CN114574529A - 一种乙醇酸在酶的作用下生成目标产物的方法 - Google Patents

一种乙醇酸在酶的作用下生成目标产物的方法 Download PDF

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CN114574529A
CN114574529A CN202011382345.XA CN202011382345A CN114574529A CN 114574529 A CN114574529 A CN 114574529A CN 202011382345 A CN202011382345 A CN 202011382345A CN 114574529 A CN114574529 A CN 114574529A
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江会锋
逯晓云
杨巧玉
初斋林
卢丽娜
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Tianjin National Synthetic Biotechnology Innovation Center Co.,Ltd.
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

本发明公开了一种乙醇酸在酶的作用下生成至少一种目标产物的方法。为解决C3植物中天然乙醇酸再利用过程需损失25％已固定的有机碳的关键问题，本申请设计了包括乙酸激酶、磷酸乙酰转移酶、羟乙酰辅酶A还原酶、乙酰磷酸合酶的乙醇酸代谢途径；或者该途径包括羟乙酰辅酶A合酶、羟乙酰辅酶A还原酶、乙酰磷酸合酶、磷酸乙酰转移酶。新的乙醇酸代谢途径大幅降低了乙醇酸再利用过程造成的有机碳损失，使光合作用产生的副产物乙醇酸100％转化为乙酰辅酶A，为提高植物的光合作用提供新思路。该途径还提供了以乙醇酸为原料制备乙醇醛或乙酰辅酶A的方法。

Description

一种乙醇酸在酶的作用下生成目标产物的方法
技术领域
本发明涉及乙醇酸代谢途径，具体涉及一种乙醇酸在酶的作用下生成目标产物的方法。
背景技术
植物细胞在光照条件下不仅能够进行光合作用，同化CO2，释放O2并合成有机物，同时还会发生一种吸收O2，释放CO2的代谢途径，这种代谢途径被称为光呼吸。在小麦，水稻和大豆等植物中，光呼吸作用使光合作用效率降低20％-50％，其主要原因在于，核酮糖-1,5-二磷酸羧化酶/加氧酶(RuBisCO)固定O2时，生成的磷酸乙醇酸再利用效率低。磷酸乙醇酸对于卡尔文循环的关键基因——丙糖磷酸异构酶和景天庚酮糖二磷酸酶具有抑制作用，可抑制核酮糖-1,5-二磷酸再生，降低光合作用效率。磷酸乙醇酸必须通过去磷酸化形成乙醇酸，并通过光呼吸途径再循环到3-磷酸甘油酸，重新进入卡尔文循环，才可以驱动可持续的光合作用。乙醇酸的再利用对光呼吸途径至关重要，然而，天然乙醇酸再利用途径除了浪费能量和还原力，每两分子乙醇酸再循环会释放一分子CO2，最终只有75％的有机碳进入卡尔文循环。光呼吸不但会降低植物光合效率，而且其释放的CO2对大气的碳排放的贡献也不容小觑；据估测，植物呼吸作用每年能向大气排放30Gt左右的碳，其中大部分来自光呼吸。因此，构建非天然新型光呼吸途径，提高乙醇酸的再利用效率，对于提高光合作用效率具有重要科学意义。
近十几年来，科学家们通过在拟南芥、烟草、水稻等植物叶绿体中导入新的乙醇酸代谢途径，将乙醇酸全部或部分氧化为CO2，增加叶绿体中CO2浓度，提高RuBisCO羧化活性。此外，还通过弱化自身光呼吸途径代谢流通量，减少能量和还原力损失，最终提高了农作物产率。然而，这些途径均无法避免损失已固定的有机碳。
发明内容
为解决植物中天然乙醇酸再利用过程需损失25％已固定的有机碳的关键问题，本申请另辟蹊径，从化学反应本质出发，理性设计了天然不存在的全新的乙醇酸代谢途径，大幅降低了乙醇酸再利用过程造成的有机碳损失，使光合作用产生的副产物乙醇酸100％转化为乙酰辅酶A，为提高植物的光合作用提供新思路。
本发明的目的之一是提供一种乙醇酸在酶的作用下生成至少一种目标产物的方法。
本发明的又一目的是提供一种生产具有增加的生长或生产力的植物的方法。
在本发明的第一方面，提供了一种乙醇酸在酶的作用下生成至少一种目标产物的方法，包括如下步骤：
步骤(1)：乙醇酸在乙酸激酶(AckA)的作用下生成羟乙酰磷酸，进一步在磷酸乙酰转移酶(PTA)的作用下生成羟乙酰辅酶A；或者乙醇酸在羟乙酰辅酶A合酶(ACS)作用下生成羟乙酰辅酶A；
步骤(2)：羟乙酰辅酶A进一步在羟乙酰辅酶A还原酶(GCR)作用下生成乙醇醛；
任选的步骤(3)：乙醇醛进一步在乙酰磷酸合酶(ACPS)的作用下生成乙酰磷酸；以及
任选的步骤(4)：乙酰磷酸进一步在磷酸乙酰转移酶(PTA)的作用下生成乙酰辅酶A。
优选地，所述方法中步骤(1)具体为：乙醇酸在ATP、乙酸激酶(AckA)的作用下生成羟乙酰磷酸，进一步在NAD(P)H和磷酸乙酰转移酶(PTA)的作用下生成羟乙酰辅酶A；或者乙醇酸在ATP、NADP(H)、辅酶A、羟乙酰辅酶A合酶(ACS)作用下生成羟乙酰辅酶A。
优选地，所述方法中步骤(2)具体为：羟乙酰辅酶A进一步在辅酶A、羟乙酰辅酶A还原酶(GCR)作用下生成乙醇醛。
优选地，所述方法中步骤(4)具体为：乙酰磷酸进一步在磷酸乙酰转移酶(PTA)、辅酶A的作用下生成乙酰辅酶A。
在另一优选例中，上述方法中步骤(1)至(4)的转化反应在10～40℃下进行，优选地在16-37℃，更优选的在30℃。
优选地，所述方法中乙酸激酶与SEQ ID NO:1所示的氨基酸序列至少95％相同，所述磷酸乙酰转移酶与SEQ ID NO:2所示的氨基酸序列至少95％相同，所述羟乙酰辅酶A还原酶与SEQ ID NO:3所示的氨基酸序列至少95％相同，所述乙酰磷酸合酶与SEQ ID NO:4所示的氨基酸序列至少95％相同，所述羟乙酰辅酶A合酶与SEQ ID NO:5所示的氨基酸序列至少95％相同。
优选地，所述方法中乙酸激酶包含如SEQ ID NO:1所示的氨基酸序列，所述磷酸乙酰转移酶包含如SEQ ID NO:2所示的氨基酸序列，所述羟乙酰辅酶A还原酶包含如SEQ IDNO:3所示的氨基酸序列，所述乙酰磷酸合酶包含如SEQ ID NO:4所示的氨基酸序列，所述羟乙酰辅酶A合酶包含如SEQ ID NO:5所示的氨基酸序列。
在另一优选例中，所述目标产物为乙酰辅酶A，所述方法包括步骤(1)、(2)、(3)和(4)。
进一步地，所述方法中的步骤(1)、步骤(2)、步骤(3)以及步骤(4)中任两个步骤、三个步骤或者四个步骤可以同时进行。
在另一优选例中，所述目标产物为乙醇醛，所述方法包括步骤(1)和(2)。
进一步地，所述方法中的步骤(1)、步骤(2)可以同时进行。
在本发明的第二方面，提供一种生产具有增加的生长或生产力的植物的方法，包括以下步骤：在所述植物的至少一部分叶绿体中将乙醇酸转化为乙酰辅酶A，此步骤包括在叶绿体中产生转基因的乙酸激酶(AckA)、磷酸乙酰转移酶(PTA)、羟乙酰辅酶A还原酶(GCR)和乙酰磷酸合酶(ACPS)的步骤；或者包括在叶绿体中产生转基因羟乙酰辅酶A合酶(ACS)、羟乙酰辅酶A还原酶(GCR)、乙酰磷酸合酶(ACPS)和磷酸乙酰转移酶(PTA)的步骤。
在另一优选例中，所述方法，其中所述乙酸激酶(AckA)与SEQ ID NO:1所示的氨基酸序列至少95％相同，所述磷酸乙酰转移酶(PTA)与SEQ ID NO:2所示的氨基酸序列至少95％相同，所述羟乙酰辅酶A还原酶(GCR)与SEQ ID NO:3所示的氨基酸序列至少95％相同，所述乙酰磷酸合酶(ACPS)与SEQ ID NO:4所示的氨基酸序列至少95％相同，所述羟乙酰辅酶A合酶(ACS)与SEQ ID NO:5所示的氨基酸序列至少95％相同。
在另一优选例中，所述方法，其中所述乙酸激酶(AckA)包含如SEQ ID NO:1所示的氨基酸序列，所述磷酸乙酰转移酶(PTA)包含如SEQ ID NO:2所示的氨基酸序列，所述羟乙酰辅酶A还原酶(GCR)包含如SEQ ID NO:3所示的氨基酸序列，所述乙酰磷酸合酶(ACPS)包含如SEQ ID NO:4所示的氨基酸序列，所述羟乙酰辅酶A合酶(ACS)包含如SEQ ID NO:5所示的氨基酸序列。
在另一优选例中，上述方法中的植物，可以是任何C3植物。例如，在一些实施方式中，本公开的植物是稻、大豆、马铃薯、豇豆、大麦、小麦或木薯。
上述制备乙醇醛或乙酰辅酶A的方法中NAD(P)H、ATP、CoA等辅因子的浓度没有特别的限定，AckA、PTA、GCR、ACS等蛋白添加量没有特别的限定，本领域技术人员可根据催化反应速率确定上述辅因子或蛋白的反应浓度。
作为示例性的实施方案，所述乙醇酸与NAD(P)H，ATP，CoA的摩尔比为(1-20):1:1:1，AckA、PTA、GCR和ACPS的质量比为1:1:1:1，ACS、GCR和ACPS的质量比为1:1:1:1。本发明方法包括但不局限于示例性的实施方案中的具体条件，可达到等同的催化功效即可。
本发明中NAD(P)H、ATP、CoA、AckA、PTA、GCR、ACPS、CS等辅因子或酶的来源或种类没有特别的限定，可以是本领域已知的具有该酶应有的催化活性的来源或种类。例如，AckA可以来自于大肠杆菌，也可以来自于乳酸菌。AckA酶可以由不同宿主菌表达，如大肠杆菌，酵母菌，芽孢杆菌等。本发明中使用的各种酶可以是但不限于示例性的实施方案中所使用的，具有等同催化功能的酶即可。
本文中相关英文简写含义如下：
CoA：辅酶A；
NAD(P)H：还原型辅酶Ⅱ，还原型烟酰胺腺嘌呤二核苷酸磷酸；
ATP：腺嘌呤核苷三磷酸；
AckA：乙酸激酶，在ATP存在的情况下，具有催化乙醇酸合成羟乙酰磷酸的功能；
PTA：磷酸乙酰转移酶，在CoA存在的情况下，具有催化羟乙酰磷酸合成羟乙酰辅酶A的功能，或具有催化乙酰磷酸合成乙酰辅酶A的功能；
GCR：羟乙酰辅酶A还原酶，具有催化羟乙酰辅酶A合成乙醇醛的功能；
ACPS：乙酰磷酸合酶，具有催化乙醇醛合成乙酰磷酸功能；
ACS：羟乙酰辅酶A合酶，在ATP、CoA存在的情况下，具有催化乙醇酸合成羟乙酰辅酶A的功能。
本发明的有益效果：
1.自然界中的乙醇酸想要被利用，只能是两分子的乙醇酸合成1分子的3-磷酸-甘油酸，该过程释放1分子的二氧化碳，会损失光合作用已经固定的有机碳。本申请提供了一种乙醇酸在酶的作用下生成至少一种目标产物的方法，属于新的乙醇酸代谢途径，可以直接将乙醇酸合成乙酰辅酶A，乙酰辅酶A是胞内代谢的重要物质，可以直接被利用，该过程无碳损失。
2.本申请提供的新的乙醇酸代谢途径的优势在于可以无碳损失地将乙醇酸代谢为乙酰辅酶A，同时代谢途径短，中间副产物积累少，可以显著提高乙醇酸的利用效率。
3.实验结果表明，转入了新的乙醇酸代谢途径的植株表现为以氨基酸为代表的生物量增加。本申请对深入阐明高光效机理、对作物与可再生能源生产、甚至对减少CO2排放均具有深远意义，在实际应用中可以将新的乙醇酸代谢途径转入不同的C3植物中以培育更加高产量的品种。
