ctt cag agt tac gat atc cca gcg gtc tcg att caa 336
Ile Ile Asp Thr Leu Gln Ser Tyr Asp Ile Pro Ala Val Ser Ile Gln
100 105 110
cct tcc agc ttt gtt gtt ttt ggc gac aaa ttg atc ttc gac acc tct 384
Pro Ser Ser Phe Val Val Phe Gly Asp Lys Leu Ile Phe Asp Thr Ser
115 120 125
gcg atc aaa gag atg ttg aaa cgc aac ctt gta ccc gtt atc cat ggg 432
Ala Ile Lys Glu Met Leu Lys Arg Asn Leu Val Pro Val Ile His Gly
130 135 140
gat atc gtc att gac gat aaa aat ggg tac cgt att atc agc ggt gac 480
Asp Ile Val Ile Asp Asp Lys Asn Gly Tyr Arg Ile Ile Ser Gly Asp
145 150 155 160
gac atc gtg cca tat tta gcc aat gaa ctg aag gca gat tta atc ctt 528
Asp Ile Val Pro Tyr Leu Ala Asn Glu Leu Lys Ala Asp Leu Ile Leu
165 170 175
tat gca acc gac gtg gac ggc gta ttg att gac aac aag ccc att aaa 576
Tyr Ala Thr Asp Val Asp Gly Val Leu Ile Asp Asn Lys Pro Ile Lys
180 185 190
cgc att gat aag aat aat atc tac aag att ttg aat tat ctt tcg ggt 624
Arg Ile Asp Lys Asn Asn Ile Tyr Lys Ile Leu Asn Tyr Leu Ser Gly
195 200 205
agc aat tca att gac gtc acg ggg ggg atg aaa tac aag atc gac atg 672
Ser Asn Ser Ile Asp Val Thr Gly Gly Met Lys Tyr Lys Ile Asp Met
210 215 220
atc cgt aaa aac aaa tgc cgt ggt ttc gtg ttt aat ggc aac aag gca 720
Ile Arg Lys Asn Lys Cys Arg Gly Phe Val Phe Asn Gly Asn Lys Ala
225 230 235 240
aac aac att tat aag gcg ctg ctt ggg gaa gtc gag ggt acc gaa atc 768
Asn Asn Ile Tyr Lys Ala Leu Leu Gly Glu Val Glu Gly Thr Glu Ile
245 250 255
gac ttt tct gaa taa 783
Asp Phe Ser Glu
260
<210> 59
<211> 260
<212> PRT
<213> 詹氏甲烷暖球菌
<400> 59
Met Leu Thr Ile Leu Lys Leu Gly Gly Ser Ile Leu Ser Asp Lys Asn
1 5 10 15
Val Pro Tyr Ser Ile Lys Trp Asp Asn Leu Glu Arg Ile Ala Met Glu
20 25 30
Ile Lys Asn Ala Leu Asp Tyr Tyr Lys Asn Gln Asn Lys Glu Ile Lys
35 40 45
Leu Ile Leu Val His Gly Gly Gly Ala Phe Gly His Pro Val Ala Lys
50 55 60
Lys Tyr Leu Lys Ile Glu Asp Gly Lys Lys Ile Phe Ile Asn Met Glu
65 70 75 80
Lys Gly Phe Trp Glu Ile Gln Arg Ala Met Arg Arg Phe Asn Asn Ile
85 90 95
Ile Ile Asp Thr Leu Gln Ser Tyr Asp Ile Pro Ala Val Ser Ile Gln
100 105 110
Pro Ser Ser Phe Val Val Phe Gly Asp Lys Leu Ile Phe Asp Thr Ser
115 120 125
Ala Ile Lys Glu Met Leu Lys Arg Asn Leu Val Pro Val Ile His Gly
130 135 140
Asp Ile Val Ile Asp Asp Lys Asn Gly Tyr Arg Ile Ile Ser Gly Asp
145 150 155 160
Asp Ile Val Pro Tyr Leu Ala Asn Glu Leu Lys Ala Asp Leu Ile Leu
165 170 175
Tyr Ala Thr Asp Val Asp Gly Val Leu Ile Asp Asn Lys Pro Ile Lys
180 185 190
Arg Ile Asp Lys Asn Asn Ile Tyr Lys Ile Leu Asn Tyr Leu Ser Gly
195 200 205
Ser Asn Ser Ile Asp Val Thr Gly Gly Met Lys Tyr Lys Ile Asp Met
210 215 220
Ile Arg Lys Asn Lys Cys Arg Gly Phe Val Phe Asn Gly Asn Lys Ala
225 230 235 240
Asn Asn Ile Tyr Lys Ala Leu Leu Gly Glu Val Glu Gly Thr Glu Ile
245 250 255
Asp Phe Ser Glu
260
<210> 60
<211> 744
<212> DNA
<213> 嗜热乙酸甲烷丝菌
<220>
<221> CDS
<222> (1)..(744)
<400> 60
tta aag att ttg aaa ttg ggc ggt agc att att acg gat aag agc cgc 48
Leu Lys Ile Leu Lys Leu Gly Gly Ser Ile Ile Thr Asp Lys Ser Arg
1 5 10 15
tta gct act gca cgt ctg gat caa att tca cgt atc gca cac gaa atc 96
Leu Ala Thr Ala Arg Leu Asp Gln Ile Ser Arg Ile Ala His Glu Ile
20 25 30
tca ggc atc gag aac ctg att gtt gtt cac gga gcc ggt tct ttt ggt 144
Ser Gly Ile Glu Asn Leu Ile Val Val His Gly Ala Gly Ser Phe Gly
35 40 45
cac atc cat gcc aaa aat ttc ggt ctt ccg gaa cgt ttc tca gga gaa 192
His Ile His Ala Lys Asn Phe Gly Leu Pro Glu Arg Phe Ser Gly Glu
50 55 60
ggg tta ctg aaa aca cat ctg tcg gtc tcg gat ttg aat cgt atc gtc 240
Gly Leu Leu Lys Thr His Leu Ser Val Ser Asp Leu Asn Arg Ile Val
65 70 75 80
gtt gaa gct ctt cat gat gca ggg gtg gac gcg ctg ccc ttg cac ccc 288
Val Glu Ala Leu His Asp Ala Gly Val Asp Ala Leu Pro Leu His Pro
85 90 95
tta tca agt gta gtc ctt cgt gac gga cgc atc cac cat atg tct acc 336
Leu Ser Ser Val Val Leu Arg Asp Gly Arg Ile His His Met Ser Thr
100 105 110
gag gtc att acg gaa atg ctt cgt cgt gat gta gtg ccg gta tta cat 384
Glu Val Ile Thr Glu Met Leu Arg Arg Asp Val Val Pro Val Leu His
115 120 125
ggg gat gtt gcg atg gac ctg tca aag ggt gcc ggc att gta agt gga 432
Gly Asp Val Ala Met Asp Leu Ser Lys Gly Ala Gly Ile Val Ser Gly
130 135 140
gac cag ttg gtt tcg tat atg gca cgt act ctg gga gct ggt atg gtc 480
Asp Gln Leu Val Ser Tyr Met Ala Arg Thr Leu Gly Ala Gly Met Val
145 150 155 160
gct atg ggg acc gat gtc gac ggg gtt atg atc gat ggt cgt gtc ctt 528
Ala Met Gly Thr Asp Val Asp Gly Val Met Ile Asp Gly Arg Val Leu
165 170 175
agt tgc att aca cct aat gac atg cac tct ttg gag agt cac tta tta 576
Ser Cys Ile Thr Pro Asn Asp Met His Ser Leu Glu Ser His Leu Leu
180 185 190
ccc gca aaa ggg gta gac gtc acg ggt gga atg cgc ggt aaa ctg gcg 624
Pro Ala Lys Gly Val Asp Val Thr Gly Gly Met Arg Gly Lys Leu Ala
195 200 205
gaa tta gta gag ctg gca ggc att gga att gat tcg cgt att ttt aat 672
Glu Leu Val Glu Leu Ala Gly Ile Gly Ile Asp Ser Arg Ile Phe Asn
210 215 220
gcc ggc gtt gct ggt aat gta cgc cgt gct ttg tct ggg gag tcg tta 720
Ala Gly Val Ala Gly Asn Val Arg Arg Ala Leu Ser Gly Glu Ser Leu
225 230 235 240
gga act ttg att act gga cgc taa 744
Gly Thr Leu Ile Thr Gly Arg
245
<210> 61
<211> 247
<212> PRT
<213> 嗜热乙酸甲烷丝菌
<400> 61
Leu Lys Ile Leu Lys Leu Gly Gly Ser Ile Ile Thr Asp Lys Ser Arg
1 5 10 15
Leu Ala Thr Ala Arg Leu Asp Gln Ile Ser Arg Ile Ala His Glu Ile
20 25 30
Ser Gly Ile Glu Asn Leu Ile Val Val His Gly Ala Gly Ser Phe Gly
35 40 45
His Ile His Ala Lys Asn Phe Gly Leu Pro Glu Arg Phe Ser Gly Glu
50 55 60
Gly Leu Leu Lys Thr His Leu Ser Val Ser Asp Leu Asn Arg Ile Val
65 70 75 80
Val Glu Ala Leu His Asp Ala Gly Val Asp Ala Leu Pro Leu His Pro
85 90 95
Leu Ser Ser Val Val Leu Arg Asp Gly Arg Ile His His Met Ser Thr
100 105 110
Glu Val Ile Thr Glu Met Leu Arg Arg Asp Val Val Pro Val Leu His
115 120 125
Gly Asp Val Ala Met Asp Leu Ser Lys Gly Ala Gly Ile Val Ser Gly
130 135 140
Asp Gln Leu Val Ser Tyr Met Ala Arg Thr Leu Gly Ala Gly Met Val
145 150 155 160
Ala Met Gly Thr Asp Val Asp Gly Val Met Ile Asp Gly Arg Val Leu
165 170 175
Ser Cys Ile Thr Pro Asn Asp Met His Ser Leu Glu Ser His Leu Leu
180 185 190
Pro Ala Lys Gly Val Asp Val Thr Gly Gly Met Arg Gly Lys Leu Ala
195 200 205
Glu Leu Val Glu Leu Ala Gly Ile Gly Ile Asp Ser Arg Ile Phe Asn
210 215 220
Ala Gly Val Ala Gly Asn Val Arg Arg Ala Leu Ser Gly Glu Ser Leu
225 230 235 240
Gly Thr Leu Ile Thr Gly Arg
245
<210> 62
<211> 543
<212> DNA
<213> 大肠杆菌
<220>
<221> CDS
<222> (1)..(543)
<400> 62
atg caa acc gag cat gtc att tta ttg gac gag caa gga gaa cca att 48
Met Gln Thr Glu His Val Ile Leu Leu Asp Glu Gln Gly Glu Pro Ile
1 5 10 15
gga act tta gaa aaa tac gct gca cat aca gcg gac acc cgc tta cat 96
Gly Thr Leu Glu Lys Tyr Ala Ala His Thr Ala Asp Thr Arg Leu His
20 25 30
ctt gct ttt tct agt tgg ctg ttt aac gat aag ggt caa tta tta gtg 144
Leu Ala Phe Ser Ser Trp Leu Phe Asn Asp Lys Gly Gln Leu Leu Val
35 40 45
acg cgc cgt gcg ctg agc aaa aaa gca tgg ccg ggt gtt tgg acg aac 192
Thr Arg Arg Ala Leu Ser Lys Lys Ala Trp Pro Gly Val Trp Thr Asn
50 55 60
agt gtt tgc gga cac ccc caa ctg gga gaa tcc aat gag gat gcg gta 240
Ser Val Cys Gly His Pro Gln Leu Gly Glu Ser Asn Glu Asp Ala Val
65 70 75 80
att cgc cgt tgt cgc tat gaa ttg ggt gtg gag att acg cca ccg aca 288
Ile Arg Arg Cys Arg Tyr Glu Leu Gly Val Glu Ile Thr Pro Pro Thr
85 90 95
ccg atc tac cct gat ttc cgt tat cgc gct acg gat cct tca ggt att 336
Pro Ile Tyr Pro Asp Phe Arg Tyr Arg Ala Thr Asp Pro Ser Gly Ile
100 105 110
gtt gaa aat gaa gta tgc cca gtg ttt gcc gcg cgc aca act tct gcg 384
Val Glu Asn Glu Val Cys Pro Val Phe Ala Ala Arg Thr Thr Ser Ala
115 120 125
ctt caa atc aac cca gac gag gtc atg gat tac caa tgg tgt gat ctt 432
Leu Gln Ile Asn Pro Asp Glu Val Met Asp Tyr Gln Trp Cys Asp Leu
130 135 140
gct gac gta ctg cac ggg att gac gcg aca ccg tgg gct ttt agt ccc 480
Ala Asp Val Leu His Gly Ile Asp Ala Thr Pro Trp Ala Phe Ser Pro
145 150 155 160
tgg atg gtt atg caa gcg aca aat gaa gaa gca cgt aag cgc ctt cag 528
Trp Met Val Met Gln Ala Thr Asn Glu Glu Ala Arg Lys Arg Leu Gln
165 170 175
gcg ttt act cag taa 543
Ala Phe Thr Gln
180
<210> 63
<211> 180
<212> PRT
<213> 大肠杆菌
<400> 63
Met Gln Thr Glu His Val Ile Leu Leu Asp Glu Gln Gly Glu Pro Ile
1 5 10 15
Gly Thr Leu Glu Lys Tyr Ala Ala His Thr Ala Asp Thr Arg Leu His
20 25 30
Leu Ala Phe Ser Ser Trp Leu Phe Asn Asp Lys Gly Gln Leu Leu Val