附图说明
图1为乙醇酸在酶的作用下生成目标产物的过程。
图2为实施例1中乙醇醛的气相质谱检测结果，其中a为不同组的气相检测结果(a中从上到下依次为标准品，样品和对照品的检测结果)，b为气相检测中样品组中经衍生化的乙醇醛的分子式。
图3为实施例2中乙酰辅酶A的液相质谱检测结果，其中a为不同组的液相检测结果(a中从上到下依次为标准品，样品和对照品的检测结果)，b为液相检测中样品组中乙酰辅酶A的一级谱图。
图4为实施例3转基因水稻和野生型水稻叶片代谢物含量的分析结果，方框表示的为转基因水稻中含量上调的代谢物(主要为氨基酸)，P值低于0.01表示在不同菌株中代谢物含量差异极显著。
具体实施方式
本文中示出和描述了本公开的优选实施方式。对于本领域技术人员显而易见的是，这些实施方式仅作为示例提供。在不脱离本公开的情况下，本领域技术人员将想到许多变化、改变和替换。在实践本公开时可以采用本文描述的本公开的实施方式的各种替代方案。所附权利要求旨在限定本公开的范围，并且由此覆盖这些权利要求及其等同物范围内的方法和结构。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
定义与说明：
无论是否具体说明，术语“约”定义为所述值的正负百分之十。例如，大约1.0g意味着0.9g至1.1g及该范围内的所有值。
出于本公开的目的，以百分比表示的两个相关核苷酸或氨基酸序列的“序列同一性”是指两个最佳比对序列中具有相同残基(x100)除以比较的位置的数目。间隙，即在一个序列中存在残基而在另一个序列中不存在的比对中的位置被认为是具有不相同残基的位置。通过Needleman和Wunsch算法(Needleman和Wunsch,J Mol Biol,(1970)48:3,443-53)进行两个序列的比对。计算机辅助序列比对可以使用标准软件程序如GAP方便地进行，GAP是Wisconsin Package Version 10.1(Genetics Computer Group，Ma dison，Wisconsin，美国)的一部分，使用缺省评分矩阵，其中缺口产生罚分为50分和缺口延长罚分为3分。
在两个或更多个多核苷酸或多肽序列的上下文中，术语“相同”或“同一性”百分比及其语法变体是指，当在使用序列比较算法或通过手动比对和目视检查测量的指定区域上比较和以最大对应性比对时，两个或更多个序列或子序列相同或具有特定百分比的相同的核苷酸或氨基酸(分别地)(例如，80％、85％同一性、90％同一性、99％或100％同一性)。在两个多核苷酸或多肽的上下文中，短语“高百分比相同”或“高百分比同一性”及其语法变体是指，当在使用序列比较算法或通过手动比对和目视检查测量的指定区域上比较和以最大对应性比对时，两个或更多个序列或子序列具有至少约80％的同一性，至少约81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％核苷酸或氨基酸同一性。
本文所用的术语“代谢途径”是指生物体代谢中的化学反应是在酶的催化下进行的，而且许多酶连续地按顺序地起作用，形成多酶体系，使第一个酶促反应产物变成第二个酶促反应的底物，依此类推。本申请中实施例3“新的乙醇酸的代谢途径”涉及的酶如表1所示：
表1.新的乙醇酸代谢途径涉及的酶
Figure BDA0002809840870000061
本申请示例性实施方式中采用的酶，基因均由金维智参照表1所示的核苷酸序列合成，均构建至pET-28a载体，位于酶切位点NdeI和XhoI之间，并在大肠杆菌中表达纯化蛋白。
当重组核酸用于特定序列的表达、克隆或复制时，为引入宿主细胞而制备的DNA构建体通常将包含宿主识别的复制系统(即载体)，所述复制系统包括编码所需多肽的预期DNA片段，并且所述DNA构建体还可以包括可操作地连接至编码多肽的片段的转录和翻译起始调控序列。另外，此类构建体可包括细胞定位信号(例如叶绿体定位信号)。在优选实施方式中，将此类DNA构建体引入宿主细胞的基因组DNA、叶绿体DNA或线粒体DNA中。
在一些实施方式中，非整合表达系统可用于诱导一种或多种导入基因的表达。表达系统(表达载体)可包括，例如复制起点或自主复制序列(ARS)和表达控制序列、启动子、增强子和必需的加工信息位点，例如核糖体结合位点、RNA剪接位点、多腺苷酸化位点、转录终止子序列和mRNA稳定序列。适当时，还可包含来自相同或相关物种的分泌多肽的信号肽，这些信号肽可使蛋白质穿过和/或滞留在细胞膜、细胞壁中或从细胞中分泌出来。
本申请示例性实施方式中所用的辅因子包括但不限于如下购买来源：
ATP：购自索莱宝；NAD(P)H：购自索莱宝；ATP：购自索莱宝。
如本文所用，术语“增加生长”和“增加生产力”及其语法变体是指在给定时间点生长速率或植物大小或生物量(如氨基酸含量)增加，或与同一物种的未改变植物相比遗传改变植物的光合作用效率增加。
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
实施例1：以乙醇酸为原料制备乙醇醛
样品1：向200μL体系中添加20mM乙醇酸，1mM NAD(P)H，1mM ATP，1mM CoA，ACS，GCR各2mg/mL。样品2：向200μL体系中添加20mM乙醇酸，1mM NAD(P)H，1mM ATP，1mM CoA，AckA，PTA，GCR各2mg/mL。对照：向200μL体系中添加20mM乙醇酸，1mM NAD(P)H，1mM ATP，1mM CoA，无酶。30℃下反应1h后，反应结束后，冻干反应体系。然后加入60μL五氟苯盐酸羟胺(PFBOA，200mM)，涡旋，室温孵育1小时。加入300μL己烷，室温静置5min。吸取有机层的100μL样品，加入30μL含有1％三甲基氯硅烷的三甲基甲硅烷基三氟乙酰胺和20μL吡啶，使PFBOA衍生物甲硅烷基化。样品中被衍生化的乙醇醛利用GC-MS检测，检测结果如图2所示。由图2可知，利用图1中辅因子和酶，可以实现利用乙醇酸合成乙醇醛。
GC-MS检测方法：检测系统是安捷伦气相色谱仪7890A；检测条件为：安捷伦色谱柱19091S-433，30m×250μm×0.25μm；起始温度设定为50℃，保留时间1min，以15℃/min的线性增长速率升温到150℃，然后以30℃/min的线性增长速率升温到300℃，保留时间1min；进样口温度为250℃，GC-MS接口温度280℃。氦气作为载体气体，1.2mL/min流速。进样量1μL，检测有5min的溶剂延迟。
本实施例所采用的方法及体系仅为示例性方案，所采用的的酶可来自于其余物种，所添加的酶浓度、底物浓度、反应时间、反应条件、衍生条件、检测条件均可做调整，可达到与本实施例同等功效即可，各种调节均应在本发明保护保护范围之内。
实施例2：以乙醇酸为原料制备乙酰辅酶A
样品1：向200μL体系中添加20mM乙醇酸，1mM NAD(P)H，1mM ATP，1mM CoA，ACS，PTA，GCR，ACPS各2mg/mL。样品2：向200μL体系中添加20mM乙醇酸，1mM NAD(P)H，1mM ATP，1mM CoA，AckA，PTA，GCR，ACPS各2mg/mL。对照：向200μL体系中添加20mM乙醇酸，1mM NAD(P)H，1mM ATP，1mM CoA，无酶。30℃下反应1h后，利用液相质谱检测乙酰辅酶A，结果如图3所示。由图3可知，利用图1中辅因子和酶，可以实现乙醇酸合成乙酰辅酶A，构建了完整的乙醇酸利用途径。
液相质谱检测条件：LC条件：仪器：岛津LC-30A；色谱柱：Merck zic-HILIC(100mm×2.1mm,3.5μm)；流动相A为10mM醋酸铵，B为100％乙腈。梯度液相条件为：0-3min，90％B；3-25min，90％-60％B；25-30min，60％B；30-38min，90％B；流速为：0.3mL/min。MS条件：仪器：ABSciex TripleTOF5600；ESI源；正离子检测模式；电压5500V；离子源温度600℃；GS1气压：55psi；GS2气压：55psi；气帘气气压：35psi；IDA采集模式，一级扫描范围50-1200Da，二级扫描范围30-1200Da。
本实施例所采用的方法及体系仅为示例性方案，所采用的酶可来自于其余物种，所添加的酶浓度、底物浓度、反应时间、反应条件、衍生条件、检测条件均可做调整，可达到与本实施例同等功效即可，各种调节均应在本发明保护保护范围之内。
实施例3：乙醇酸合成乙酰辅酶A途径在植物中的应用
利用农杆菌转染方式将乙醇酸合成乙酰辅酶A途径相关基因转入水稻中，使AckA，PTA、GCR和ACPS或者ACS、PTA、GCR和ACPS蛋白在水稻叶绿体定位表达。将转基因植物再传一代，收集种子并培养获得T1纯合植株。分别取野生型植株(6株)和转基因植株(6株)叶片各1g，由北京诺禾致源科技股份有限公司提取并检测分析叶片中的代谢物组学。结果如图4所示。
结果显示，与野生型植株相比，转基因植株中所含有的氨基酸浓度更高。显著性差异分析结果显示，P值均低于0.01，表明野生型与转基因植株氨基酸含量的差异极显著。存在显著含量差异的氨基酸包括组氨酸(His)、苯丙氨酸(Phe)、酪氨酸(Tyr)、色氨酸(Trp)、甲硫氨酸(Met)、脯氨酸(Pro)、赖氨酸(Lys)、谷氨酰胺(Gln)、天冬氨酸(Asp)、天冬酰胺(Asn)、丝氨酸(Ser)、丙氨酸(Ala)。由于乙酰辅酶A是合成上述氨基酸的重要前体物质，因此上述氨基酸的胞内含量显著增加可以说明转基因植物合成了更多的乙酰辅酶A，即转基因植物利用新型乙醇酸代谢途径成功催化乙醇酸合成乙酰辅酶A。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 一种乙醇酸在酶的作用下生成目标产物的方法
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 400
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 1
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Lys Phe Ala Ile Ile Asp Ala Val Asn Gly Asp Glu Tyr Leu Ser Gly
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Leu Ala Glu Cys Phe His Leu Pro Glu Ala Arg Ile Lys Trp Lys Met
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Asp Gly Ser Lys Gln Glu Ala Ala Leu Gly Ala Gly Ala Ala His Ser