35 40 45
Thr Arg Arg Ala Leu Ser Lys Lys Ala Trp Pro Gly Val Trp Thr Asn
50 55 60
Ser Val Cys Gly His Pro Gln Leu Gly Glu Ser Asn Glu Asp Ala Val
65 70 75 80
Ile Arg Arg Cys Arg Tyr Glu Leu Gly Val Glu Ile Thr Pro Pro Thr
85 90 95
Pro Ile Tyr Pro Asp Phe Arg Tyr Arg Ala Thr Asp Pro Ser Gly Ile
100 105 110
Val Glu Asn Glu Val Cys Pro Val Phe Ala Ala Arg Thr Thr Ser Ala
115 120 125
Leu Gln Ile Asn Pro Asp Glu Val Met Asp Tyr Gln Trp Cys Asp Leu
130 135 140
Ala Asp Val Leu His Gly Ile Asp Ala Thr Pro Trp Ala Phe Ser Pro
145 150 155 160
Trp Met Val Met Gln Ala Thr Asn Glu Glu Ala Arg Lys Arg Leu Gln
165 170 175
Ala Phe Thr Gln
180
<210> 64
<211> 894
<212> DNA
<213> 嗜热脂肪土芽孢杆菌
<220>
<221> CDS
<222> (1)..(894)
<400> 64
atg gcg cag ctt tca gtt gaa cag ttt ctc aac gag caa aaa cag gcg 48
Met Ala Gln Leu Ser Val Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala
1 5 10 15
gtg gaa aca gcg ctc tcc cgt tat ata gag cgc tta gaa ggg ccg gcg 96
Val Glu Thr Ala Leu Ser Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala
20 25 30
aag ctg aaa aag gcg atg gcg tac tca ttg gag gcc ggc ggc aaa cga 144
Lys Leu Lys Lys Ala Met Ala Tyr Ser Leu Glu Ala Gly Gly Lys Arg
35 40 45
atc cgt ccg ttg ctg ctt ctg tcc acc gtt cgg gcg ctc ggc aaa gac 192
Ile Arg Pro Leu Leu Leu Leu Ser Thr Val Arg Ala Leu Gly Lys Asp
50 55 60
ccg gcg gtc gga ttg ccc gtc gcc tgc gcg att gaa atg atc cat acg 240
Pro Ala Val Gly Leu Pro Val Ala Cys Ala Ile Glu Met Ile His Thr
65 70 75 80
tac ttt ttg atc cat gat gat ttg ccg agc atg gac aac gat gat ttg 288
Tyr Phe Leu Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Leu
85 90 95
cgg cgc ggc aag ccg acg aac cat aaa gtg ttc ggc gag gcg atg gcc 336
Arg Arg Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Met Ala
100 105 110
atc ttg gcg ggg gac ggg ttg ttg acg tac gcg ttt caa ttg atc acc 384
Ile Leu Ala Gly Asp Gly Leu Leu Thr Tyr Ala Phe Gln Leu Ile Thr
115 120 125
gaa atc gac gat gag cgc atc cct cct tcc gtc cgg ctt cgg ctc atc 432
Glu Ile Asp Asp Glu Arg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile
130 135 140
gaa cgg ctg gcg aaa gcg gcc ggt ccg gaa ggg atg gtc gcc ggt cag 480
Glu Arg Leu Ala Lys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln
145 150 155 160
gca gcc gat atg gaa gga gag ggg aaa acg ctg acg ctt tcg gag ctc 528
Ala Ala Asp Met Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu
165 170 175
gaa tac att cat cgg cat aaa acc ggg aaa atg ctg caa tac agc gtg 576
Glu Tyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val
180 185 190
cac gcc ggc gcc ttg atc ggc ggc gct gat gcc cgg caa acg cgg gag 624
His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu
195 200 205
ctt gac gaa ttc gcc gcc cat cta ggc ctt gcc ttt caa att cgc gat 672
Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp
210 215 220
gat att ctc gat att gaa ggg gca gaa gaa aaa atc ggc aag ccg gtc 720
Asp Ile Leu Asp Ile Glu Gly Ala Glu Glu Lys Ile Gly Lys Pro Val
225 230 235 240
ggc agc gac caa agc aac aac aaa gcg acg tat cca gcg ttg ctg tcg 768
Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala Leu Leu Ser
245 250 255
ctt gcc ggc gcg aag gaa aag ttg gcg ttc cat atc gag gcg gcg cag 816
Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile Glu Ala Ala Gln
260 265 270
cgc cat tta cgg aac gct gac gtt gac ggc gcc gcg ctc gcc tat att 864
Arg His Leu Arg Asn Ala Asp Val Asp Gly Ala Ala Leu Ala Tyr Ile
275 280 285
tgc gaa ctg gtc gcc gcc cgc gac cat taa 894
Cys Glu Leu Val Ala Ala Arg Asp His
290 295
<210> 65
<211> 297
<212> PRT
<213> 嗜热脂肪土芽孢杆菌
<400> 65
Met Ala Gln Leu Ser Val Glu Gln Phe Leu Asn Glu Gln Lys Gln Ala
1 5 10 15
Val Glu Thr Ala Leu Ser Arg Tyr Ile Glu Arg Leu Glu Gly Pro Ala
20 25 30
Lys Leu Lys Lys Ala Met Ala Tyr Ser Leu Glu Ala Gly Gly Lys Arg
35 40 45
Ile Arg Pro Leu Leu Leu Leu Ser Thr Val Arg Ala Leu Gly Lys Asp
50 55 60
Pro Ala Val Gly Leu Pro Val Ala Cys Ala Ile Glu Met Ile His Thr
65 70 75 80
Tyr Phe Leu Ile His Asp Asp Leu Pro Ser Met Asp Asn Asp Asp Leu
85 90 95
Arg Arg Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Met Ala
100 105 110
Ile Leu Ala Gly Asp Gly Leu Leu Thr Tyr Ala Phe Gln Leu Ile Thr
115 120 125
Glu Ile Asp Asp Glu Arg Ile Pro Pro Ser Val Arg Leu Arg Leu Ile
130 135 140
Glu Arg Leu Ala Lys Ala Ala Gly Pro Glu Gly Met Val Ala Gly Gln
145 150 155 160
Ala Ala Asp Met Glu Gly Glu Gly Lys Thr Leu Thr Leu Ser Glu Leu
165 170 175
Glu Tyr Ile His Arg His Lys Thr Gly Lys Met Leu Gln Tyr Ser Val
180 185 190
His Ala Gly Ala Leu Ile Gly Gly Ala Asp Ala Arg Gln Thr Arg Glu
195 200 205
Leu Asp Glu Phe Ala Ala His Leu Gly Leu Ala Phe Gln Ile Arg Asp
210 215 220
Asp Ile Leu Asp Ile Glu Gly Ala Glu Glu Lys Ile Gly Lys Pro Val
225 230 235 240
Gly Ser Asp Gln Ser Asn Asn Lys Ala Thr Tyr Pro Ala Leu Leu Ser
245 250 255
Leu Ala Gly Ala Lys Glu Lys Leu Ala Phe His Ile Glu Ala Ala Gln
260 265 270
Arg His Leu Arg Asn Ala Asp Val Asp Gly Ala Ala Leu Ala Tyr Ile
275 280 285
Cys Glu Leu Val Ala Ala Arg Asp His
290 295
<210> 66
<211> 924
<212> DNA
<213> 人工序列
<220>
<223> NphB M23 cDNA
<400> 66
atgtccgagg cggccgacgt ggagcgtgtt tatgctgcta tggaagaagc tgcgggtctg 60
ctgggagtgg catgtgctcg tgataagatt tatccccttc tttcaacctt ccaggataca 120
ttggttgaag gtggcagtgt ggtagtgttt agcatggcta gtggacgcca cagcacggaa 180
ctggacttta gtatttcagt acccacgtcc catggtgacc catacgcaac tgtcgtcgaa 240
aaggggctgt tccctgcaac aggccatcct gttgacgatc ttttggctga tacgcagaag 300
cacctgcctg tttctatgtt cgccattgat ggagaagtca ccggaggttt caaaaaaact 360
tatgctttct ttccaactga taatatgcca ggtgtggccg agttgagtgc catccccagt 420
atgccaccgg cggtcgcgga aaacgccgaa ttattcgcgc gttatgggtt agataaggtg 480
cagatgacgt caatggacta caagaagcgc caggtcaatt tgtacttctc tgagttaagt 540
gcacagactt tagaagccga gtctgtcctt gcgcttgttc gtgaactggg tttgcacgtg 600
ccgaacgaac tgggtcttaa attttgcaag cgctcctttt ccgtttatcc gacactgaac 660
tgggaaacag ggaaaattga tcgtttatgt tttgcggtga tttcaaacga ccctaccttg 720
gtaccaagtt cggacgaagg ggacattgaa aaatttcaca actacgcgac gaaggcgccg 780
tacgcatacg tcggcgaaaa gcgtacgctg gtttacgggt tgacgctgag tcccaaagag 840
gaatactata aattaagcgc agcgtaccat atcaccgatg tacaacgcgg actgctgaag 900
gcctttgata gccttgaaga ctaa 924
<210> 67
<211> 924
<212> DNA
<213> 人工序列
<220>
<223> NphB M31 cDNA
<400> 67
atgtccgagg cggccgacgt ggagcgtgtt tatgctgcta tggaagaagc tgcgggtctg 60
ctgggagtgg catgtgctcg tgataagatt tatccccttc tttcaacctt ccaggataca 120
ttggttgaag gtggcagtgt ggtagtgttt agcatggcta gtggacgcca cagcacggaa 180
ctggacttta gtatttcagt acccacgtcc catggtgacc catacgcaac tgtcgtcgaa 240
aaggggctgt tccctgcaac aggccatcct gttgacgatc ttttggctga tacgcagaag 300
cacctgcctg tttctatgtt cgccattgat ggagaagtca ccggaggttt caaaaaaact 360
tatgctttct ttccaactga taatatgcca ggtgtggccg agttgagtgc catccccagt 420
atgccaccgg cggtcgcgga aaacgccgaa ttattcgcgc gttatgggtt agataaggtg 480
cagatgacgt caatggacta caagaagcgc caggtcaatt tgtacttctc tgagttaagt 540
gcacagactt tagaagccga gtctgtcctt gcgcttgttc gtgaactggg tttgcacgtg 600
ccgaacgaac tgggtcttaa attttgcaag cgctcctttt ccgtttatcc gacactgaac 660
tgggaaacag ggaaaattga tcgtttatgt tttagcgtga tttcaaacga ccctaccttg 720
gtaccaagtt cggacgaagg ggacattgaa aaatttcaca actacgcgac gaaggcgccg 780
tacgcatacg tcggcgaaaa gcgtacgctg gtttacgggt tgacgctgag tcccaaagag 840
gaatactata aattaggcgc agtgtaccat atcaccgatg tacaacgcgg actgctgaag 900
gcctttgata gccttgaaga ctaa 924
<210> 68
<211> 924
<212> DNA
<213> 人工序列
<220>
<223> NpHB M31 Pross 10 cDNA
<220>
<221> CDS
<222> (1)..(924)
<400> 68
atg tcg gaa gct gcc gat gta gaa cgt gtc tac gcc gcc atc gaa gaa 48
Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Ile Glu Glu
1 5 10 15
gcc gca ggt ttg ttg ggg gtc gca tgc gca cgc gat aag att tgg ccc 96
Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Trp Pro
20 25 30
ttg ctg tca aca ttc cag gat acc ttg gtt gag ggt gga agc gta gtt 144
Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val
35 40 45
gtt ttt agc atg gcc tcg ggg cgt cac tca acg gag ctg gac ttc tca 192
Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser
50 55 60
att tcc gtc ccg cct agt cat ggc gat ccg tac gcg att gtg gtg gaa 240
Ile Ser Val Pro Pro Ser His Gly Asp Pro Tyr Ala Ile Val Val Glu
65 70 75 80
aag ggc ttg ttc ccg gca act gga cat cca gtt gat gac ctt ctg gcg 288
Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala
85 90 95
gac att cag aag cat ctt ccc gta tct atg ttt gcg att gac ggg gaa 336
Asp Ile Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu
100 105 110
gtt acc ggg ggg ttc aaa aaa act tat gcg ttc ttc ccg acc gat aac 384
Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn
115 120 125
atg ccc ggt gtc gcg gaa ctg gcg gcc atc cca tcg atg cct cct gca 432
Met Pro Gly Val Ala Glu Leu Ala Ala Ile Pro Ser Met Pro Pro Ala
130 135 140
gtc gct gaa aat gct gaa ctg ttc gcg cgt tat ggc ctg gac aag gta 480
Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val
145 150 155 160
caa atg acc tcg atg gat tat aaa aaa cgt caa gtg aac ctg tat ttc 528
Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe
165 170 175
tcc gaa ctg tcg gct cag acg ctg gag gct gaa tca gta ctt gct tta 576
Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu
180 185 190
gtg cgt gaa ctg ggt ctt cat gtc cca aac gag ctg ggt ctg aaa ttt 624
Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe
195 200 205
tgc aaa cgc tcc ttc tca gta tac cca aca tta aac tgg gac acc tcg 672
Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Asp Thr Ser
210 215 220
aag att gac cgc ctt tgc ttc tct gta atc agt aca gat ccg aca ctt 720
Lys Ile Asp Arg Leu Cys Phe Ser Val Ile Ser Thr Asp Pro Thr Leu
225 230 235 240
gta cct agc tca gac gag gga gac att gaa aaa ttt cac aat tac gct 768
Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala
245 250 255
aca aag gcc ccc tat gca tat gtt gga gaa aag cgt aca ctt gtt tac 816
Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr
260 265 270
ggc ttg act tta tct ccc aaa gag gag tat tat aaa ttg ggt gcc gtt 864
Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Val
275 280 285
tac cac att act gac gta caa cgc aaa ctt ttg aag gcg ttc gac agc 912
Tyr His Ile Thr Asp Val Gln Arg Lys Leu Leu Lys Ala Phe Asp Ser
290 295 300
ctt gag gat taa 924
Leu Glu Asp
305
<210> 69
<211> 307
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建体
<400> 69
Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Ile Glu Glu
1 5 10 15
Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Trp Pro
20 25 30
Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val
35 40 45
Val Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser
50 55 60
Ile Ser Val Pro Pro Ser His Gly Asp Pro Tyr Ala Ile Val Val Glu
65 70 75 80
Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala
85 90 95
Asp Ile Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu
100 105 110
Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn
115 120 125
Met Pro Gly Val Ala Glu Leu Ala Ala Ile Pro Ser Met Pro Pro Ala
130 135 140
Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val
145 150 155 160
Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe
165 170 175
Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu
180 185 190
Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe
195 200 205
Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Asp Thr Ser
210 215 220
Lys Ile Asp Arg Leu Cys Phe Ser Val Ile Ser Thr Asp Pro Thr Leu
225 230 235 240
Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala
245 250 255
Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr
260 265 270
Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Val
275 280 285
Tyr His Ile Thr Asp Val Gln Arg Lys Leu Leu Lys Ala Phe Asp Ser
290 295 300
Leu Glu Asp
305
<210> 70
<211> 1632
<212> DNA
<213> 大麻
<220>
<221> CDS
<222> (1)..(1632)
<400> 70
atg gaa aag agt ggc tac gga cgc gac ggt att tac cgt agc ctg cgt 48
Met Glu Lys Ser Gly Tyr Gly Arg Asp Gly Ile Tyr Arg Ser Leu Arg
1 5 10 15
cct cct tta cac ctg cca aac aat aac aat ttg agt atg gtc tca ttc 96
Pro Pro Leu His Leu Pro Asn Asn Asn Asn Leu Ser Met Val Ser Phe
20 25 30
ctg ttc cgt aac agc agc agc tat cca cag aaa ccg gcg ttg atc gat 144
Leu Phe Arg Asn Ser Ser Ser Tyr Pro Gln Lys Pro Ala Leu Ile Asp
35 40 45
agc gag act aat caa att tta tct ttt agt cat ttt aaa agc acc gtg 192
Ser Glu Thr Asn Gln Ile Leu Ser Phe Ser His Phe Lys Ser Thr Val
50 55 60
atc aag gtc tcc cat ggc ttc tta aac ctg ggg atc aaa aag aat gac 240
Ile Lys Val Ser His Gly Phe Leu Asn Leu Gly Ile Lys Lys Asn Asp
65 70 75 80
gtg gtt tta atc tac gca ccc aat tcg atc cac ttt ccc gta tgc ttc 288
Val Val Leu Ile Tyr Ala Pro Asn Ser Ile His Phe Pro Val Cys Phe
85 90 95
ctt ggc att att gct tct ggg gcg atc gcc act act tca aat cca tta 336
Leu Gly Ile Ile Ala Ser Gly Ala Ile Ala Thr Thr Ser Asn Pro Leu
100 105 110
tac acc gtg agt gag ttg tcg aaa caa gta aag gac tcg aac cct aaa 384
Tyr Thr Val Ser Glu Leu Ser Lys Gln Val Lys Asp Ser Asn Pro Lys
115 120 125
ttg att atc aca gtc cct cag tta ttg gaa aag gtc aag ggt ttc aat 432
Leu Ile Ile Thr Val Pro Gln Leu Leu Glu Lys Val Lys Gly Phe Asn
130 135 140
ctg cca act atc ctt atc ggc cct gat tct gag cag gaa tcg tct agt 480
Leu Pro Thr Ile Leu Ile Gly Pro Asp Ser Glu Gln Glu Ser Ser Ser
145 150 155 160
gat aaa gta atg act ttc aat gat ctg gtc aat ctg gga gga agt tcg 528
Asp Lys Val Met Thr Phe Asn Asp Leu Val Asn Leu Gly Gly Ser Ser
165 170 175
ggt agc gaa ttc cct atc gtc gac gat ttc aag caa tcc gac acc gcc 576
Gly Ser Glu Phe Pro Ile Val Asp Asp Phe Lys Gln Ser Asp Thr Ala
180 185 190
gca ctg ttg tac tca agt ggc acg aca ggt atg agc aag ggg gtc gtt 624
Ala Leu Leu Tyr Ser Ser Gly Thr Thr Gly Met Ser Lys Gly Val Val
195 200 205
ctg acg cac aaa aat ttt att gcc tca tcg ttg atg gta aca atg gaa 672
Leu Thr His Lys Asn Phe Ile Ala Ser Ser Leu Met Val Thr Met Glu
210 215 220
cag gac ttg gtc ggc gag atg gac aat gtg ttc ctg tgt ttc ctt cct 720
Gln Asp Leu Val Gly Glu Met Asp Asn Val Phe Leu Cys Phe Leu Pro
225 230 235 240
atg ttt cac gtc ttt ggc tta gcc att att acg tat gct cag tta cag 768
Met Phe His Val Phe Gly Leu Ala Ile Ile Thr Tyr Ala Gln Leu Gln
245 250 255
cgc ggt aat acc gtg att tca atg gcc cgc ttt gac ttg gaa aag atg 816
Arg Gly Asn Thr Val Ile Ser Met Ala Arg Phe Asp Leu Glu Lys Met
260 265 270
tta aaa gat gtt gaa aag tac aaa gtt acc cac ctt tgg gtc gta ccc 864
Leu Lys Asp Val Glu Lys Tyr Lys Val Thr His Leu Trp Val Val Pro
275 280 285
cca gtt atc tta gcg ttg tcg aag aac tca atg gtg aaa aaa ttc aat 912
Pro Val Ile Leu Ala Leu Ser Lys Asn Ser Met Val Lys Lys Phe Asn
290 295 300
ttg tca tcc atc aag tat att ggt tca ggc gct gcg cca tta gga aag 960
Leu Ser Ser Ile Lys Tyr Ile Gly Ser Gly Ala Ala Pro Leu Gly Lys
305 310 315 320
gat ctg atg gaa gaa tgc tct aag gtg gtt cct tac gga atc gtg gct 1008
Asp Leu Met Glu Glu Cys Ser Lys Val Val Pro Tyr Gly Ile Val Ala
325 330 335
caa gga tat ggc atg acg gaa acg tgc gga atc gta tcc atg gaa gac 1056
Gln Gly Tyr Gly Met Thr Glu Thr Cys Gly Ile Val Ser Met Glu Asp
340 345 350
atc cgc ggc ggg aaa cgc aat tca ggg tcg gcc gga atg ttg gca agt 1104
Ile Arg Gly Gly Lys Arg Asn Ser Gly Ser Ala Gly Met Leu Ala Ser
355 360 365
ggg gta gaa gct cag atc gtg agt gtg gac acc tta aaa ccc ctt ccc 1152
Gly Val Glu Ala Gln Ile Val Ser Val Asp Thr Leu Lys Pro Leu Pro
370 375 380
ccg aat caa tta ggg gaa atc tgg gta aaa ggt cca aat atg atg caa 1200
Pro Asn Gln Leu Gly Glu Ile Trp Val Lys Gly Pro Asn Met Met Gln
385 390 395 400
ggc tat ttc aac aat cct caa gcg acc aaa ctt acc att gat aaa aag 1248
Gly Tyr Phe Asn Asn Pro Gln Ala Thr Lys Leu Thr Ile Asp Lys Lys
405 410 415
ggt tgg gtt cat act ggc gac ttg ggg tat ttc gac gaa gac gga cac 1296
Gly Trp Val His Thr Gly Asp Leu Gly Tyr Phe Asp Glu Asp Gly His
420 425 430
tta tat gtt gta gac cgt att aag gag ctt att aaa tac aag gga ttc 1344
Leu Tyr Val Val Asp Arg Ile Lys Glu Leu Ile Lys Tyr Lys Gly Phe
435 440 445
caa gtt gcg cct gcg gaa ctg gag gga tta tta gtt agt cac ccc gag 1392
Gln Val Ala Pro Ala Glu Leu Glu Gly Leu Leu Val Ser His Pro Glu
450 455 460
atc tta gac gcg gta gtt att ccc ttc ccc gat gct gag gca ggc gaa 1440
Ile Leu Asp Ala Val Val Ile Pro Phe Pro Asp Ala Glu Ala Gly Glu
465 470 475 480
gtc ccg gtg gca tac gtt gtt cgc tcg cct aac agt tcg ttg acc gaa 1488
Val Pro Val Ala Tyr Val Val Arg Ser Pro Asn Ser Ser Leu Thr Glu
485 490 495
aat gac gtt aaa aaa ttc atc gcc ggt cag gtc gcc tcc ttt aag cgt 1536
Asn Asp Val Lys Lys Phe Ile Ala Gly Gln Val Ala Ser Phe Lys Arg
500 505 510
ctg cgc aag gtt act ttt att aat tcc gtc ccc aag agc gca agt ggg 1584
Leu Arg Lys Val Thr Phe Ile Asn Ser Val Pro Lys Ser Ala Ser Gly
515 520 525
aag att ctg cgc cgc gag ctt att caa aag gtt cgc tct aac atg taa 1632
Lys Ile Leu Arg Arg Glu Leu Ile Gln Lys Val Arg Ser Asn Met