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Glu Ala Leu Asn Phe Ile Val Asn Thr Ile Leu Ala Gln Lys Pro Glu
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Leu Ser Ala Gln Leu Thr Ala Ile Gly His Arg Ile Val His Gly Gly
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Glu Lys Tyr Thr Ser Ser Val Val Ile Asp Glu Ser Val Ile Gln Gly
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Ile Lys Asp Ser Ala Ser Phe Ala Pro Leu His Asn Pro Ala His Leu
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Ile Gly Ile Ala Glu Ala Leu Lys Ser Phe Pro Gln Leu Lys Asp Lys
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Ser Tyr Leu Tyr Ala Leu Pro Tyr Ser Leu Tyr Lys Glu His Gly Val
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Arg Arg Tyr Gly Ala His Gly Thr Ser His Phe Tyr Val Thr Gln Glu
180 185 190
Ala Ala Lys Met Leu Asn Lys Pro Val Glu Glu Leu Asn Ile Ile Thr
195 200 205
Cys His Leu Gly Asn Gly Gly Ser Val Ser Ala Ile Arg Asn Gly Lys
210 215 220
Cys Val Asp Thr Ser Met Gly Leu Thr Pro Leu Glu Gly Leu Val Met
225 230 235 240
Gly Thr Arg Ser Gly Asp Ile Asp Pro Ala Ile Ile Phe His Leu His
245 250 255
Asp Thr Leu Gly Met Ser Val Asp Gln Ile Asn Lys Met Leu Thr Lys
260 265 270
Glu Ser Gly Leu Leu Gly Leu Thr Glu Val Thr Ser Asp Cys Arg Tyr
275 280 285
Val Glu Asp Asn Tyr Ala Thr Lys Glu Asp Ala Lys Arg Ala Met Asp
290 295 300
Val Tyr Cys His Arg Leu Ala Lys Tyr Ile Gly Ser Tyr Thr Ala Leu
305 310 315 320
Met Asp Gly Arg Leu Asp Ala Val Val Phe Thr Gly Gly Ile Gly Glu
325 330 335
Asn Ala Ala Met Val Arg Glu Leu Ser Leu Gly Lys Leu Gly Val Leu
340 345 350
Gly Phe Glu Val Asp His Glu Arg Asn Leu Ala Ala Arg Phe Gly Lys
355 360 365
Ser Gly Phe Ile Asn Lys Glu Gly Thr Arg Pro Ala Val Val Ile Pro
370 375 380
Thr Asn Glu Glu Leu Val Ile Ala Gln Asp Ala Ser Arg Leu Thr Ala
385 390 395 400
<210> 2
<211> 717
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 2
Met Ala Ser Val Ser Arg Ile Ile Met Leu Ile Pro Thr Gly Thr Ser
1 5 10 15
Val Gly Leu Thr Ser Val Ser Leu Gly Val Ile Arg Ala Met Glu Arg
20 25 30
Lys Gly Val Arg Leu Ser Val Phe Lys Pro Ile Ala Gln Pro Arg Thr
35 40 45
Gly Gly Asp Ala Pro Asp Gln Thr Thr Thr Ile Val Arg Ala Asn Ser
50 55 60
Ser Thr Thr Thr Ala Ala Glu Pro Leu Lys Met Ser Tyr Val Glu Gly
65 70 75 80
Leu Leu Ser Ser Asn Gln Lys Asp Val Leu Met Glu Glu Ile Val Ala
85 90 95
Asn Tyr His Ala Asn Thr Lys Asp Ala Glu Val Val Leu Val Glu Gly
100 105 110
Leu Val Pro Thr Arg Lys His Gln Phe Ala Gln Ser Leu Asn Tyr Glu
115 120 125
Ile Ala Lys Thr Leu Asn Ala Glu Ile Val Phe Val Met Ser Gln Gly
130 135 140
Thr Asp Thr Pro Glu Gln Leu Lys Glu Arg Ile Glu Leu Thr Arg Asn
145 150 155 160
Ser Phe Gly Gly Ala Lys Asn Thr Asn Ile Thr Gly Val Ile Val Asn
165 170 175
Lys Leu Asn Ala Pro Val Asp Glu Gln Gly Arg Thr Arg Pro Asp Leu
180 185 190
Ser Glu Ile Phe Asp Asp Ser Ser Lys Ala Lys Val Asn Asn Val Asp
195 200 205
Pro Ala Lys Leu Gln Glu Ser Ser Pro Leu Pro Val Leu Gly Ala Val
210 215 220
Pro Trp Ser Phe Asp Leu Ile Ala Thr Arg Ala Ile Asp Met Ala Arg
225 230 235 240
His Leu Asn Ala Thr Ile Ile Asn Glu Gly Asp Ile Asn Thr Arg Arg
245 250 255
Val Lys Ser Val Thr Phe Cys Ala Arg Ser Ile Pro His Met Leu Glu
260 265 270
His Phe Arg Ala Gly Ser Leu Leu Val Thr Ser Ala Asp Arg Pro Asp
275 280 285
Val Leu Val Ala Ala Cys Leu Ala Ala Met Asn Gly Val Glu Ile Gly
290 295 300
Ala Leu Leu Leu Thr Gly Gly Tyr Glu Met Asp Ala Arg Ile Ser Lys
305 310 315 320
Leu Cys Glu Arg Ala Phe Ala Thr Gly Leu Pro Val Phe Met Val Asn
325 330 335
Thr Asn Thr Trp Gln Thr Ser Leu Ser Leu Gln Ser Phe Asn Leu Glu
340 345 350
Val Pro Val Asp Asp His Glu Arg Ile Glu Lys Val Gln Glu Tyr Val
355 360 365
Ala Asn Tyr Ile Asn Ala Asp Trp Ile Glu Ser Leu Thr Ala Thr Ser
370 375 380
Glu Arg Ser Arg Arg Leu Ser Pro Pro Ala Phe Arg Tyr Gln Leu Thr
385 390 395 400
Glu Leu Ala Arg Lys Ala Gly Lys Arg Ile Val Leu Pro Glu Gly Asp
405 410 415
Glu Pro Arg Thr Val Lys Ala Ala Ala Ile Cys Ala Glu Arg Gly Ile
420 425 430
Ala Thr Cys Val Leu Leu Gly Asn Pro Ala Glu Ile Asn Arg Val Ala
435 440 445
Ala Ser Gln Gly Val Glu Leu Gly Ala Gly Ile Glu Ile Val Asp Pro
450 455 460
Glu Val Val Arg Glu Ser Tyr Val Gly Arg Leu Val Glu Leu Arg Lys
465 470 475 480
Asn Lys Gly Met Thr Glu Thr Val Ala Arg Glu Gln Leu Glu Asp Asn
485 490 495
Val Val Leu Gly Thr Leu Met Leu Glu Gln Asp Glu Val Asp Gly Leu
500 505 510
Val Ser Gly Ala Val His Thr Thr Ala Asn Thr Ile Arg Pro Pro Leu
515 520 525
Gln Leu Ile Lys Thr Ala Pro Gly Ser Ser Leu Val Ser Ser Val Phe
530 535 540
Phe Met Leu Leu Pro Glu Gln Val Tyr Val Tyr Gly Asp Cys Ala Ile
545 550 555 560
Asn Pro Asp Pro Thr Ala Glu Gln Leu Ala Glu Ile Ala Ile Gln Ser
565 570 575
Ala Asp Ser Ala Ala Ala Phe Gly Ile Glu Pro Arg Val Ala Met Leu
580 585 590
Ser Tyr Ser Thr Gly Thr Ser Gly Ala Gly Ser Asp Val Glu Lys Val
595 600 605
Arg Glu Ala Thr Arg Leu Ala Gln Glu Lys Arg Pro Asp Leu Met Ile
610 615 620
Asp Gly Pro Leu Gln Tyr Asp Ala Ala Val Met Ala Asp Val Ala Lys
625 630 635 640
Ser Lys Ala Pro Asn Ser Pro Val Ala Gly Arg Ala Thr Val Phe Ile
645 650 655
Phe Pro Asp Leu Asn Thr Gly Asn Thr Thr Tyr Lys Ala Val Gln Arg
660 665 670
Ser Ala Asp Leu Ile Ser Ile Gly Pro Met Leu Gln Gly Met Arg Lys
675 680 685
Pro Val Asn Asp Leu Ser Arg Gly Ala Leu Val Asp Asp Ile Val Tyr
690 695 700
Thr Ile Ala Leu Thr Ala Ile Gln Ser Ala Gln Gln Gln
705 710 715
<210> 3
<211> 522
<212> PRT
<213> 假单胞菌(Pseudomonas aeruginosa)
<400> 3
Met Ala His His His His His His Val Gly Thr Asn Asp Ala Asn Ile
1 5 10 15
Ala Asp Val Val Thr Lys Val Leu Gly Glu Tyr Gly Ala Pro Gly Ala
20 25 30
Val Ser Val Ala Ala Leu Thr Ala Lys Ser Pro Asp Gly Lys Ser Asn
35 40 45
Ser Ser Ala Asp Ala Asp Val Val Ala Arg Met Val Ala Lys Ala Ile
50 55 60
Arg Asp His Ala Gly Thr Ala Gln Pro Ser Gly Asn Ala Ala Thr Ser
65 70 75 80
Ser Ala Ala Val Ser Asp Gly Val Phe Glu Thr Met Asp Ala Ala Val
85 90 95
Glu Ala Ala Ala Leu Ala Gln Gln Gln Tyr Leu Leu Cys Ser Met Ser
100 105 110
Asp Arg Ala Arg Phe Val Gln Gly Ile Arg Asp Val Ile Leu Asn Gln
115 120 125
Asp Thr Leu Glu Lys Met Ser Arg Met Ala Val Glu Glu Thr Gly Met
130 135 140
Gly Asn Tyr Glu His Lys Leu Ile Lys Asn Arg Leu Ala Gly Glu Lys
145 150 155 160
Thr Pro Gly Ile Glu Asp Leu Thr Thr Asp Ala Phe Ser Gly Asp Asn
165 170 175
Gly Leu Thr Leu Val Glu Tyr Ser Pro Phe Gly Val Ile Gly Ala Ile
180 185 190
Thr Pro Thr Thr Asn Pro Thr Glu Thr Ile Val Cys Asn Ser Ile Gly
195 200 205
Met Leu Ala Ala Gly Asn Ser Val Val Phe Ser Pro His Gly Arg Ala
210 215 220
Arg Gln Val Ser Leu Leu Leu Val Arg Leu Ile Asn Gln Lys Leu Ala
225 230 235 240
Ala Leu Gly Ala Pro Glu Asn Leu Val Val Thr Val Glu Lys Pro Ser
245 250 255
Arg Glu Asn Thr Leu Ala Met Met Ala His Pro Lys Val Arg Met Leu
260 265 270
Val Ala Thr Gly Gly Pro Ala Leu Val Lys Ala Val Leu Ser Thr Gly
275 280 285
Lys Lys Ala Ile Gly Ala Gly Ala Gly Asn Pro Pro Val Val Val Asp
290 295 300
Glu Thr Ala Asn Ile Glu Lys Ala Ala Cys Asp Ile Val Asn Gly Cys
305 310 315 320
Ser Phe Asp Asn Asn Ile Thr Cys Thr Ala Glu Lys Glu Ile Ile Ala
325 330 335
Val Ala Gln Ile Ala Asp Tyr Leu Ile Phe Asn Leu Lys Lys Asn Gly
340 345 350
Ala Tyr Glu Ile Lys Asp Pro Ala Val Leu Gln Gln Leu Gln Asp Leu
355 360 365
Val Leu Thr Ala Lys Gly Gly Pro Gln Thr Lys Cys Val Gly Lys Ser
370 375 380
Ala Val Trp Leu Leu Ser Gln Ile Gly Ile Ser Val Asp Ala Ser Ile
385 390 395 400
Lys Ile Ile Leu Met Glu Val Pro Arg Glu His Pro Phe Val Gln Glu
405 410 415
Glu Leu Met Met Pro Ile Leu Pro Leu Val Arg Val Glu Thr Val Asp
420 425 430
Asp Ala Ile Asp Leu Ala Ile Glu Val Glu His Asp Asn Arg His Thr
435 440 445
Ala Ile Met His Ser Thr Asp Val Arg Lys Leu Thr Lys Met Ala Lys
450 455 460
Leu Ile Gln Thr Thr Ile Phe Val Lys Asn Gly Pro Ser Tyr Ala Gly
465 470 475 480
His Gly Ala Gly Gly Glu Gly Tyr Ser Thr Phe Thr Ile Ala Gly Pro
485 490 495
Thr Gly Glu Gly Leu Thr Ser Ala Lys Ser Phe Ala Arg Arg Arg Lys
500 505 510
Cys Val Met Val Glu Ala Leu Asn Ile Arg
515 520
<210> 4
<211> 825
<212> PRT
<213> 双歧杆菌(Bifidobacterium)
<400> 4
Met Thr Ser Pro Val Ile Gly Thr Pro Trp Lys Lys Leu Asn Ala Pro
1 5 10 15
Val Ser Glu Glu Ala Ile Glu Gly Val Asp Lys Tyr Trp Arg Ala Ala
20 25 30
Asn Tyr Leu Ser Ile Gly Gln Ile Tyr Leu Arg Ser Asn Pro Leu Met
35 40 45
Lys Glu Pro Phe Thr Arg Glu Asp Val Lys His Arg Leu Val Gly His
50 55 60
Trp Gly Thr Thr Pro Gly Leu Asn Phe Leu Ile Gly His Ile Asn Arg
65 70 75 80
Leu Ile Ala Asp His Gln Gln Asn Thr Val Ile Ile Met Gly Pro Gly
85 90 95
His Gly Gly Pro Ala Gly Thr Ala Gln Ser Tyr Leu Asp Gly Thr Tyr
100 105 110
Thr Glu Tyr Phe Pro Asn Ile Thr Lys Asp Glu Ala Gly Leu Gln Lys
115 120 125
Phe Phe Arg Gln Phe Ser Tyr Pro Gly Gly Ile Pro Ser His Tyr Ala
130 135 140
Pro Glu Thr Pro Gly Ser Ile His Glu Gly Gly Glu Leu Gly Tyr Ala
145 150 155 160
Leu Ser His Ala Tyr Gly Ala Val Met Asn Asn Pro Ser Leu Phe Val
165 170 175
Pro Ala Ile Val Gly Asp Gly Glu Ala Glu Thr Gly Pro Leu Ala Thr
180 185 190
Gly Trp Gln Ser Asn Lys Leu Ile Asn Pro Arg Thr Asp Gly Ile Val
195 200 205
Leu Pro Ile Leu His Leu Asn Gly Tyr Lys Ile Ala Asn Pro Thr Ile
210 215 220
Leu Ser Arg Ile Ser Asp Glu Glu Leu His Glu Phe Phe His Gly Met
225 230 235 240
Gly Tyr Glu Pro Tyr Glu Phe Val Ala Gly Phe Asp Asn Glu Asp His
245 250 255
Leu Ser Ile His Arg Arg Phe Ala Glu Leu Phe Glu Thr Val Phe Asp
260 265 270
Glu Ile Cys Asp Ile Lys Ala Ala Ala Gln Thr Asp Asp Met Thr Arg
275 280 285
Pro Phe Tyr Pro Met Ile Ile Phe Arg Thr Pro Lys Gly Trp Thr Cys
290 295 300
Pro Lys Phe Ile Asp Gly Lys Lys Thr Glu Gly Ser Trp Arg Ser His
305 310 315 320