530 535 540
<210> 71
<211> 543
<212> PRT
<213> 大麻
<400> 71
Met Glu Lys Ser Gly Tyr Gly Arg Asp Gly Ile Tyr Arg Ser Leu Arg
1 5 10 15
Pro Pro Leu His Leu Pro Asn Asn Asn Asn Leu Ser Met Val Ser Phe
20 25 30
Leu Phe Arg Asn Ser Ser Ser Tyr Pro Gln Lys Pro Ala Leu Ile Asp
35 40 45
Ser Glu Thr Asn Gln Ile Leu Ser Phe Ser His Phe Lys Ser Thr Val
50 55 60
Ile Lys Val Ser His Gly Phe Leu Asn Leu Gly Ile Lys Lys Asn Asp
65 70 75 80
Val Val Leu Ile Tyr Ala Pro Asn Ser Ile His Phe Pro Val Cys Phe
85 90 95
Leu Gly Ile Ile Ala Ser Gly Ala Ile Ala Thr Thr Ser Asn Pro Leu
100 105 110
Tyr Thr Val Ser Glu Leu Ser Lys Gln Val Lys Asp Ser Asn Pro Lys
115 120 125
Leu Ile Ile Thr Val Pro Gln Leu Leu Glu Lys Val Lys Gly Phe Asn
130 135 140
Leu Pro Thr Ile Leu Ile Gly Pro Asp Ser Glu Gln Glu Ser Ser Ser
145 150 155 160
Asp Lys Val Met Thr Phe Asn Asp Leu Val Asn Leu Gly Gly Ser Ser
165 170 175
Gly Ser Glu Phe Pro Ile Val Asp Asp Phe Lys Gln Ser Asp Thr Ala
180 185 190
Ala Leu Leu Tyr Ser Ser Gly Thr Thr Gly Met Ser Lys Gly Val Val
195 200 205
Leu Thr His Lys Asn Phe Ile Ala Ser Ser Leu Met Val Thr Met Glu
210 215 220
Gln Asp Leu Val Gly Glu Met Asp Asn Val Phe Leu Cys Phe Leu Pro
225 230 235 240
Met Phe His Val Phe Gly Leu Ala Ile Ile Thr Tyr Ala Gln Leu Gln
245 250 255
Arg Gly Asn Thr Val Ile Ser Met Ala Arg Phe Asp Leu Glu Lys Met
260 265 270
Leu Lys Asp Val Glu Lys Tyr Lys Val Thr His Leu Trp Val Val Pro
275 280 285
Pro Val Ile Leu Ala Leu Ser Lys Asn Ser Met Val Lys Lys Phe Asn
290 295 300
Leu Ser Ser Ile Lys Tyr Ile Gly Ser Gly Ala Ala Pro Leu Gly Lys
305 310 315 320
Asp Leu Met Glu Glu Cys Ser Lys Val Val Pro Tyr Gly Ile Val Ala
325 330 335
Gln Gly Tyr Gly Met Thr Glu Thr Cys Gly Ile Val Ser Met Glu Asp
340 345 350
Ile Arg Gly Gly Lys Arg Asn Ser Gly Ser Ala Gly Met Leu Ala Ser
355 360 365
Gly Val Glu Ala Gln Ile Val Ser Val Asp Thr Leu Lys Pro Leu Pro
370 375 380
Pro Asn Gln Leu Gly Glu Ile Trp Val Lys Gly Pro Asn Met Met Gln
385 390 395 400
Gly Tyr Phe Asn Asn Pro Gln Ala Thr Lys Leu Thr Ile Asp Lys Lys
405 410 415
Gly Trp Val His Thr Gly Asp Leu Gly Tyr Phe Asp Glu Asp Gly His
420 425 430
Leu Tyr Val Val Asp Arg Ile Lys Glu Leu Ile Lys Tyr Lys Gly Phe
435 440 445
Gln Val Ala Pro Ala Glu Leu Glu Gly Leu Leu Val Ser His Pro Glu
450 455 460
Ile Leu Asp Ala Val Val Ile Pro Phe Pro Asp Ala Glu Ala Gly Glu
465 470 475 480
Val Pro Val Ala Tyr Val Val Arg Ser Pro Asn Ser Ser Leu Thr Glu
485 490 495
Asn Asp Val Lys Lys Phe Ile Ala Gly Gln Val Ala Ser Phe Lys Arg
500 505 510
Leu Arg Lys Val Thr Phe Ile Asn Ser Val Pro Lys Ser Ala Ser Gly
515 520 525
Lys Ile Leu Arg Arg Glu Leu Ile Gln Lys Val Arg Ser Asn Met
530 535 540
<210> 72
<211> 2163
<212> DNA
<213> 大麻
<220>
<221> CDS
<222> (1)..(2163)
<400> 72
atg ggt aag aat tac aag tcc ctg gac tct gtt gtg gcc tct gac ttc 48
Met Gly Lys Asn Tyr Lys Ser Leu Asp Ser Val Val Ala Ser Asp Phe
1 5 10 15
ata gcc cta ggt atc acc tct gaa gtt gct gag aca ctc cat ggt aga 96
Ile Ala Leu Gly Ile Thr Ser Glu Val Ala Glu Thr Leu His Gly Arg
20 25 30
ctg gcc gag atc gtg tgt aat tat ggc gct gcc act ccc caa aca tgg 144
Leu Ala Glu Ile Val Cys Asn Tyr Gly Ala Ala Thr Pro Gln Thr Trp
35 40 45
atc aat att gcc aac cat att ctg tcg cct gac ctc ccc ttc tcc ctg 192
Ile Asn Ile Ala Asn His Ile Leu Ser Pro Asp Leu Pro Phe Ser Leu
50 55 60
cac cag atg ctc ttc tat ggt tgc tat aaa gac ttt gga cct gcc cct 240
His Gln Met Leu Phe Tyr Gly Cys Tyr Lys Asp Phe Gly Pro Ala Pro
65 70 75 80
cct gct tgg ata ccc gac ccg gag aaa gta aag tcc acc aat ctg ggc 288
Pro Ala Trp Ile Pro Asp Pro Glu Lys Val Lys Ser Thr Asn Leu Gly
85 90 95
gca ctt ttg gag aag cga gga aaa gag ttt ttg gga gtc aag tat aag 336
Ala Leu Leu Glu Lys Arg Gly Lys Glu Phe Leu Gly Val Lys Tyr Lys
100 105 110
gat ccc att tca agc ttt tct cat ttc caa gaa ttt tct gta aga aac 384
Asp Pro Ile Ser Ser Phe Ser His Phe Gln Glu Phe Ser Val Arg Asn
115 120 125
cct gag gtg tat tgg aga aca gta cta atg gat gag atg aag ata agt 432
Pro Glu Val Tyr Trp Arg Thr Val Leu Met Asp Glu Met Lys Ile Ser
130 135 140
ttt tca aag gat cca gaa tgt ata ttg cgt aga gat gat att aat aat 480
Phe Ser Lys Asp Pro Glu Cys Ile Leu Arg Arg Asp Asp Ile Asn Asn
145 150 155 160
cca ggg ggt agt gaa tgg ctt cca gga ggt tat ctt aac tca gca aag 528
Pro Gly Gly Ser Glu Trp Leu Pro Gly Gly Tyr Leu Asn Ser Ala Lys
165 170 175
aat tgc ttg aat gta aat agt aac aag aaa ttg aat gat aca atg att 576
Asn Cys Leu Asn Val Asn Ser Asn Lys Lys Leu Asn Asp Thr Met Ile
180 185 190
gta tgg cgt gat gaa gga aat gat gat ttg cct cta aac aaa ttg aca 624
Val Trp Arg Asp Glu Gly Asn Asp Asp Leu Pro Leu Asn Lys Leu Thr
195 200 205
ctt gac caa ttg cgt aaa cgt gtt tgg tta gtt ggt tat gca ctt gaa 672
Leu Asp Gln Leu Arg Lys Arg Val Trp Leu Val Gly Tyr Ala Leu Glu
210 215 220
gaa atg ggt ttg gag aag ggt tgt gca att gca att gat atg cca atg 720
Glu Met Gly Leu Glu Lys Gly Cys Ala Ile Ala Ile Asp Met Pro Met
225 230 235 240
cat gtg gat gct gtg gtt atc tat cta gct att gtt ctt gcg gga tat 768
His Val Asp Ala Val Val Ile Tyr Leu Ala Ile Val Leu Ala Gly Tyr
245 250 255
gta gtt gtt tct att gct gat agt ttt tct gct cct gaa ata tca aca 816
Val Val Val Ser Ile Ala Asp Ser Phe Ser Ala Pro Glu Ile Ser Thr
260 265 270
aga ctt cga cta tca aaa gca aaa gcc att ttt aca cag gat cat att 864
Arg Leu Arg Leu Ser Lys Ala Lys Ala Ile Phe Thr Gln Asp His Ile
275 280 285
att cgt ggg aag aag cgt att ccc tta tac agt aga gtt gtg gaa gcc 912
Ile Arg Gly Lys Lys Arg Ile Pro Leu Tyr Ser Arg Val Val Glu Ala
290 295 300
aag tct ccc atg gcc att gtt att cct tgt agt ggc tct aat att ggt 960
Lys Ser Pro Met Ala Ile Val Ile Pro Cys Ser Gly Ser Asn Ile Gly
305 310 315 320
gca gaa ttg cgt gat ggc gat att tct tgg gat tac ttt cta gaa aga 1008
Ala Glu Leu Arg Asp Gly Asp Ile Ser Trp Asp Tyr Phe Leu Glu Arg
325 330 335
gca aaa gag ttt aaa aat tgt gaa ttt act gct aga gaa caa cca gtt 1056
Ala Lys Glu Phe Lys Asn Cys Glu Phe Thr Ala Arg Glu Gln Pro Val
340 345 350
gat gcc tat aca aac atc ctc ttc tca tct gga aca aca ggg gag cca 1104
Asp Ala Tyr Thr Asn Ile Leu Phe Ser Ser Gly Thr Thr Gly Glu Pro
355 360 365
aag gca att cca tgg act caa gca act cct tta aaa gca gct gca gat 1152
Lys Ala Ile Pro Trp Thr Gln Ala Thr Pro Leu Lys Ala Ala Ala Asp
370 375 380
ggg tgg agc cat ttg gac att agg aaa ggt gat gtc att gtt tgg ccc 1200
Gly Trp Ser His Leu Asp Ile Arg Lys Gly Asp Val Ile Val Trp Pro
385 390 395 400
act aat ctt ggt tgg atg atg ggt cct tgg ctg gtc tat gct tca ctc 1248
Thr Asn Leu Gly Trp Met Met Gly Pro Trp Leu Val Tyr Ala Ser Leu
405 410 415
ctt aat ggg gct tct att gcc ttg tat aat gga tca cca ctt gtt tct 1296
Leu Asn Gly Ala Ser Ile Ala Leu Tyr Asn Gly Ser Pro Leu Val Ser
420 425 430
ggc ttt gcc aaa ttt gtg cag gat gct aaa gta aca atg cta ggt gtg 1344
Gly Phe Ala Lys Phe Val Gln Asp Ala Lys Val Thr Met Leu Gly Val
435 440 445
gtc cct agt att gtt cga tca tgg aaa agt acc aat tgt gtt agt ggc 1392
Val Pro Ser Ile Val Arg Ser Trp Lys Ser Thr Asn Cys Val Ser Gly
450 455 460
tat gat tgg tcc acc atc cgt tgc ttt tcc tct tct ggt gaa gca tct 1440
Tyr Asp Trp Ser Thr Ile Arg Cys Phe Ser Ser Ser Gly Glu Ala Ser
465 470 475 480
aat gta gat gaa tac cta tgg ttg atg ggg aga gca aac tac aag cct 1488
Asn Val Asp Glu Tyr Leu Trp Leu Met Gly Arg Ala Asn Tyr Lys Pro
485 490 495
gtt atc gaa atg tgt ggt ggc aca gaa att ggt ggt gca ttt tct gct 1536
Val Ile Glu Met Cys Gly Gly Thr Glu Ile Gly Gly Ala Phe Ser Ala
500 505 510
ggc tct ttc tta caa gct caa tca tta tct tca ttt agt tca caa tgt 1584
Gly Ser Phe Leu Gln Ala Gln Ser Leu Ser Ser Phe Ser Ser Gln Cys
515 520 525
atg ggt tgc act tta tac ata ctt gac aag aat ggt tat cca atg cct 1632
Met Gly Cys Thr Leu Tyr Ile Leu Asp Lys Asn Gly Tyr Pro Met Pro
530 535 540
aaa aac aaa cca gga att ggt gaa tta gcg ctt ggt cca gtc atg ttt 1680
Lys Asn Lys Pro Gly Ile Gly Glu Leu Ala Leu Gly Pro Val Met Phe
545 550 555 560
gga gca tcg aag act ctg ttg aat ggt aat cac cat gat gtt tat ttt 1728
Gly Ala Ser Lys Thr Leu Leu Asn Gly Asn His His Asp Val Tyr Phe
565 570 575
aag gga atg cct aca ttg aat gga gag gtt tta agg agg cat ggg gac 1776
Lys Gly Met Pro Thr Leu Asn Gly Glu Val Leu Arg Arg His Gly Asp
580 585 590
att ttt gag ctt aca tct aat ggt tat tat cat gca cat ggt cgt gca 1824
Ile Phe Glu Leu Thr Ser Asn Gly Tyr Tyr His Ala His Gly Arg Ala
595 600 605