Gln Val Pro Leu Ala Ser Ala Arg Asp Thr Glu Ala His Phe Glu Val
325 330 335
Leu Lys Asn Trp Leu Glu Ser Tyr Lys Pro Glu Glu Leu Phe Asp Glu
340 345 350
Asn Gly Ala Val Lys Pro Glu Val Thr Ala Phe Met Pro Thr Gly Glu
355 360 365
Leu Arg Ile Gly Glu Asn Pro Asn Ala Asn Gly Gly Arg Ile Arg Glu
370 375 380
Glu Leu Lys Leu Pro Lys Leu Glu Asp Tyr Glu Val Lys Glu Val Ala
385 390 395 400
Glu Tyr Gly His Gly Trp Gly Gln Leu Glu Ala Thr Arg Arg Leu Gly
405 410 415
Val Tyr Thr Arg Asp Ile Ile Lys Asn Asn Pro Asp Ser Phe Arg Ile
420 425 430
Phe Gly Pro Asp Glu Thr Ala Ser Asn Arg Leu Gln Ala Ala Tyr Asp
435 440 445
Val Thr Asn Lys Gln Trp Asp Ala Gly Tyr Leu Ser Ala Gln Val Asp
450 455 460
Glu His Met Ala Val Thr Gly Gln Val Thr Glu Gln Leu Ser Glu His
465 470 475 480
Gln Met Glu Gly Phe Leu Glu Gly Tyr Leu Leu Thr Gly Arg His Gly
485 490 495
Ile Trp Ser Ser Tyr Glu Ser Phe Val His Val Ile Asp Ser Met Leu
500 505 510
Asn Gln His Ala Lys Trp Leu Glu Ala Thr Val Arg Glu Ile Pro Trp
515 520 525
Arg Lys Pro Ile Ser Ser Met Asn Leu Leu Val Ser Ser His Val Trp
530 535 540
Arg Gln Asp His Asn Gly Phe Ser His Gln Asp Pro Gly Val Thr Ser
545 550 555 560
Val Leu Leu Asn Lys Cys Phe Asn Asn Asp His Val Ile Gly Ile Tyr
565 570 575
Phe Pro Val Asp Ser Asn Met Leu Leu Ala Val Ala Glu Lys Cys Tyr
580 585 590
Lys Ser Thr Asn Lys Ile Asn Ala Ile Ile Ala Gly Lys Gln Pro Ala
595 600 605
Ala Thr Trp Leu Thr Leu Asp Glu Ala Arg Ala Glu Leu Glu Lys Gly
610 615 620
Ala Ala Glu Trp Lys Trp Ala Ser Asn Val Lys Ser Asn Asp Glu Ala
625 630 635 640
Gln Ile Val Leu Ala Ala Thr Gly Asp Val Pro Thr Gln Glu Ile Met
645 650 655
Ala Ala Ala Asp Lys Leu Asp Ala Met Gly Ile Lys Phe Lys Val Val
660 665 670
Asn Val Val Asp Leu Val Lys Leu Gln Ser Ala Lys Glu Asn Asn Glu
675 680 685
Ala Leu Ser Asp Glu Glu Phe Ala Glu Leu Phe Thr Glu Asp Lys Pro
690 695 700
Val Leu Phe Ala Tyr His Ser Tyr Ala Arg Asp Val Arg Gly Leu Ile
705 710 715 720
Tyr Asp Arg Pro Asn His Asp Asn Phe Asn Val His Gly Tyr Glu Glu
725 730 735
Gln Gly Ser Thr Thr Thr Pro Tyr Asp Met Val Arg Val Asn Asn Ile
740 745 750
Asp Arg Tyr Glu Leu Gln Ala Glu Ala Leu Arg Met Ile Asp Ala Asp
755 760 765
Lys Tyr Ala Asp Lys Ile Asn Glu Leu Glu Ala Phe Arg Gln Glu Ala
770 775 780
Phe Gln Phe Ala Val Asp Asn Gly Tyr Asp His Pro Asp Tyr Thr Asp
785 790 795 800
Trp Val Tyr Ser Gly Val Asn Thr Asn Lys Gln Gly Ala Ile Ser Ala
805 810 815
Thr Ala Ala Thr Ala Gly Asp Asn Glu
820 825
<210> 5
<211> 660
<212> PRT
<213> 沙门氏菌(Salmonella enterica)
<400> 5
Met Ala Gln Thr His Lys His Pro Val Pro Pro Asn Ile Ala Ala Asn
1 5 10 15
Ala Trp Ile Asn Pro Glu Gln Tyr Glu Ala Met Tyr Lys Gln Ser Ile
20 25 30
Asn Asp Pro Asp Thr Phe Trp Arg Glu Gln Gly Lys Ile Leu Asp Trp
35 40 45
Ile Lys Pro Tyr Thr Arg Val Lys Asn Thr Ser Phe Glu Pro Gly Asn
50 55 60
Val Ser Ile Arg Trp Tyr Glu Asp Gly Thr Leu Asn Leu Ala Tyr Asn
65 70 75 80
Cys Leu Asp Arg His Leu Glu Glu Arg Gly Asp Gln Thr Ala Ile Ile
85 90 95
Trp Glu Gly Asp Asp Pro Ser Glu Ser Arg His Ile Thr Tyr Arg Glu
100 105 110
Leu His Arg Asp Val Cys Arg Phe Ala Asn Val Leu Lys Ala Leu Gly
115 120 125
Ile Lys Lys Gly Asp Val Val Ala Ile Tyr Met Pro Met Val Pro Glu
130 135 140
Ala Ala Val Ala Met Leu Ala Cys Ala Arg Ile Gly Ala Ile His Ser
145 150 155 160
Val Ile Phe Gly Gly Phe Ser Pro Glu Ala Val Ala Gly Arg Ile Ile
165 170 175
Asp Ser Asn Ser Arg Leu Val Ile Thr Ala Asp Glu Gly Val Arg Ala
180 185 190
Gly Arg Ser Ile Pro Leu Lys Lys Asn Val Asp Glu Ala Leu Lys Asn
195 200 205
Pro Asn Val Thr Ser Val Glu His Val Ile Val Phe Lys Arg Thr Gly
210 215 220
Gly Asp Ile Asp Trp Gln Glu Gly Arg Asp Leu Trp Trp Arg Asp Leu
225 230 235 240
Ile Glu Lys Ala Ser Pro Glu His Gln Pro Glu Pro Met Asn Ala Glu
245 250 255
Asp Pro Leu Phe Ile Leu Tyr Thr Ser Gly Ser Thr Gly Lys Pro Lys
260 265 270
Gly Val Leu His Thr Thr Gly Gly Tyr Leu Val Tyr Ala Ala Thr Thr
275 280 285
Phe Lys Tyr Val Phe Asp Tyr His Pro Gly Asp Ile Tyr Trp Cys Thr
290 295 300
Ala Asp Val Gly Trp Ile Thr Gly His Ala Tyr Leu Leu Tyr Gly Pro
305 310 315 320
Leu Ala Cys Gly Ala Thr Thr Leu Met Phe Glu Gly Val Pro Asn Trp
325 330 335
Pro Thr Pro Ser Arg Met Ala Gln Val Val Asp Lys His Gln Val Asn
340 345 350
Ile Leu Phe Thr Ala Pro Thr Ala Ile Arg Ala Leu Met Ala Glu Gly
355 360 365
Asp Lys Ala Ile Glu Gly Thr Asp Arg Ser Ser Leu Arg Ile Leu Gly
370 375 380
Ser Thr Gly Glu Pro Ile Asn Pro Glu Ala Trp Glu Trp Tyr Trp Lys
385 390 395 400
Lys Ile Gly Asn Glu Arg Cys Pro Val Val Asp Thr Trp Trp Gln Thr
405 410 415
Glu Thr Gly Gly Cys Met Ile Thr Pro Leu Pro Gly Ala Ile Glu Leu
420 425 430
Lys Pro Gly Ser Ala Thr Arg Pro Phe Phe Gly Val Gln Pro Ala Leu
435 440 445
Val Asp Asn Glu Gly Asn Pro Gln Glu Gly Ala Thr Glu Gly Asn Leu
450 455 460
Val Ile Thr Asp Ser Trp Pro Gly Gln Ala Arg Thr Leu Tyr Gly Asp
465 470 475 480
His Glu Arg Phe Glu Gln Thr Tyr Phe Ser Thr Phe Lys Gly Met Tyr
485 490 495
Phe Ser Gly Asp Gly Ala Arg Arg Asp Glu Asp Gly Tyr Tyr Trp Ile
500 505 510
Thr Gly Arg Val Asp Asp Val Leu Asn Val Ser Gly His Arg Leu Gly
515 520 525
Thr Ala Glu Ile Glu Ser Ala Leu Val Ala His Pro Lys Ile Ala Glu
530 535 540
Ala Ala Val Val Gly Ile Pro His Asp Ile Lys Gly Gln Ala Ile Tyr
545 550 555 560
Ala Tyr Val Thr Leu Asn His Gly Glu Glu Pro Ser Pro Glu Leu Tyr
565 570 575
Lys Glu Val Arg Asn Trp Val Arg Lys Glu Ile Gly Pro Leu Ala Thr
580 585 590
Pro Asp Val Leu His Trp Thr Asp Gly Leu Pro Lys Thr Arg Ser Gly
595 600 605
Lys Ile Met Arg Arg Ile Leu Arg Lys Ile Ala Ala Gly Asp Thr Ser
610 615 620
Asn Leu Gly Asp Thr Ser Thr Leu Ala Asp Pro Ser Val Val Asp Lys
625 630 635 640
Pro Ile Glu Glu Lys Gln Ala Ile Ala Met Pro Ser Leu Glu His His
645 650 655
His His His His
660
<210> 6
<211> 1200
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 6
atgtcttcta aactggttct ggttctgaac tgcggttctt cttctctgaa attcgctatc 60
atcgacgctg ttaacggtga cgaatacctg tctggtctgg ctgaatgctt ccacctgccg 120
gaagctcgta tcaaatggaa aatggacggt tctaaacagg aagctgctct gggtgctggt 180
gctgctcact ctgaagctct gaacttcatc gttaacacca tcctggctca gaaaccggaa 240
ctgtctgctc agctgaccgc tatcggtcac cgtatcgttc acggtggtga aaaatacacc 300
tcttctgttg ttatcgacga atctgttatc cagggtatca aagactctgc ttctttcgct 360
ccgctgcaca acccggctca cctgatcggt atcgctgaag ctctgaaatc tttcccgcag 420
ctgaaagaca aaaacgttgc tgttttcgac accgctttcc accagaccat gccggaagaa 480
tcttacctgt acgctctgcc gtactctctg tacaaagaac acggtgttcg tcgttacggt 540
gctcacggta cctctcactt ctacgttacc caggaagctg ctaaaatgct gaacaaaccg 600
gttgaagaac tgaacatcat cacctgccac ctgggtaacg gtggttctgt ttctgctatc 660
cgtaacggta aatgcgttga cacctctatg ggtctgaccc cgctggaagg tctggttatg 720
ggtacccgtt ctggtgacat cgacccggct atcatcttcc acctgcacga caccctgggt 780
atgtctgttg accagatcaa caaaatgctg accaaagaat ctggtctgct gggtctgacc 840
gaagttacct ctgactgccg ttacgttgaa gacaactacg ctaccaaaga agacgctaaa 900
cgtgctatgg acgtttactg ccaccgtctg gctaaataca tcggttctta caccgctctg 960
atggacggtc gtctggacgc tgttgttttc accggtggta tcggtgaaaa cgctgctatg 1020
gttcgtgaac tgtctctggg taaactgggt gttctgggtt tcgaagttga ccacgaacgt 1080
aacctggctg ctcgtttcgg taaatctggt ttcatcaaca aagaaggtac ccgtccggct 1140
gttgttatcc cgaccaacga agaactggtt atcgctcagg acgcttctcg tctgaccgct 1200
<210> 7
<211> 2154
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 7
atggctagcg tgtcccgtat tattatgctg atccctaccg gaaccagcgt cggtctgacc 60
agcgtcagcc ttggcgtgat ccgtgcaatg gaacgcaaag gcgttcgtct gagcgttttc 120
aaacctatcg ctcagccgcg taccggtggc gatgcgcccg atcagactac gactatcgtg 180
cgtgcgaact cttccaccac gacggccgct gaaccgctga aaatgagcta cgttgaaggt 240
ctgctttcca gcaatcagaa agatgtgctg atggaagaga tcgtcgcaaa ctaccacgct 300
aacaccaaag acgctgaagt cgttctggtt gaaggtctgg tcccgacacg taagcaccag 360
tttgcccagt ctctgaacta cgaaatcgct aaaacgctga atgcggaaat cgtcttcgtt 420
atgtctcagg gcactgacac cccggaacag ctgaaagagc gtatcgaact gacccgcaac 480
agcttcggcg gtgccaaaaa caccaacatc accggcgtta tcgttaacaa actgaacgca 540
ccggttgatg aacagggtcg tactcgcccg gatctgtccg agattttcga cgactcttcc 600
aaagctaaag taaacaatgt tgatccggcg aagctgcaag aatccagccc gctgccggtt 660
ctcggcgctg tgccgtggag ctttgacctg atcgcgactc gtgcgatcga tatggctcgc 720
cacctgaatg cgaccatcat caacgaaggc gacatcaata ctcgccgcgt taaatccgtc 780
actttctgcg cacgcagcat tccgcacatg ctggagcact tccgtgccgg ttctctgctg 840
gtgacttccg cagaccgtcc tgacgtgctg gtggccgctt gcctggcagc catgaacggc 900
gtagaaatcg gtgccctgct gctgactggc ggttacgaaa tggacgcgcg catttctaaa 960
ctgtgcgaac gtgctttcgc taccggcctg ccggtattta tggtgaacac caacacctgg 1020
cagacctctc tgagcctgca gagcttcaac ctggaagttc cggttgacga tcacgaacgt 1080
atcgagaaag ttcaggaata cgttgctaac tacatcaacg ctgactggat cgaatctctg 1140
actgccactt ctgagcgcag ccgtcgtctg tctccgcctg cgttccgtta tcagctgact 1200
gaacttgcgc gcaaagcggg caaacgtatc gtactgccgg aaggtgacga accgcgtacc 1260
gttaaagcag ccgctatctg tgctgaacgt ggtatcgcaa cttgcgtact gctgggtaat 1320
ccggcagaga tcaaccgtgt tgcagcgtct cagggtgtag aactgggtgc agggattgaa 1380
atcgttgatc cagaagtggt tcgcgaaagc tatgttggtc gtctggtcga actgcgtaag 1440
aacaaaggca tgaccgaaac cgttgcccgc gaacagctgg aagacaacgt ggtgctcggt 1500
acgctgatgc tggaacagga tgaagttgat ggtctggttt ccggtgctgt tcacactacc 1560
gcaaacacca tccgtccgcc gctgcagctg atcaaaactg caccgggcag ctccctggta 1620
tcttccgtgt tcttcatgct gctgccggaa caggtttacg tttacggtga ctgtgcgatc 1680
aacccggatc cgaccgctga acagctggca gaaatcgcga ttcagtccgc tgattccgct 1740
gcggccttcg gtatcgaacc gcgcgttgct atgctctcct actccaccgg tacttctggt 1800
gcaggtagcg acgtagaaaa agttcgcgaa gcaactcgtc tggcgcagga aaaacgtcct 1860