gat gat aca atg aat att gga ggc atc aag att agt tcc ata gag att 1872
Asp Asp Thr Met Asn Ile Gly Gly Ile Lys Ile Ser Ser Ile Glu Ile
610 615 620
gaa cga gtt tgt aat gaa gtt gat gac aga gtt ttc gag aca act gct 1920
Glu Arg Val Cys Asn Glu Val Asp Asp Arg Val Phe Glu Thr Thr Ala
625 630 635 640
att gga gtg cca cct ttg ggc ggt gga cct gag caa tta gta att ttc 1968
Ile Gly Val Pro Pro Leu Gly Gly Gly Pro Glu Gln Leu Val Ile Phe
645 650 655
ttt gta tta aaa gat tca aat gat aca act att gac tta aat caa ttg 2016
Phe Val Leu Lys Asp Ser Asn Asp Thr Thr Ile Asp Leu Asn Gln Leu
660 665 670
agg tta tct ttc aac ttg ggt tta cag aag aaa cta aat cct ctg ttc 2064
Arg Leu Ser Phe Asn Leu Gly Leu Gln Lys Lys Leu Asn Pro Leu Phe
675 680 685
aag gtc act cgt gtt gtg cct ctt tca tca ctt ccg aga aca gca acc 2112
Lys Val Thr Arg Val Val Pro Leu Ser Ser Leu Pro Arg Thr Ala Thr
690 695 700
aac aag atc atg aga agg gtt ttg cgc cag caa ttt tct cac ttt gaa 2160
Asn Lys Ile Met Arg Arg Val Leu Arg Gln Gln Phe Ser His Phe Glu
705 710 715 720
tga 2163
<210> 73
<211> 720
<212> PRT
<213> 大麻
<400> 73
Met Gly Lys Asn Tyr Lys Ser Leu Asp Ser Val Val Ala Ser Asp Phe
1 5 10 15
Ile Ala Leu Gly Ile Thr Ser Glu Val Ala Glu Thr Leu His Gly Arg
20 25 30
Leu Ala Glu Ile Val Cys Asn Tyr Gly Ala Ala Thr Pro Gln Thr Trp
35 40 45
Ile Asn Ile Ala Asn His Ile Leu Ser Pro Asp Leu Pro Phe Ser Leu
50 55 60
His Gln Met Leu Phe Tyr Gly Cys Tyr Lys Asp Phe Gly Pro Ala Pro
65 70 75 80
Pro Ala Trp Ile Pro Asp Pro Glu Lys Val Lys Ser Thr Asn Leu Gly
85 90 95
Ala Leu Leu Glu Lys Arg Gly Lys Glu Phe Leu Gly Val Lys Tyr Lys
100 105 110
Asp Pro Ile Ser Ser Phe Ser His Phe Gln Glu Phe Ser Val Arg Asn
115 120 125
Pro Glu Val Tyr Trp Arg Thr Val Leu Met Asp Glu Met Lys Ile Ser
130 135 140
Phe Ser Lys Asp Pro Glu Cys Ile Leu Arg Arg Asp Asp Ile Asn Asn
145 150 155 160
Pro Gly Gly Ser Glu Trp Leu Pro Gly Gly Tyr Leu Asn Ser Ala Lys
165 170 175
Asn Cys Leu Asn Val Asn Ser Asn Lys Lys Leu Asn Asp Thr Met Ile
180 185 190
Val Trp Arg Asp Glu Gly Asn Asp Asp Leu Pro Leu Asn Lys Leu Thr
195 200 205
Leu Asp Gln Leu Arg Lys Arg Val Trp Leu Val Gly Tyr Ala Leu Glu
210 215 220
Glu Met Gly Leu Glu Lys Gly Cys Ala Ile Ala Ile Asp Met Pro Met
225 230 235 240
His Val Asp Ala Val Val Ile Tyr Leu Ala Ile Val Leu Ala Gly Tyr
245 250 255
Val Val Val Ser Ile Ala Asp Ser Phe Ser Ala Pro Glu Ile Ser Thr
260 265 270
Arg Leu Arg Leu Ser Lys Ala Lys Ala Ile Phe Thr Gln Asp His Ile
275 280 285
Ile Arg Gly Lys Lys Arg Ile Pro Leu Tyr Ser Arg Val Val Glu Ala
290 295 300
Lys Ser Pro Met Ala Ile Val Ile Pro Cys Ser Gly Ser Asn Ile Gly
305 310 315 320
Ala Glu Leu Arg Asp Gly Asp Ile Ser Trp Asp Tyr Phe Leu Glu Arg
325 330 335
Ala Lys Glu Phe Lys Asn Cys Glu Phe Thr Ala Arg Glu Gln Pro Val
340 345 350
Asp Ala Tyr Thr Asn Ile Leu Phe Ser Ser Gly Thr Thr Gly Glu Pro
355 360 365
Lys Ala Ile Pro Trp Thr Gln Ala Thr Pro Leu Lys Ala Ala Ala Asp
370 375 380
Gly Trp Ser His Leu Asp Ile Arg Lys Gly Asp Val Ile Val Trp Pro
385 390 395 400
Thr Asn Leu Gly Trp Met Met Gly Pro Trp Leu Val Tyr Ala Ser Leu
405 410 415
Leu Asn Gly Ala Ser Ile Ala Leu Tyr Asn Gly Ser Pro Leu Val Ser
420 425 430
Gly Phe Ala Lys Phe Val Gln Asp Ala Lys Val Thr Met Leu Gly Val
435 440 445
Val Pro Ser Ile Val Arg Ser Trp Lys Ser Thr Asn Cys Val Ser Gly
450 455 460
Tyr Asp Trp Ser Thr Ile Arg Cys Phe Ser Ser Ser Gly Glu Ala Ser
465 470 475 480
Asn Val Asp Glu Tyr Leu Trp Leu Met Gly Arg Ala Asn Tyr Lys Pro
485 490 495
Val Ile Glu Met Cys Gly Gly Thr Glu Ile Gly Gly Ala Phe Ser Ala
500 505 510
Gly Ser Phe Leu Gln Ala Gln Ser Leu Ser Ser Phe Ser Ser Gln Cys
515 520 525
Met Gly Cys Thr Leu Tyr Ile Leu Asp Lys Asn Gly Tyr Pro Met Pro
530 535 540
Lys Asn Lys Pro Gly Ile Gly Glu Leu Ala Leu Gly Pro Val Met Phe
545 550 555 560
Gly Ala Ser Lys Thr Leu Leu Asn Gly Asn His His Asp Val Tyr Phe
565 570 575
Lys Gly Met Pro Thr Leu Asn Gly Glu Val Leu Arg Arg His Gly Asp
580 585 590
Ile Phe Glu Leu Thr Ser Asn Gly Tyr Tyr His Ala His Gly Arg Ala
595 600 605
Asp Asp Thr Met Asn Ile Gly Gly Ile Lys Ile Ser Ser Ile Glu Ile
610 615 620
Glu Arg Val Cys Asn Glu Val Asp Asp Arg Val Phe Glu Thr Thr Ala
625 630 635 640
Ile Gly Val Pro Pro Leu Gly Gly Gly Pro Glu Gln Leu Val Ile Phe
645 650 655
Phe Val Leu Lys Asp Ser Asn Asp Thr Thr Ile Asp Leu Asn Gln Leu
660 665 670
Arg Leu Ser Phe Asn Leu Gly Leu Gln Lys Lys Leu Asn Pro Leu Phe
675 680 685
Lys Val Thr Arg Val Val Pro Leu Ser Ser Leu Pro Arg Thr Ala Thr
690 695 700
Asn Lys Ile Met Arg Arg Val Leu Arg Gln Gln Phe Ser His Phe Glu
705 710 715 720
<210> 74
<211> 1695
<212> DNA
<213> 大麻
<220>
<221> CDS
<222> (1)..(1695)
<400> 74
atg gaa gta ctg aag gag gtt gcg aag gaa ggt agc gca gcc cgt gaa 48
Met Glu Val Leu Lys Glu Val Ala Lys Glu Gly Ser Ala Ala Arg Glu
1 5 10 15
ggt gtc gct att cgc gcc gac cag aaa tcg tac agc tat aag caa ttg 96
Gly Val Ala Ile Arg Ala Asp Gln Lys Ser Tyr Ser Tyr Lys Gln Leu
20 25 30
atc tcc tcc gcg cag tcg atc tgc tca ctg tta tgc ggt act gaa ctt 144
Ile Ser Ser Ala Gln Ser Ile Cys Ser Leu Leu Cys Gly Thr Glu Leu
35 40 45
aaa gcg att cac gaa gcc ggg aaa caa gct cgt cct agc gcg tct atc 192
Lys Ala Ile His Glu Ala Gly Lys Gln Ala Arg Pro Ser Ala Ser Ile
50 55 60
aat ggg gcc ggg ggt cac ggc cac ttg gga gga gct cgt att gga att 240
Asn Gly Ala Gly Gly His Gly His Leu Gly Gly Ala Arg Ile Gly Ile
65 70 75 80
gtt gct aag ccg tcg gca gaa ttt gta gcc ggt gtt tta ggt acg tgg 288
Val Ala Lys Pro Ser Ala Glu Phe Val Ala Gly Val Leu Gly Thr Trp
85 90 95
tta tct ggt gga gtt gcg gtt ccc ctt gca ctg tct tac ccg gag gcg 336
Leu Ser Gly Gly Val Ala Val Pro Leu Ala Leu Ser Tyr Pro Glu Ala
100 105 110
gaa tta ctg cat gtc atg aac gat tca gat atc agc atg atc ttg agc 384
Glu Leu Leu His Val Met Asn Asp Ser Asp Ile Ser Met Ile Leu Ser
115 120 125
acg gaa gac cat caa gaa ctg atg caa aat att gct gcc aag act tcc 432
Thr Glu Asp His Gln Glu Leu Met Gln Asn Ile Ala Ala Lys Thr Ser
130 135 140
gca cag ttt tcc tta att cca tct gtg ccg tcg tcg tgc tca caa gaa 480
Ala Gln Phe Ser Leu Ile Pro Ser Val Pro Ser Ser Cys Ser Gln Glu
145 150 155 160
gta gcg gtc gat cat cgt cag acc ggt gac atc tct acc gac tct atc 528
Val Ala Val Asp His Arg Gln Thr Gly Asp Ile Ser Thr Asp Ser Ile
165 170 175
ttg ctt aac cgc gag atc tct aac gag aat ccc gca ctt atc gtc tat 576
Leu Leu Asn Arg Glu Ile Ser Asn Glu Asn Pro Ala Leu Ile Val Tyr
180 185 190
acg tcg ggg acg aca ggc aag ccg aag ggc gtc gtt cac aca cac caa 624
Thr Ser Gly Thr Thr Gly Lys Pro Lys Gly Val Val His Thr His Gln
195 200 205
tca att tct gca cag gtt cag acg tta gcc aag gca tgg gag tat act 672
Ser Ile Ser Ala Gln Val Gln Thr Leu Ala Lys Ala Trp Glu Tyr Thr
210 215 220
cct gcc gat caa ttc tta cac tgc tta ccg ctg cat cat gtg cat ggg 720
Pro Ala Asp Gln Phe Leu His Cys Leu Pro Leu His His Val His Gly
225 230 235 240
ctg ttt aac gca ctg ttc gcg ccc ctt tac gcg cgt tca aca gtt gaa 768
Leu Phe Asn Ala Leu Phe Ala Pro Leu Tyr Ala Arg Ser Thr Val Glu
245 250 255
ttt ctg ccg aaa ttt tct gtc cgc ggt att tgg caa cgc tgg cgc gaa 816
Phe Leu Pro Lys Phe Ser Val Arg Gly Ile Trp Gln Arg Trp Arg Glu
260 265 270
tcc tac cca acg tca gag acg aaa gcc aat gac tgc att acg gta ttt 864
Ser Tyr Pro Thr Ser Glu Thr Lys Ala Asn Asp Cys Ile Thr Val Phe
275 280 285
aca gga gtt ccc acc atg tac acg cgt ctg att caa gga tat gaa gct 912
Thr Gly Val Pro Thr Met Tyr Thr Arg Leu Ile Gln Gly Tyr Glu Ala
290 295 300
atg gat cca gag tta aaa gag gcc tct gca tct gct gct aag cag ctg 960
Met Asp Pro Glu Leu Lys Glu Ala Ser Ala Ser Ala Ala Lys Gln Leu
305 310 315 320
cgc ctt atg atg tgt ggt tcc tct gcg ctg cca gtt cct gtc atg cag 1008
Arg Leu Met Met Cys Gly Ser Ser Ala Leu Pro Val Pro Val Met Gln
325 330 335
cag tgg caa acc atc acc ggc cac cgt ctt ctg gaa cgt tac gga atg 1056
Gln Trp Gln Thr Ile Thr Gly His Arg Leu Leu Glu Arg Tyr Gly Met
340 345 350
acc gaa ttt gtc atg gca att tct aac ccc ttg aaa ggt gag cgc aaa 1104
Thr Glu Phe Val Met Ala Ile Ser Asn Pro Leu Lys Gly Glu Arg Lys
355 360 365
tcc ggt act gtc gga aag ccg ttt cca ggt gta gag gtg cgc att tta 1152
Ser Gly Thr Val Gly Lys Pro Phe Pro Gly Val Glu Val Arg Ile Leu
370 375 380
gca gag gat gaa aac ggc gat gat gct acc ggg gtg gga gag ctg tgc 1200
Ala Glu Asp Glu Asn Gly Asp Asp Ala Thr Gly Val Gly Glu Leu Cys
385 390 395 400
gta cgc agt ccg tcc ctt ttc aaa gag tat tgg cgt ttg ccc gag gtc 1248