gacctgatga tcgacggtcc gctgcagtac gacgctgcgg taatggctga cgttgcgaaa 1920
tccaaagcgc cgaactctcc ggttgcaggt cgcgctaccg tgttcatctt cccggatctg 1980
aacaccggta acaccaccta caaagcggta cagcgttctg ccgacctgat ctccatcggg 2040
ccgatgctgc agggtatgcg caagccggtt aacgacctgt cccgtggcgc actggttgac 2100
gatatcgtct acaccatcgc gctgactgcg attcagtctg cacagcagca gtaa 2154
<210> 8
<211> 1569
<212> DNA
<213> 假单胞菌(Pseudomonas aeruginosa)
<400> 8
atggcgcacc atcaccacca tcatgttggc accaacgatg cgaacatcgc cgatgttgtg 60
acgaaggttc tgggtgaata tggcgcgccg ggcgccgtta gtgttgccgc gctcaccgcc 120
aaaagtccgg atggtaagag caatagcagc gcggatgccg atgtggttgc gcgtatggtt 180
gccaaggcca ttcgcgatca cgcgggtacc gcccaaccaa gcggtaatgc cgccaccagt 240
agtgccgcgg ttagtgacgg cgtgtttgaa acgatggatg ccgccgttga agcggcggcg 300
ctggcccagc agcaatatct gctgtgcagc atgagtgatc gtgcccgctt cgtgcaaggc 360
attcgtgacg tgattctgaa tcaagatacg ctggagaaga tgagtcgtat ggccgttgaa 420
gagaccggca tgggcaacta cgagcacaag ctcatcaaga accgtctggc gggcgaaaaa 480
acgccgggca tcgaagatct gaccacggac gcgttcagcg gtgataacgg tctgacgctg 540
gttgaataca gcccattcgg cgttatcggc gccattaccc caacgaccaa tccgaccgaa 600
accatcgttt gcaacagcat cggcatgctc gccgccggta atagcgttgt gttcagtccg 660
catggtcgtg cccgtcaagt tagtctgctg ctggtgcgtc tgatcaatca gaaactcgcg 720
gcgctgggtg ccccggaaaa tctggtggtg acggtggaaa aaccgagccg cgaaaatacg 780
ctggccatga tggcgcaccc gaaagtgcgc atgctggttg ccacgggtgg tccagcgctg 840
gtgaaagccg ttctcagcac cggtaagaaa gccatcggtg ccggtgcggg taacccaccg 900
gttgtggtgg atgaaaccgc caacatcgag aaagcggcgt gcgacatcgt gaacggctgc 960
agctttgaca acaatatcac gtgcaccgcc gagaaggaga tcattgccgt ggcccagatt 1020
gccgactatc tgatcttcaa tctgaagaaa aatggcgcct acgaaatcaa agacccggcg 1080
gtgctgcagc agctgcaaga tctggtgctg acggccaaag gtggtccgca gaccaagtgt 1140
gtgggtaaaa gcgcggtttg gctgctgagc caaattggca tcagcgtgga cgccagcatc 1200
aagatcattc tgatggaggt tccgcgcgag catccgttcg tgcaagaaga actgatgatg 1260
ccgattctgc cactggtgcg tgtggaaacc gttgatgatg ccattgatct ggcgatcgaa 1320
gtggaacacg acaatcgcca tacggcgatc atgcacagca ccgacgtgcg taaactgacc 1380
aagatggcga agctgattca gacgaccatc ttcgtgaaaa acggtccaag ctacgccggt 1440
catggtgcgg gtggcgaagg ctatagtacg tttaccatcg ccggcccaac cggcgaaggt 1500
ctgacgagcg cgaaaagctt tgcgcgtcgt cgcaagtgtg tgatggttga ggccctcaat 1560
atccgctaa 1569
<210> 9
<211> 2478
<212> DNA
<213> 双歧杆菌(Bifidobacterium)
<400> 9
atgacctctc cggttatcgg taccccgtgg aaaaaactga acgcgccggt ttctgaagaa 60
gcgatcgaag gtgttgacaa atactggcgt gcggcgaact acctgtctat cggtcagatc 120
tacctgcgtt ctaacccgct gatgaaagaa ccgttcaccc gtgaagacgt taaacaccgt 180
ctggttggtc actggggtac caccccgggt ctgaacttcc tgatcggtca catcaaccgt 240
ctgatcgcgg accaccagca gaacaccgtt atcatcatgg gtccgggtca cggtggtccg 300
gcgggtaccg cgcagtctta cctggacggt acctacaccg aatacttccc gaacatcacc 360
aaagacgaag cgggtctgca gaaattcttc cgtcagttct cttacccggg tggtatcccg 420
tctcactacg cgccggaaac cccgggttct atccacgaag gtggtgaact gggttacgcg 480
ctgtctcacg cgtacggtgc ggttatgaac aacccgtctc tgttcgttcc ggcgatcgtt 540
ggtgacggtg aagcggaaac cggtccgctg gcgaccggtt ggcagtctaa caaactgatc 600
aacccgcgta ccgacggtat cgttctgccg atcctgcacc tgaacggtta caaaatcgcg 660
aacccgacca tcctgtctcg tatctctgac gaagaactgc acgagttctt ccacggtatg 720
ggttacgaac cgtacgagtt cgttgcgggt ttcgacaacg aagaccacct gtctatccac 780
cgtcgtttcg cggaactgtt cgaaaccgtt ttcgacgaaa tctgcgacat caaagcggcg 840
gcgcagaccg acgacatgac ccgtccgttc tacccgatga tcatcttccg taccccgaaa 900
ggttggacct gcccgaaatt catcgacggt aaaaaaaccg aaggttcttg gcgttctcac 960
caggttccgc tggcgtctgc gcgtgacacc gaagcgcact tcgaagttct gaaaaactgg 1020
ctggaatctt acaaaccgga agaactgttc gacgaaaacg gtgcggttaa accggaagtt 1080
accgcgttca tgccgaccgg tgaactgcgt atcggtgaaa acccgaacgc gaacggtggt 1140
cgtatccgtg aagaactgaa actgccgaaa ctggaagact acgaagttaa agaagttgcg 1200
gaatacggtc acggttgggg tcagctggaa gcgacccgtc gtctgggtgt ttacacccgt 1260
gacatcatca aaaacaaccc ggactctttc cgtatcttcg gtccggacga aaccgcgtct 1320
aaccgtctgc aggcggcgta cgacgttacc aacaaacagt gggacgcggg ttacctgtct 1380
gcgcaggttg acgaacacat ggcggttacc ggtcaggtta ccgaacagct gtctgaacac 1440
cagatggaag gtttcctgga aggttacctg ctgaccggtc gtcacggtat ctggtcttct 1500
tacgaatctt tcgttcacgt tatcgactct atgctgaacc agcacgcgaa atggctggaa 1560
gcgaccgttc gtgaaatccc gtggcgtaaa ccgatctctt ctatgaacct gctggtttct 1620
tctcacgttt ggcgtcagga ccacaacggt ttctctcacc aggacccggg tgttacctct 1680
gttctgctga acaaatgctt caacaacgac cacgttatcg gtatctactt cccggttgac 1740
tctaacatgc tgctggcggt tgcggaaaaa tgctacaaat ctaccaacaa aatcaacgcg 1800
atcatcgcgg gtaaacagcc ggcggcgacc tggctgaccc tggacgaagc gcgtgcggaa 1860
ctggaaaaag gtgcggcgga atggaaatgg gcgtctaacg ttaaatctaa cgacgaagcg 1920
cagatcgttc tggcggcgac cggtgacgtt ccgacccagg aaatcatggc ggcggcggac 1980
aaactggacg cgatgggtat caaattcaaa gttgttaacg ttgttgacct ggttaaactg 2040
cagtctgcga aagaaaacaa cgaagcgctg tctgacgaag agttcgcgga actgttcacc 2100
gaagacaaac cggttctgtt cgcgtaccac tcttacgcgc gtgacgttcg tggtctgatc 2160
tacgaccgtc cgaaccacga caacttcaac gttcacggtt acgaagaaca gggttctacc 2220
accaccccgt acgacatggt tcgtgttaac aacatcgacc gttacgaact gcaggcggaa 2280
gcgctgcgta tgatcgacgc ggacaaatac gcggacaaaa tcaacgaact ggaagcgttc 2340
cgtcaggaag cgttccagtt cgcggttgac aacggttacg accacccgga ctacaccgac 2400
tgggtttact ctggtgttaa caccaacaaa cagggtgcga tctctgcgac cgcggcgacc 2460
gcgggtgaca acgaatga 2478
<210> 10
<211> 1983
<212> DNA
<213> 沙门氏菌(Salmonella enterica)
<400> 10
atggctcaga cccacaaaca cccggttccg ccgaacatcg cggcgaacgc gtggatcaac 60
ccggaacagt acgaagcgat gtacaaacag tctatcaacg acccggacac cttctggcgt 120
gaacagggta aaatcctgga ctggatcaaa ccgtacaccc gtgttaaaaa cacctctttc 180
gaaccgggta acgtttctat ccgttggtac gaagacggta ccctgaacct ggcgtacaac 240
tgcctggacc gtcacctgga agaacgtggt gaccagaccg cgatcatctg ggaaggtgac 300
gacccgtctg aatctcgtca catcacctac cgtgaactgc accgtgacgt ttgccgtttc 360
gcgaacgttc tgaaagcgct gggtatcaaa aaaggtgacg ttgttgcgat ctacatgccg 420
atggttccgg aagcggcggt tgcgatgctg gcgtgcgcgc gtatcggtgc gatccactct 480
gttatcttcg gtggtttctc tccggaagcg gttgcgggtc gtatcatcga ctctaactct 540
cgtctggtta tcaccgcgga cgaaggtgtt cgtgcgggtc gttctatccc gctgaaaaag 600
aacgttgacg aagcgctgaa aaacccgaac gttacctctg ttgaacacgt tatcgttttc 660
aaacgtaccg gtggtgacat cgactggcag gaaggtcgtg acctgtggtg gcgtgacctg 720
atcgaaaaag cgtctccgga acaccagccg gaaccgatga acgcggaaga cccgctgttc 780
atcctgtaca cctctggttc taccggtaaa ccgaaaggtg ttctgcacac caccggtggt 840
tacctggttt acgcggcgac caccttcaaa tacgttttcg actaccaccc gggtgacatc 900
tactggtgca ccgcggacgt tggttggatt accggtcacg cgtatctgct gtacggtccg 960
ctggcgtgcg gtgcgaccac cctgatgttc gaaggtgttc cgaactggcc gaccccgtct 1020
cgtatggcgc aggttgttga caaacaccag gttaacatcc tgtttaccgc gccgaccgcg 1080
atccgtgcgc tgatggcgga aggtgacaaa gcgatcgaag gtaccgaccg ttcttctctg 1140
cgtatcctgg gttctaccgg tgaaccgatc aacccggaag cgtgggaatg gtactggaaa 1200
aaaatcggta acgaacgttg cccggttgtt gacacctggt ggcagaccga aaccggtggt 1260
tgcatgatca ccccgctgcc gggtgcgatc gaactgaaac cgggttctgc gacccgtccg 1320
ttcttcggtg ttcagccggc gctggttgac aacgaaggta acccgcagga aggtgcgacc 1380
gaaggtaacc tggttatcac cgactcttgg ccgggtcagg cgcgtaccct gtacggtgac 1440
cacgaacgtt tcgaacagac ctacttctct accttcaaag gtatgtactt ctctggtgac 1500
ggtgcgcgtc gtgacgaaga cggttactac tggatcaccg gtcgtgttga cgacgttctg 1560
aacgtttctg gtcaccgtct gggtaccgcg gaaatcgaat ctgcgctggt tgcgcacccg 1620
aaaatcgcgg aagcggcggt tgttggtatc ccgcacgaca tcaaaggtca ggcgatctac 1680
gcgtacgtta ccctgaacca cggtgaagaa ccgtctccgg aactgtacaa agaagttcgt 1740
aactgggttc gtaaagaaat cggtccgctg gcgaccccgg acgttctgca ctggaccgac 1800
ggtctgccga aaacccgttc tggtaaaatc atgcgtcgta tcctgcgtaa aatcgcggcg 1860
ggtgacacct ctaacctggg tgacacctct accctggcgg acccgtctgt tgttgacaaa 1920
ccgatcgaag aaaaacaggc gatcgcgatg ccgtctctcg agcaccacca ccaccaccac 1980
tga 1983

Claims (8)

1.一种乙醇酸在酶的作用下生成至少一种目标产物的方法，其特征在于，包括如下步骤：
步骤(1)：乙醇酸在乙酸激酶(AckA)的作用下生成羟乙酰磷酸，进一步在磷酸乙酰转移酶的作用下生成羟乙酰辅酶A；或者乙醇酸在羟乙酰辅酶A合酶作用下生成羟乙酰辅酶A；
步骤(2)：羟乙酰辅酶A进一步在羟乙酰辅酶A还原酶作用下生成乙醇醛；
任选的步骤(3)：乙醇醛进一步在乙酰磷酸合酶的作用下生成乙酰磷酸；以及
任选的步骤(4)：乙酰磷酸进一步在磷酸乙酰转移酶的作用下生成乙酰辅酶A；
优选地，所述方法中步骤(1)具体为：乙醇酸在ATP、乙酸激酶的作用下生成羟乙酰磷酸，进一步在NAD(P)H和磷酸乙酰转移酶的作用下生成羟乙酰辅酶A；或者乙醇酸在ATP、NADP(H)、辅酶A、羟乙酰辅酶A合酶作用下生成羟乙酰辅酶A；
优选地，所述方法中步骤(2)具体为：羟乙酰辅酶A进一步在辅酶A、羟乙酰辅酶A还原酶作用下生成乙醇醛；
优选地，所述方法中步骤(4)具体为：乙酰磷酸进一步在磷酸乙酰转移酶、辅酶A的作用下生成乙酰辅酶A；
优选地，所述方法中步骤(1)至(4)的转化反应在10～40℃下进行，进一步优选地在16-37℃，更优选的在30℃。
2.根据权利要求1所述的方法，其特征在于，所述方法中乙酸激酶与SEQ ID NO:1所示的氨基酸序列至少95％相同，所述磷酸乙酰转移酶与SEQ ID NO:2所示的氨基酸序列至少95％相同，所述羟乙酰辅酶A还原酶与SEQ ID NO:3所示的氨基酸序列至少95％相同，所述乙酰磷酸合酶与SEQ ID NO:4所示的氨基酸序列至少95％相同，所述羟乙酰辅酶A合酶与SEQ ID NO:5所示的氨基酸序列至少95％相同；
优选地，所述方法中乙酸激酶包含如SEQ ID NO:1所示的氨基酸序列，所述磷酸乙酰转移酶包含如SEQ ID NO:2所示的氨基酸序列，所述羟乙酰辅酶A还原酶包含如SEQ ID NO:3所示的氨基酸序列，所述乙酰磷酸合酶包含如SEQ ID NO:4所示的氨基酸序列，所述羟乙酰辅酶A合酶包含如SEQ ID NO:5所示的氨基酸序列。
3.根据权利要求1或2所述的方法，其特征在于，所述目标产物为乙酰辅酶A时，所述方法包括步骤(1)、(2)、(3)和(4)；
优选地，所述方法中的步骤(1)、步骤(2)、步骤(3)以及步骤(4)中任两个步骤、三个步骤或者四个步骤同时进行。
4.根据权利要求1或2所述的方法，其特征在于，所述目标产物为乙醇醛时，所述方法包括步骤(1)和(2)；
优选地，所述方法中的步骤(1)、步骤(2)同时进行。
5.一种生产具有增加的生长或生产力的植物的方法，其特征在于，包括以下步骤：在所述植物的至少一部分叶绿体中将乙醇酸转化为乙酰辅酶A；此步骤包括在叶绿体中产生转基因的乙酸激酶、磷酸乙酰转移酶、羟乙酰辅酶A还原酶和乙酰磷酸合酶的步骤；或者包括在叶绿体中产生转基因羟乙酰辅酶A合酶、羟乙酰辅酶A还原酶、乙酰磷酸合酶和磷酸乙酰转移酶的步骤。
6.根据权利要求5所述的方法，其特征在于，所述方法，其中所述乙酸激酶与SEQ IDNO:1所示的氨基酸序列至少95％相同，所述磷酸乙酰转移酶与SEQ ID NO:2所示的氨基酸序列至少95％相同，所述羟乙酰辅酶A还原酶与SEQ ID NO:3所示的氨基酸序列至少95％相同，所述乙酰磷酸合酶与SEQ ID NO:4所示的氨基酸序列至少95％相同，所述羟乙酰辅酶A合酶与SEQ ID NO:5所示的氨基酸序列至少95％相同。
7.根据权利要求5所述的方法，其特征在于，所述方法，其中所述乙酸激酶包含如SEQID NO:1所示的氨基酸序列，所述磷酸乙酰转移酶(PTA)包含如SEQ ID NO:2所示的氨基酸序列，所述羟乙酰辅酶A还原酶包含如SEQ ID NO:3所示的氨基酸序列，所述乙酰磷酸合酶包含如SEQ ID NO:4所示的氨基酸序列，所述羟乙酰辅酶A合酶包含如SEQ ID NO:5所示的氨基酸序列。
8.根据权利要求5-7任一项所述的方法，其特征在于，所述植物是C3植物，优选为稻、大豆、马铃薯、豇豆、大麦、小麦或木薯。
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