Val Arg Ser Pro Ser Leu Phe Lys Glu Tyr Trp Arg Leu Pro Glu Val
405 410 415
aca aaa gcc tcc ttt aca gac gac ggc ttt ttc aaa acc ggc gac gca 1296
Thr Lys Ala Ser Phe Thr Asp Asp Gly Phe Phe Lys Thr Gly Asp Ala
420 425 430
ggc aag gtc gat gag gac ggt tac tac gtg att ctg ggc cgt act agc 1344
Gly Lys Val Asp Glu Asp Gly Tyr Tyr Val Ile Leu Gly Arg Thr Ser
435 440 445
gca gat att atg aaa gtt gga ggc tat aag ctg tct gct ctg gaa atc 1392
Ala Asp Ile Met Lys Val Gly Gly Tyr Lys Leu Ser Ala Leu Glu Ile
450 455 460
gag tcg gtc ctt ctg gaa cac ccg act gtc gag gaa tgc tgt gtc ttg 1440
Glu Ser Val Leu Leu Glu His Pro Thr Val Glu Glu Cys Cys Val Leu
465 470 475 480
gga ctt ccc gac aag gat tat ggg gaa gcc gta tcc gca atc att gta 1488
Gly Leu Pro Asp Lys Asp Tyr Gly Glu Ala Val Ser Ala Ile Ile Val
485 490 495
ccg gca gcc gag gcg aag aag aaa cgc gaa gag gag tca cgc ccc gcc 1536
Pro Ala Ala Glu Ala Lys Lys Lys Arg Glu Glu Glu Ser Arg Pro Ala
500 505 510
att agt ctg gag gaa ctg ttc tca tgg gca cag cac aaa ctt gcc ccc 1584
Ile Ser Leu Glu Glu Leu Phe Ser Trp Ala Gln His Lys Leu Ala Pro
515 520 525
tac aaa ctg ccc acg cgt tta ttc ctg tgg gac tct tta cct cgc aac 1632
Tyr Lys Leu Pro Thr Arg Leu Phe Leu Trp Asp Ser Leu Pro Arg Asn
530 535 540
gca atg ggg aaa gtc aac aaa aaa gag ctg aag aaa aaa ctg aca gtt 1680
Ala Met Gly Lys Val Asn Lys Lys Glu Leu Lys Lys Lys Leu Thr Val
545 550 555 560
gag caa ggt att taa 1695
Glu Gln Gly Ile
<210> 75
<211> 564
<212> PRT
<213> 大麻
<400> 75
Met Glu Val Leu Lys Glu Val Ala Lys Glu Gly Ser Ala Ala Arg Glu
1 5 10 15
Gly Val Ala Ile Arg Ala Asp Gln Lys Ser Tyr Ser Tyr Lys Gln Leu
20 25 30
Ile Ser Ser Ala Gln Ser Ile Cys Ser Leu Leu Cys Gly Thr Glu Leu
35 40 45
Lys Ala Ile His Glu Ala Gly Lys Gln Ala Arg Pro Ser Ala Ser Ile
50 55 60
Asn Gly Ala Gly Gly His Gly His Leu Gly Gly Ala Arg Ile Gly Ile
65 70 75 80
Val Ala Lys Pro Ser Ala Glu Phe Val Ala Gly Val Leu Gly Thr Trp
85 90 95
Leu Ser Gly Gly Val Ala Val Pro Leu Ala Leu Ser Tyr Pro Glu Ala
100 105 110
Glu Leu Leu His Val Met Asn Asp Ser Asp Ile Ser Met Ile Leu Ser
115 120 125
Thr Glu Asp His Gln Glu Leu Met Gln Asn Ile Ala Ala Lys Thr Ser
130 135 140
Ala Gln Phe Ser Leu Ile Pro Ser Val Pro Ser Ser Cys Ser Gln Glu
145 150 155 160
Val Ala Val Asp His Arg Gln Thr Gly Asp Ile Ser Thr Asp Ser Ile
165 170 175
Leu Leu Asn Arg Glu Ile Ser Asn Glu Asn Pro Ala Leu Ile Val Tyr
180 185 190
Thr Ser Gly Thr Thr Gly Lys Pro Lys Gly Val Val His Thr His Gln
195 200 205
Ser Ile Ser Ala Gln Val Gln Thr Leu Ala Lys Ala Trp Glu Tyr Thr
210 215 220
Pro Ala Asp Gln Phe Leu His Cys Leu Pro Leu His His Val His Gly
225 230 235 240
Leu Phe Asn Ala Leu Phe Ala Pro Leu Tyr Ala Arg Ser Thr Val Glu
245 250 255
Phe Leu Pro Lys Phe Ser Val Arg Gly Ile Trp Gln Arg Trp Arg Glu
260 265 270
Ser Tyr Pro Thr Ser Glu Thr Lys Ala Asn Asp Cys Ile Thr Val Phe
275 280 285
Thr Gly Val Pro Thr Met Tyr Thr Arg Leu Ile Gln Gly Tyr Glu Ala
290 295 300
Met Asp Pro Glu Leu Lys Glu Ala Ser Ala Ser Ala Ala Lys Gln Leu
305 310 315 320
Arg Leu Met Met Cys Gly Ser Ser Ala Leu Pro Val Pro Val Met Gln
325 330 335
Gln Trp Gln Thr Ile Thr Gly His Arg Leu Leu Glu Arg Tyr Gly Met
340 345 350
Thr Glu Phe Val Met Ala Ile Ser Asn Pro Leu Lys Gly Glu Arg Lys
355 360 365
Ser Gly Thr Val Gly Lys Pro Phe Pro Gly Val Glu Val Arg Ile Leu
370 375 380
Ala Glu Asp Glu Asn Gly Asp Asp Ala Thr Gly Val Gly Glu Leu Cys
385 390 395 400
Val Arg Ser Pro Ser Leu Phe Lys Glu Tyr Trp Arg Leu Pro Glu Val
405 410 415
Thr Lys Ala Ser Phe Thr Asp Asp Gly Phe Phe Lys Thr Gly Asp Ala
420 425 430
Gly Lys Val Asp Glu Asp Gly Tyr Tyr Val Ile Leu Gly Arg Thr Ser
435 440 445
Ala Asp Ile Met Lys Val Gly Gly Tyr Lys Leu Ser Ala Leu Glu Ile
450 455 460
Glu Ser Val Leu Leu Glu His Pro Thr Val Glu Glu Cys Cys Val Leu
465 470 475 480
Gly Leu Pro Asp Lys Asp Tyr Gly Glu Ala Val Ser Ala Ile Ile Val
485 490 495
Pro Ala Ala Glu Ala Lys Lys Lys Arg Glu Glu Glu Ser Arg Pro Ala
500 505 510
Ile Ser Leu Glu Glu Leu Phe Ser Trp Ala Gln His Lys Leu Ala Pro
515 520 525
Tyr Lys Leu Pro Thr Arg Leu Phe Leu Trp Asp Ser Leu Pro Arg Asn
530 535 540
Ala Met Gly Lys Val Asn Lys Lys Glu Leu Lys Lys Lys Leu Thr Val
545 550 555 560
Glu Gln Gly Ile
<210> 76
<211> 1158
<212> DNA
<213> 大麻
<220>
<221> CDS
<222> (1)..(1158)
<400> 76
atg aat cat ctg cgt gct gaa gga cca gct tcc gta ttg gca att gga 48
Met Asn His Leu Arg Ala Glu Gly Pro Ala Ser Val Leu Ala Ile Gly
1 5 10 15
aca gct aac cct gag aac att ctt ctt cag gat gag ttt ccc gac tat 96
Thr Ala Asn Pro Glu Asn Ile Leu Leu Gln Asp Glu Phe Pro Asp Tyr
20 25 30
tac ttc cgc gtg aca aag agc gaa cac atg aca cag ctt aaa gag aag 144
Tyr Phe Arg Val Thr Lys Ser Glu His Met Thr Gln Leu Lys Glu Lys
35 40 45
ttc cgt aag atc tgt gac aaa agc atg atc cgc aaa cgt aac tgc ttc 192
Phe Arg Lys Ile Cys Asp Lys Ser Met Ile Arg Lys Arg Asn Cys Phe
50 55 60
ctt aac gag gag cat ctg aag cag aat ccc cgt ctt gtt gaa cat gag 240
Leu Asn Glu Glu His Leu Lys Gln Asn Pro Arg Leu Val Glu His Glu
65 70 75 80
atg cag acc ttg gat gct cgc cag gac atg ttg gtt gtt gag gtc cct 288
Met Gln Thr Leu Asp Ala Arg Gln Asp Met Leu Val Val Glu Val Pro
85 90 95
aag ctg ggc aaa gat gcg tgt gca aaa gcg att aaa gag tgg ggg cag 336
Lys Leu Gly Lys Asp Ala Cys Ala Lys Ala Ile Lys Glu Trp Gly Gln
100 105 110
cct aaa agc aaa att act cat ctg att ttc aca agc gcc agt aca acc 384
Pro Lys Ser Lys Ile Thr His Leu Ile Phe Thr Ser Ala Ser Thr Thr
115 120 125
gat atg ccc ggt gcg gac tac cat tgt gca aaa tta ttg ggt tta tcg 432
Asp Met Pro Gly Ala Asp Tyr His Cys Ala Lys Leu Leu Gly Leu Ser
130 135 140
cct tca gta aaa cgt gtt atg atg tac cag tta gga tgc tac ggt ggt 480
Pro Ser Val Lys Arg Val Met Met Tyr Gln Leu Gly Cys Tyr Gly Gly
145 150 155 160
ggc acc gta ctt cgt att gcg aag gac atc gcc gag aac aac aaa gga 528
Gly Thr Val Leu Arg Ile Ala Lys Asp Ile Ala Glu Asn Asn Lys Gly
165 170 175
gcc cgt gta ctt gct gta tgt tgt gat atc atg gcg tgc ctt ttt cgc 576
Ala Arg Val Leu Ala Val Cys Cys Asp Ile Met Ala Cys Leu Phe Arg
180 185 190
ggc ccc agc gag agt gac ctt gag tta ctt gtg ggg cag gcc atc ttc 624
Gly Pro Ser Glu Ser Asp Leu Glu Leu Leu Val Gly Gln Ala Ile Phe
195 200 205
gga gac ggt gcc gca gcc gtc att gtt ggc gca gag ccc gat gaa tcc 672
Gly Asp Gly Ala Ala Ala Val Ile Val Gly Ala Glu Pro Asp Glu Ser
210 215 220
gtt ggc gag cgc ccg atc ttt gag ctt gta agt aca gga caa act atc 720
Val Gly Glu Arg Pro Ile Phe Glu Leu Val Ser Thr Gly Gln Thr Ile
225 230 235 240
ttg ccc aac tct gag ggg act atc ggc gga cat att cgt gag gcg ggc 768
Leu Pro Asn Ser Glu Gly Thr Ile Gly Gly His Ile Arg Glu Ala Gly
245 250 255
ttg att ttt gac ctt cac aag gat gtt cca atg ctt atc tcc aat aat 816
Leu Ile Phe Asp Leu His Lys Asp Val Pro Met Leu Ile Ser Asn Asn
260 265 270
att gaa aaa tgt ctt atc gaa gca ttc act ccg att ggt atc tcc gat 864
Ile Glu Lys Cys Leu Ile Glu Ala Phe Thr Pro Ile Gly Ile Ser Asp
275 280 285
tgg aat tcg att ttt tgg atc acc cat cct ggt ggg aaa gct att tta 912
Trp Asn Ser Ile Phe Trp Ile Thr His Pro Gly Gly Lys Ala Ile Leu
290 295 300
gac aag gtg gag gag aaa tta cat ctt aag tca gat aag ttt gtc gac 960
Asp Lys Val Glu Glu Lys Leu His Leu Lys Ser Asp Lys Phe Val Asp
305 310 315 320
agt cgc cac gtg ttg tcg gaa cat ggc aac atg tca tcg tca acc gtc 1008
Ser Arg His Val Leu Ser Glu His Gly Asn Met Ser Ser Ser Thr Val
325 330 335
ttg ttc gtt atg gac gaa tta cgt aaa cgc agt tta gaa gag ggt aag 1056
Leu Phe Val Met Asp Glu Leu Arg Lys Arg Ser Leu Glu Glu Gly Lys
340 345 350
agt acg acg ggg gac ggg ttc gag tgg gga gtc tta ttc ggg ttc ggt 1104
Ser Thr Thr Gly Asp Gly Phe Glu Trp Gly Val Leu Phe Gly Phe Gly
355 360 365
cca gga ttg aca gtg gaa cgc gtc gtg gtt cgc agt gtc ccc att aag 1152
Pro Gly Leu Thr Val Glu Arg Val Val Val Arg Ser Val Pro Ile Lys
370 375 380
tac taa 1158
Tyr
385
<210> 77
<211> 385
<212> PRT
<213> 大麻
<400> 77
Met Asn His Leu Arg Ala Glu Gly Pro Ala Ser Val Leu Ala Ile Gly
1 5 10 15
Thr Ala Asn Pro Glu Asn Ile Leu Leu Gln Asp Glu Phe Pro Asp Tyr
20 25 30
Tyr Phe Arg Val Thr Lys Ser Glu His Met Thr Gln Leu Lys Glu Lys
35 40 45
Phe Arg Lys Ile Cys Asp Lys Ser Met Ile Arg Lys Arg Asn Cys Phe
50 55 60
Leu Asn Glu Glu His Leu Lys Gln Asn Pro Arg Leu Val Glu His Glu
65 70 75 80
Met Gln Thr Leu Asp Ala Arg Gln Asp Met Leu Val Val Glu Val Pro
85 90 95
Lys Leu Gly Lys Asp Ala Cys Ala Lys Ala Ile Lys Glu Trp Gly Gln
100 105 110
Pro Lys Ser Lys Ile Thr His Leu Ile Phe Thr Ser Ala Ser Thr Thr
115 120 125
Asp Met Pro Gly Ala Asp Tyr His Cys Ala Lys Leu Leu Gly Leu Ser
130 135 140
Pro Ser Val Lys Arg Val Met Met Tyr Gln Leu Gly Cys Tyr Gly Gly
145 150 155 160
Gly Thr Val Leu Arg Ile Ala Lys Asp Ile Ala Glu Asn Asn Lys Gly
165 170 175
Ala Arg Val Leu Ala Val Cys Cys Asp Ile Met Ala Cys Leu Phe Arg
180 185 190
Gly Pro Ser Glu Ser Asp Leu Glu Leu Leu Val Gly Gln Ala Ile Phe
195 200 205
Gly Asp Gly Ala Ala Ala Val Ile Val Gly Ala Glu Pro Asp Glu Ser
210 215 220
Val Gly Glu Arg Pro Ile Phe Glu Leu Val Ser Thr Gly Gln Thr Ile
225 230 235 240
Leu Pro Asn Ser Glu Gly Thr Ile Gly Gly His Ile Arg Glu Ala Gly
245 250 255
Leu Ile Phe Asp Leu His Lys Asp Val Pro Met Leu Ile Ser Asn Asn
260 265 270
Ile Glu Lys Cys Leu Ile Glu Ala Phe Thr Pro Ile Gly Ile Ser Asp
275 280 285
Trp Asn Ser Ile Phe Trp Ile Thr His Pro Gly Gly Lys Ala Ile Leu
290 295 300
Asp Lys Val Glu Glu Lys Leu His Leu Lys Ser Asp Lys Phe Val Asp
305 310 315 320
Ser Arg His Val Leu Ser Glu His Gly Asn Met Ser Ser Ser Thr Val
325 330 335
Leu Phe Val Met Asp Glu Leu Arg Lys Arg Ser Leu Glu Glu Gly Lys
340 345 350
Ser Thr Thr Gly Asp Gly Phe Glu Trp Gly Val Leu Phe Gly Phe Gly
355 360 365
Pro Gly Leu Thr Val Glu Arg Val Val Val Arg Ser Val Pro Ile Lys
370 375 380
Tyr
385
<210> 78
<211> 306
<212> DNA
<213> 大麻
<220>
<221> CDS
<222> (1)..(306)
<400> 78
atg gca gtc aaa cac ttg atc gtg tta aag ttc aaa gat gaa atc aca 48
Met Ala Val Lys His Leu Ile Val Leu Lys Phe Lys Asp Glu Ile Thr
1 5 10 15
gag gct cag aag gaa gaa ttt ttc aag acg tat gta aac ctt gtt aat 96
Glu Ala Gln Lys Glu Glu Phe Phe Lys Thr Tyr Val Asn Leu Val Asn
20 25 30
atc atc ccc gct atg aag gat gtg tat tgg ggt aaa gac gtg aca cag 144
Ile Ile Pro Ala Met Lys Asp Val Tyr Trp Gly Lys Asp Val Thr Gln
35 40 45
aag aac aaa gag gaa ggc tac acg cac atc gta gag gtc aca ttt gag 192
Lys Asn Lys Glu Glu Gly Tyr Thr His Ile Val Glu Val Thr Phe Glu
50 55 60
agc gtc gaa act att cag gat tac atc att cat ccc gca cac gtt gga 240
Ser Val Glu Thr Ile Gln Asp Tyr Ile Ile His Pro Ala His Val Gly
65 70 75 80
ttc ggg gat gtg tat cgc tct ttc tgg gaa aaa ttg ctg atc ttc gac 288
Phe Gly Asp Val Tyr Arg Ser Phe Trp Glu Lys Leu Leu Ile Phe Asp
85 90 95
tat aca ccg cgt aag taa 306
Tyr Thr Pro Arg Lys
100
<210> 79
<211> 101
<212> PRT
<213> 大麻
<400> 79
Met Ala Val Lys His Leu Ile Val Leu Lys Phe Lys Asp Glu Ile Thr
1 5 10 15
Glu Ala Gln Lys Glu Glu Phe Phe Lys Thr Tyr Val Asn Leu Val Asn
20 25 30
Ile Ile Pro Ala Met Lys Asp Val Tyr Trp Gly Lys Asp Val Thr Gln
35 40 45
Lys Asn Lys Glu Glu Gly Tyr Thr His Ile Val Glu Val Thr Phe Glu
50 55 60
Ser Val Glu Thr Ile Gln Asp Tyr Ile Ile His Pro Ala His Val Gly
65 70 75 80
Phe Gly Asp Val Tyr Arg Ser Phe Trp Glu Lys Leu Leu Ile Phe Asp
85 90 95
Tyr Thr Pro Arg Lys
100
<210> 80
<211> 654
<212> DNA
<213> 嗜热脱氮土芽孢杆菌
<220>
<221> CDS
<222> (1)..(654)
<400> 80
atg aat tta gtg ctg atg ggg ctg cca ggt gcc ggc aaa ggc acg caa 48
Met Asn Leu Val Leu Met Gly Leu Pro Gly Ala Gly Lys Gly Thr Gln
1 5 10 15
gcc gag aaa atc gta gaa aca tat gga atc cca cat att tca acc ggg 96
Ala Glu Lys Ile Val Glu Thr Tyr Gly Ile Pro His Ile Ser Thr Gly
20 25 30
gat atg ttt cgg gcg gcg atg aaa gaa ggc aca ccg tta gga ttg cag 144
Asp Met Phe Arg Ala Ala Met Lys Glu Gly Thr Pro Leu Gly Leu Gln
35 40 45
gca aaa gaa tat atc gac cgt ggt gat ctt gtt ccg gat gag gtg acg 192
Ala Lys Glu Tyr Ile Asp Arg Gly Asp Leu Val Pro Asp Glu Val Thr
50 55 60
atc ggt atc gtc cgt gaa cgg tta agc aaa gac gac tgc caa aac ggc 240
Ile Gly Ile Val Arg Glu Arg Leu Ser Lys Asp Asp Cys Gln Asn Gly
65 70 75 80
ttt ttg ctt gac gga ttc cca cgc acg gtt gcc caa gcg gag gcg ctg 288
Phe Leu Leu Asp Gly Phe Pro Arg Thr Val Ala Gln Ala Glu Ala Leu
85 90 95
gaa gcg atg ctg gct gaa atc ggc cgc aag ctt gac tat gtc atc cat 336
Glu Ala Met Leu Ala Glu Ile Gly Arg Lys Leu Asp Tyr Val Ile His
100 105 110
atc gat gtt cgc caa gat gtg tta atg gag cgc ctc aca ggc aga cga 384
Ile Asp Val Arg Gln Asp Val Leu Met Glu Arg Leu Thr Gly Arg Arg
115 120 125
att tgt cgc aac tgc gga gcg aca tac cat ctt gtt ttt cac cca ccg 432
Ile Cys Arg Asn Cys Gly Ala Thr Tyr His Leu Val Phe His Pro Pro
130 135 140
gct cag cca ggc gta tgt gat aaa tgc ggt ggc gag ctt tat cag cgc 480
Ala Gln Pro Gly Val Cys Asp Lys Cys Gly Gly Glu Leu Tyr Gln Arg
145 150 155 160
cct gac gat aat gaa gca aca gtg gcg aat cgg ctt gag gtg aat acg 528
Pro Asp Asp Asn Glu Ala Thr Val Ala Asn Arg Leu Glu Val Asn Thr
165 170 175
aaa caa atg aag cca ttg ctc gat ttc tat gag caa aaa ggc tat ttg 576
Lys Gln Met Lys Pro Leu Leu Asp Phe Tyr Glu Gln Lys Gly Tyr Leu
180 185 190
cgt cac att aac ggc gaa caa gaa atg gaa aaa gtg ttt agc gac att 624
Arg His Ile Asn Gly Glu Gln Glu Met Glu Lys Val Phe Ser Asp Ile
195 200 205
cgc gaa ttg ctc ggg gga ctt act cga taa 654
Arg Glu Leu Leu Gly Gly Leu Thr Arg
210 215
<210> 81
<211> 217
<212> PRT
<213> 嗜热脱氮土芽孢杆菌
<400> 81
Met Asn Leu Val Leu Met Gly Leu Pro Gly Ala Gly Lys Gly Thr Gln
1 5 10 15
Ala Glu Lys Ile Val Glu Thr Tyr Gly Ile Pro His Ile Ser Thr Gly
20 25 30
Asp Met Phe Arg Ala Ala Met Lys Glu Gly Thr Pro Leu Gly Leu Gln
35 40 45
Ala Lys Glu Tyr Ile Asp Arg Gly Asp Leu Val Pro Asp Glu Val Thr
50 55 60
Ile Gly Ile Val Arg Glu Arg Leu Ser Lys Asp Asp Cys Gln Asn Gly
65 70 75 80
Phe Leu Leu Asp Gly Phe Pro Arg Thr Val Ala Gln Ala Glu Ala Leu
85 90 95
Glu Ala Met Leu Ala Glu Ile Gly Arg Lys Leu Asp Tyr Val Ile His
100 105 110
Ile Asp Val Arg Gln Asp Val Leu Met Glu Arg Leu Thr Gly Arg Arg
115 120 125
Ile Cys Arg Asn Cys Gly Ala Thr Tyr His Leu Val Phe His Pro Pro
130 135 140
Ala Gln Pro Gly Val Cys Asp Lys Cys Gly Gly Glu Leu Tyr Gln Arg
145 150 155 160
Pro Asp Asp Asn Glu Ala Thr Val Ala Asn Arg Leu Glu Val Asn Thr
165 170 175
Lys Gln Met Lys Pro Leu Leu Asp Phe Tyr Glu Gln Lys Gly Tyr Leu
180 185 190
Arg His Ile Asn Gly Glu Gln Glu Met Glu Lys Val Phe Ser Asp Ile
195 200 205
Arg Glu Leu Leu Gly Gly Leu Thr Arg
210 215
<210> 82
<211> 1512
<212> DNA
<213> 沼泽红假单胞菌
<220>
<221> CDS
<222> (1)..(1512)
<400> 82
atg aac gcc aac ctg ttc gcc cgc ctg ttc gat aag ctc gac gac ccc 48
Met Asn Ala Asn Leu Phe Ala Arg Leu Phe Asp Lys Leu Asp Asp Pro
1 5 10 15
cac aag ctc gcg atc gaa acc gcg gcc ggg gac aag atc agc tac gcc 96
His Lys Leu Ala Ile Glu Thr Ala Ala Gly Asp Lys Ile Ser Tyr Ala
20 25 30
gag ctg gtg gcg cgg gcg ggc cgc gtc gcc aac gtg ctg gtg gca cgc 144
Glu Leu Val Ala Arg Ala Gly Arg Val Ala Asn Val Leu Val Ala Arg
35 40 45
ggc ctg cag gtc ggc gac cgc gtt gcg gcg caa acc gag aag tcg gtg 192
Gly Leu Gln Val Gly Asp Arg Val Ala Ala Gln Thr Glu Lys Ser Val
50 55 60
gaa gcg ctg gtg ctg tat ctc gcc acg gtg cgg gcc ggc ggc gtg tat 240
Glu Ala Leu Val Leu Tyr Leu Ala Thr Val Arg Ala Gly Gly Val Tyr
65 70 75 80
ctg ccg ctc aac acc gcc tat acg ctg cac gag ctc gat tac ttc atc 288
Leu Pro Leu Asn Thr Ala Tyr Thr Leu His Glu Leu Asp Tyr Phe Ile
85 90 95
acc gat gcc gag ccg aag atc gtg gtg tgc gat ccg tcc aag cgc gac 336
Thr Asp Ala Glu Pro Lys Ile Val Val Cys Asp Pro Ser Lys Arg Asp
100 105 110
ggg atc gcg gcg att gcc gcc aag gtc ggc gcc acg gtg gag acg ctt 384
Gly Ile Ala Ala Ile Ala Ala Lys Val Gly Ala Thr Val Glu Thr Leu
115 120 125
ggc ccc gac ggt cgg ggc tcg ctc acc gat gcg gca gct gga gcc agc 432
Gly Pro Asp Gly Arg Gly Ser Leu Thr Asp Ala Ala Ala Gly Ala Ser
130 135 140
gag gcg ttc gcc acg atc gac cgc ggc gcc gat gat ctg gcg gcg atc 480
Glu Ala Phe Ala Thr Ile Asp Arg Gly Ala Asp Asp Leu Ala Ala Ile
145 150 155 160
ctc tac acc tca ggg acg acc ggc cgc tcc aag ggc gcg atg ctc agc 528
Leu Tyr Thr Ser Gly Thr Thr Gly Arg Ser Lys Gly Ala Met Leu Ser
165 170 175
cac gac aat ttg gcg tcg aac tcg ctg acg ctg gtc gat tac tgg cgc 576
His Asp Asn Leu Ala Ser Asn Ser Leu Thr Leu Val Asp Tyr Trp Arg
180 185 190
ttc acg ccg gat gac gtg ctg atc cac gcg ctg ccg atc tat cac acc 624
Phe Thr Pro Asp Asp Val Leu Ile His Ala Leu Pro Ile Tyr His Thr
195 200 205
cat gga ttg ttc gtg gcc agc aac gtc acg ctg ttc gcg cgc gga tcg 672
His Gly Leu Phe Val Ala Ser Asn Val Thr Leu Phe Ala Arg Gly Ser
210 215 220
atg atc ttc ctg ccg aag ttc gat ccc gac aag atc ctc gac ctg atg 720
Met Ile Phe Leu Pro Lys Phe Asp Pro Asp Lys Ile Leu Asp Leu Met
225 230 235 240
gcg cgc gcc acc gtg ctg atg ggt gtg ccg acg ttc tac acg cgg ctc 768
Ala Arg Ala Thr Val Leu Met Gly Val Pro Thr Phe Tyr Thr Arg Leu
245 250 255
ttg cag agc ccg cgg ctg acc aag gag acg acg ggc cac atg agg ctg 816
Leu Gln Ser Pro Arg Leu Thr Lys Glu Thr Thr Gly His Met Arg Leu
260 265 270
ttc atc tcc ggg tcg gcg ccg ctg ctc gcc gat acg cat cgc gaa tgg 864
Phe Ile Ser Gly Ser Ala Pro Leu Leu Ala Asp Thr His Arg Glu Trp
275 280 285
tcg gcg aag acc ggt cac gcc gtg ctc gag cgc tac ggc atg acc gag 912
Ser Ala Lys Thr Gly His Ala Val Leu Glu Arg Tyr Gly Met Thr Glu
290 295 300
acc aac atg aac acc tcg aac ccg tat gac ggc gac cgc gtc ccc ggc 960
Thr Asn Met Asn Thr Ser Asn Pro Tyr Asp Gly Asp Arg Val Pro Gly
305 310 315 320
gcg gtc ggc ccg gcg ctg ccc ggc gtt tcg gcg cgc gtg acc gat ccg 1008
Ala Val Gly Pro Ala Leu Pro Gly Val Ser Ala Arg Val Thr Asp Pro
325 330 335
gaa acc ggc aag gaa ctg ccg cgc ggc gac atc ggg atg atc gag gtg 1056
Glu Thr Gly Lys Glu Leu Pro Arg Gly Asp Ile Gly Met Ile Glu Val
340 345 350
aag ggc ccg aac gtg ttc aag ggc tac tgg cgg atg ccg gag aag acc 1104
Lys Gly Pro Asn Val Phe Lys Gly Tyr Trp Arg Met Pro Glu Lys Thr
355 360 365
aag tct gaa ttc cgc gac gac ggc ttc ttc atc acc ggc gac ctc ggc 1152
Lys Ser Glu Phe Arg Asp Asp Gly Phe Phe Ile Thr Gly Asp Leu Gly
370 375 380
aag atc gac gag cgc ggc tac gtc cac atc ctc ggc cgc ggc aag gat 1200
Lys Ile Asp Glu Arg Gly Tyr Val His Ile Leu Gly Arg Gly Lys Asp
385 390 395 400
ctg gtg atc acc ggc ggc ttc aac gtc tat ccg aag gaa atc gag agc 1248
Leu Val Ile Thr Gly Gly Phe Asn Val Tyr Pro Lys Glu Ile Glu Ser
405 410 415
gag atc gac gcc atg ccg ggc gtg gtc gaa tcc gcg gtg atc ggc gtg 1296
Glu Ile Asp Ala Met Pro Gly Val Val Glu Ser Ala Val Ile Gly Val
420 425 430
ccg cac gcc gat ttc ggc gag ggc gtc act gcc gtg gtg gtg cgc gac 1344
Pro His Ala Asp Phe Gly Glu Gly Val Thr Ala Val Val Val Arg Asp
435 440 445
aag ggt gcc acg atc gac gaa gcg cag gtg ctg cac ggc ctc gac ggt 1392
Lys Gly Ala Thr Ile Asp Glu Ala Gln Val Leu His Gly Leu Asp Gly
450 455 460
cag ctc gcc aag ttc aag atg ccg aag aaa gtg atc ttc gtc gac gac 1440
Gln Leu Ala Lys Phe Lys Met Pro Lys Lys Val Ile Phe Val Asp Asp
465 470 475 480
ctg ccg cgc aac acc atg ggc aag gtc cag aag aac gtc ctg cgc gag 1488
Leu Pro Arg Asn Thr Met Gly Lys Val Gln Lys Asn Val Leu Arg Glu
485 490 495
acc tac aag gac atc tac aag taa 1512
Thr Tyr Lys Asp Ile Tyr Lys
500
<210> 83
<211> 503
<212> PRT
<213> 沼泽红假单胞菌
<400> 83
Met Asn Ala Asn Leu Phe Ala Arg Leu Phe Asp Lys Leu Asp Asp Pro
1 5 10 15
His Lys Leu Ala Ile Glu Thr Ala Ala Gly Asp Lys Ile Ser Tyr Ala
20 25 30
Glu Leu Val Ala Arg Ala Gly Arg Val Ala Asn Val Leu Val Ala Arg
35 40 45
Gly Leu Gln Val Gly Asp Arg Val Ala Ala Gln Thr Glu Lys Ser Val
50 55 60
Glu Ala Leu Val Leu Tyr Leu Ala Thr Val Arg Ala Gly Gly Val Tyr
65 70 75 80
Leu Pro Leu Asn Thr Ala Tyr Thr Leu His Glu Leu Asp Tyr Phe Ile
85 90 95
Thr Asp Ala Glu Pro Lys Ile Val Val Cys Asp Pro Ser Lys Arg Asp
100 105 110
Gly Ile Ala Ala Ile Ala Ala Lys Val Gly Ala Thr Val Glu Thr Leu
115 120 125
Gly Pro Asp Gly Arg Gly Ser Leu Thr Asp Ala Ala Ala Gly Ala Ser
130 135 140
Glu Ala Phe Ala Thr Ile Asp Arg Gly Ala Asp Asp Leu Ala Ala Ile
145 150 155 160
Leu Tyr Thr Ser Gly Thr Thr Gly Arg Ser Lys Gly Ala Met Leu Ser
165 170 175
His Asp Asn Leu Ala Ser Asn Ser Leu Thr Leu Val Asp Tyr Trp Arg
180 185 190
Phe Thr Pro Asp Asp Val Leu Ile His Ala Leu Pro Ile Tyr His Thr
195 200 205
His Gly Leu Phe Val Ala Ser Asn Val Thr Leu Phe Ala Arg Gly Ser
210 215 220
Met Ile Phe Leu Pro Lys Phe Asp Pro Asp Lys Ile Leu Asp Leu Met
225 230 235 240
Ala Arg Ala Thr Val Leu Met Gly Val Pro Thr Phe Tyr Thr Arg Leu
245 250 255
Leu Gln Ser Pro Arg Leu Thr Lys Glu Thr Thr Gly His Met Arg Leu
260 265 270
Phe Ile Ser Gly Ser Ala Pro Leu Leu Ala Asp Thr His Arg Glu Trp
275 280 285
Ser Ala Lys Thr Gly His Ala Val Leu Glu Arg Tyr Gly Met Thr Glu
290 295 300
Thr Asn Met Asn Thr Ser Asn Pro Tyr Asp Gly Asp Arg Val Pro Gly
305 310 315 320
Ala Val Gly Pro Ala Leu Pro Gly Val Ser Ala Arg Val Thr Asp Pro
325 330 335
Glu Thr Gly Lys Glu Leu Pro Arg Gly Asp Ile Gly Met Ile Glu Val
340 345 350
Lys Gly Pro Asn Val Phe Lys Gly Tyr Trp Arg Met Pro Glu Lys Thr
355 360 365
Lys Ser Glu Phe Arg Asp Asp Gly Phe Phe Ile Thr Gly Asp Leu Gly
370 375 380
Lys Ile Asp Glu Arg Gly Tyr Val His Ile Leu Gly Arg Gly Lys Asp
385 390 395 400
Leu Val Ile Thr Gly Gly Phe Asn Val Tyr Pro Lys Glu Ile Glu Ser
405 410 415
Glu Ile Asp Ala Met Pro Gly Val Val Glu Ser Ala Val Ile Gly Val
420 425 430
Pro His Ala Asp Phe Gly Glu Gly Val Thr Ala Val Val Val Arg Asp
435 440 445
Lys Gly Ala Thr Ile Asp Glu Ala Gln Val Leu His Gly Leu Asp Gly
450 455 460
Gln Leu Ala Lys Phe Lys Met Pro Lys Lys Val Ile Phe Val Asp Asp
465 470 475 480
Leu Pro Arg Asn Thr Met Gly Lys Val Gln Lys Asn Val Leu Arg Glu
485 490 495
Thr Tyr Lys Asp Ile Tyr Lys
500

Claims (25)

1.一种重组多肽，其包含选自以下的序列：
(a)SEQ ID NO:30且具有至少Y288X突变，其中X是A、N、S或V；
(b)SEQ ID NO:30，其具有至少Y288X突变，其中X是A、N、S或V，和至少一个选自V49Z1、F213Z2、A232S、I234T、V271Z3和/或G286S的其它突变，其中Z1是S、N、T或G，Z2是H、N或G且Z3是N或H；和
(c)从氨基酸21开始在SEQ ID NO:1-28或29中列举的序列，
其中(a)-(c)中的任一个的多肽能够用于执行异戊二烯化反应。
2.权利要求1的重组多肽，其中所述多肽包含SEQ ID NO:30且具有选自以下的突变：
(i)Y288A；
(ii)Y288N；
(iii)Y288A和F213H；
(iv)Y288A和F213N；
(v)Y288N和V49S；
(vi)Y288S和V49N；
(vii)Y288A和V49S；
(viii)Y288N和G286S；
(ix)Y288N、F213N和V49G；
(x)Y288A、F213N和I234T；
(xi)Y288S、F213N和V49N；
(xii)Y288A、F213N和A232S；
(xiii)Y288N、F213G和V49T；
(xiv)Y288N、F213N、V49S和V271N；
(xv)Y288N、F213G、V49T和V271H；
(xvi)Y288A和G286S；
(xvii)Y288A、G286S和A232S；
(xviii)Y288A、G286S、A232S和F213H；
(xix)Y288V和G286S；
(xx)Y288A和A232S；和
(xxi)Y288V和A232S。
3.权利要求1的重组多肽，所述重组多肽具有SEQ ID NO:30的序列且具有Y288A和G286S突变。
4.权利要求1的重组多肽，其中所述异戊二烯化反应包括从香叶基焦磷酸酯和油橄榄醇酯产生大麻萜酚酸，或从香叶基焦磷酸酯和2,4-二羟基-6-丙基苯甲酸产生次萜酚酸，或从香叶基焦磷酸酯和2,4-二羟基苯甲酸或其衍生物产生CBGXA，其中所述2,4-二羟基苯甲酸或其衍生物由式I表示：
其中X表示任何化学基团。
5.一种包含重组酶途径的组合物，其包含权利要求1的多肽和多种将葡萄糖转化为香叶基焦磷酸酯的酶。
6.权利要求5的组合物，所述重组酶途径进一步包含含有丙酮酸氧化酶和乙酰基磷酸转移酶的丙酮酸脱氢酶旁路酶途径。
7.权利要求5或6的组合物，其中所述途径包含再循环NADH/NAD的NADH氧化酶。
8.权利要求7的组合物，其中所述途径包含以下酶：
(i)己糖激酶；
(ii)葡萄糖-6-磷酸异构酶；
(iii)磷酸果糖激酶；
(iv)果糖-1,6-二磷酸醛缩酶；
(v)磷酸丙糖异构酶；
(vi)Gald-3-P脱氢酶；
(vii)突变体Gald-3-P脱氢酶；
(viii)NADH氧化酶
(ix)磷酸甘油酸激酶
(x)磷酸甘油酸酯变位酶；
(xi)烯醇化酶；
(xii)丙酮酸激酶；
(xiii)丙酮酸氧化酶；
(xiv)乙酰基磷酸转移酶；
(xv)乙酰辅酶A乙酰基转移酶；
(xvi)HMG-CoA合酶；
(xvii)HMG-CoA还原酶；
(xviii)甲羟戊酸激酶；
(xix)磷酸甲羟戊酸激酶；
(xx)二磷酸甲羟戊酸脱羧酶；
(xxi)香叶基-PP合酶或法呢基-PP合酶突变体S82F；和
(xxii)突变体芳族异戊二烯基转移酶。
9.权利要求8的组合物，其中给所述途径补充ATP和油橄榄醇酯且所述途径产生大麻素前体。
10.权利要求9的组合物，其中所述途径进一步包含大麻二酚酸合酶。
11.权利要求10的组合物，其中所述途径产生大麻二酚酸。
12.一种包含重组酶途径的组合物，其包含权利要求1的多肽和多种将戊二烯醇或异戊二烯醇转化为香叶基焦磷酸酯的酶。
13.一种生产异戊二烯化的化合物的方法，所述方法包括使底物与具有以下通用结构的异戊二烯基、与权利要求1的重组多肽接触：
其中所述异戊二烯基被添加至所述底物。
14.一种用于从葡萄糖生产异戊二烯化的化合物的无细胞酶促系统，所述系统包含一种途径，所述途径包括
(i)将丙酮酸转化成乙酰基磷酸酯的酶；
(ii)将乙酰基磷酸酯转化成乙酰辅酶A的酶；和
(iii)将甘油醛-3-磷酸转化成1,3-二磷酸甘油酸酯的第一辅因子依赖性的酶,所述酶产生不平衡的辅因子的产生和利用；
(iv)将甘油醛-3-磷酸转化成1,3-二磷酸甘油酸酯的第二辅因子依赖性的酶，其中所述第二辅因子依赖性的酶被突变成具有改变的其辅因子偏好；
(v)再循环辅因子的酶，其中所述辅因子选自NAD+/NADH、NADP+/NADPH和FAD+/FADH；和
(vi)包含权利要求1的多肽的非特异性异戊二烯基转移酶。
15.权利要求14的无细胞酶促系统，其中所述第一辅因子依赖性的酶包含使用NAD+作为辅因子的脱氢酶活性，且其中所述第二辅因子依赖性的酶包含使用NADP+作为辅因子的脱氢酶活性。
16.权利要求14或15的无细胞酶促系统，其中所述再循环辅因子的酶是NADPH/NADH氧化酶。
17.权利要求14的无细胞酶促系统，其中所述途径将3个葡萄糖转化成1个香叶基焦磷酸酯。
18.权利要求14的无细胞酶促系统，其中所述途径包含以下酶：
(i)己糖激酶；
(ii)葡萄糖-6-磷酸异构酶；
(iii)磷酸果糖激酶；
(iv)果糖-1,6-二磷酸醛缩酶；
(v)磷酸丙糖异构酶；
(vi)Gald-3-P脱氢酶；
(vii)突变体Gald-3-P脱氢酶；
(viii)NADH氧化酶；
(ix)磷酸甘油酸激酶；
(x)磷酸甘油酸酯变位酶；
(xi)烯醇化酶；
(xii)丙酮酸激酶；
(xiii)丙酮酸氧化酶；
(xiv)乙酰基磷酸转移酶；
(xv)乙酰辅酶A乙酰基转移酶；
(xvi)HMG-CoA合酶；
(xvii)HMG-CoA还原酶；
(xviii)甲羟戊酸激酶；
(xix)磷酸甲羟戊酸激酶；
(xx)二磷酸甲羟戊酸脱羧酶；和
(xxi)香叶基-PP合酶或法呢基-PP合酶突变体S82F。
19.权利要求14的无细胞酶促系统，其中所述非特异性异戊二烯基转移酶包含芳族异戊二烯基转移酶，并且其中所述芳族异戊二烯基转移酶是AtaPT或NovQ酶或其突变体，以在有合适的底物存在下将GPP转化成异戊二烯基-化合物。
20.权利要求19的无细胞酶促系统，其中所述合适的底物选自芹菜配基、油橄榄醇酸、2,4-二羟基-6-丙基苯甲酸和白藜芦醇。
21.权利要求20的无细胞酶促系统，其中所述底物是2,4-二羟基-6-丙基苯甲酸。
22.一种分离的编码多肽的多核苷酸，所述多肽选自：
(a)SEQ ID NO:30且具有至少Y288X突变，其中X是A、N、S或V；
(b)SEQ ID NO:30，其具有至少Y288X突变，其中X是A、N、S或V，和至少一个选自V49Z1、F213Z2、A232S、I234T、V271Z3和/或G286S的其它突变，其中Z1是S、N、T或G，Z2是H、N或G且Z3是N或H；和
(c)从氨基酸21开始在SEQ ID NO:1-28或29中列举的序列。
23.包含权利要求22的分离的多核苷酸的载体。
24.包含权利要求22的分离的多核苷酸的重组微生物。
25.包含权利要求23的载体的重组微生物。
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CN112789505A (zh) 2021-05-11
KR20210049805A (ko) 2021-05-06
EP3830581A4 (en) 2022-07-27
US20210309975A1 (en) 2021-10-07
CA3107544A1 (en) 2020-02-06
EP3830581A1 (en) 2021-06-09
BR112021001450A2 (pt) 2021-04-27

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