CN115181168B - 对乙醇不敏感对正丁醇特异性响应的BmoR蛋白突变体 - Google Patents
对乙醇不敏感对正丁醇特异性响应的BmoR蛋白突变体 Download PDFInfo
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- CN115181168B CN115181168B CN202110361553.XA CN202110361553A CN115181168B CN 115181168 B CN115181168 B CN 115181168B CN 202110361553 A CN202110361553 A CN 202110361553A CN 115181168 B CN115181168 B CN 115181168B
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
本发明属于生物工程技术领域，具体涉及一种对正丁醇特异性响应的BmoR蛋白突变体，及其在正丁醇检测或生物传感器中的应用。所述BmoR蛋白突变体是在序列表SEQ ID NO.1所示的野生型BmoR蛋白的基础上发生W21R和/或E54V突变获得的。上述突变体仅对正丁醇敏感，对乙醇或异丁醇无响应，解决了野生型BmoR蛋白无法区分正丁醇和异丁醇的问题；同时对正丁醇的检测范围达到0‑100mM，提高了BmoR蛋白对正丁醇的响应饱和度，可用于更高产量菌株的筛选和应用。
Description
技术领域：
本发明属于生物工程技术领域，具体涉及一种对乙醇高度不敏感对正丁醇特异性响应的BmoR蛋白突变体，及其在正丁醇检测或生物传感器中的应用。
背景技术：
微生物合成的正丁醇是重要的运输燃料，通过代谢工程合成正丁醇在许多微生物宿主中实现，而改造宿主菌株并筛选出高产宿主是实现醇工业化生产的基础和关键。
乙醇是一种可再生的生物燃料，由多种生物质材料制成。燃料乙醇原料包括淀粉和糖含量高的谷物和农作物，例如玉米，高粱，大麦，甘蔗和甜菜。乙醇也可以由草，树，农林业残留物(例如玉米芯和木料，稻草，锯末和木屑)制成。目前，生物乙醇用作公路运输车辆汽油替代品的主要燃料，主要通过糖发酵工艺生产。在目前的能源不安全状况和化石对环境安全的挑战中，生物乙醇已发展成为生物燃料生产的潜在来源。对于生物乙醇生产，已经对广泛的生物质资源进行了研究和实验。但在乙醇的生产途径中，伴随着乙醇的大量生产，各种高级醇如正丁醇、异丁醇或异戊醇的产量也在提高。同时，若在乙醇酒精中高级醇含量过多，会大大损坏酒体的口味，味苦涩，且还带有臭味。而且酒中高级醇过多会使人头疼，饮用后易上头，易喝醉。高级醇在水中的溶解度较小，在白酒降度中随着乙醇浓度的减小其溶解度也将减小，这就给白酒降度带来了一定的困难。因此实现对众多副产物的识别筛选显得尤为重要。
生物传感器能够特异性响应目标化合物从而输出便于检测的蛋白信号，因而已经被广泛应用于高通量筛选。
生物传感器由分子识别元件和信号转换器组成。当分子识别元件与被测物体结合时，所产生的信号可以通过转换器转换为光信号或电信号，可对被测物进行检测和分析。作为一种合成生物学新兴工具，可以通过设计构建生物传感器来动态的响应信号分子浓度的变化。与此同时，生物传感器的设计是为了促进微生物细胞工厂的优化和生产一系列广泛应用于工业的天然产品，如衣康酸、脂肪酸、异丁醇、正丁醇和生物碱。生物传感器主要包括RNA核酸开关、转录因子调控的生物传感器、G蛋白偶联受体以及荧光蛋白生物传感器。而荧光蛋白生物传感器的低动态范围、RNA核酸开关难以在体外进行、G蛋白偶联受体仅能在胞外进行等弊端阻碍了生物传感器在生物界的发展。
基于转录因子(TF)的生物传感器应用最为广泛。最常用的转录因子是细菌转录因子，包括配体结合结构域(LBD)或代谢结合结构域(MBD)和DNA结合结构域(DBD)。BmoR是假单胞菌正链烷烃代谢途径的转录因子，是bEBP的成员，用于调节烷烃单加氧酶的σ54依赖型启动子Pbmo，信号分子是C2-C5的直链或支链醇。但野生型转录因子BmoR存在响应特异性差，对底物正丁醇和异丁醇均会产生响应，无法对其中一种醇进行特异响应，从而满足工业需求；以及检测范围窄(0-40mM)等问题，无法广泛应用于生物传感器中。因此实现对醇的特异响应成并提高检测范围成为亟待解决的问题。
本发明通过蛋白质改造得到的BmoR蛋白突变体，不仅实现了对乙醇的高度不敏感，还可以对正丁醇产生特异响应，同时提高了其底物检测上限，为高效检测正丁醇和快速筛选高产菌株提供了一种解决方案。
发明内容：
本发明的目的在于提供一种对正丁醇能产生特异性响应，但对乙醇或异丁醇不敏感的BmoR蛋白及生物传感器，通过引入蛋白质工程方法对野生型BmoR 的N端进行修饰，利用易错PCR技术构建随机突变文库；通过外源添加乙醇，正丁醇和异丁醇，对突变库进行筛选分析，最终得到了对乙醇或异丁醇高度不敏感且对正丁醇特异性响应的BmoR突变体蛋白。
进一步地，所述BmoR突变体蛋白，是在序列表SEQ ID NO.1所示的野生型BmoR蛋白的基础上发生W21R和/或E54V突变获得的，以下简称W21R突变体、E54V突变体和W21R/E54V突变体(同时发生W21R和E54V突变)，所述突变体蛋白具体为：
(1)序列表SEQ ID NO.3、5或7所示的氨基酸序列；或
(2)SEQ ID NO.3、5或7同源性75％以上的氨基酸序列；或
(3)在SEQ ID NO.3、5或7的基础上进行一个或多个氨基酸替换，和/或缺失，和/或添加后获得的具有SEQ ID NO.3、5或7相同功能的氨基酸序列。
进一步地，本发明还提供W21R突变体、E54V突变体和W21R/E54V突变体的编码基因；
更进一步地，所述编码基因如序列表SEQ ID NO.4、6或8所示。
本发明的另一目的是提供W21R突变体、E54V突变体和W21R/E54V突变体的应用，特别是在检测含正丁醇的样品或筛选正丁醇生产菌株中的应用，更特别地，是在构建检测正丁醇的生物传感器中的应用；
进一步地，所述生物传感器是基于W21R突变体、E54V突变体或 W21R/E54V突变体的生物传感器，所述传感器是包含W21R突变体、E54V突变体或W21R/E54V突变体编码基因及其启动子、启动子Pbmo及报告基因的表达元件；启动子启动bmoR基因表达，BmoR蛋白与醇分子结合形成六聚体，进而启动下游启动子Pbmo，从而表达报告基因，产生荧光等信号；所述生物传感器能够实现对0-100mM浓度下的正丁醇的特异性响应和筛选，进一步应用于工业生产中，实现对含正丁醇的样品以及正丁醇生产菌株的特异性筛选；
进一步地，所述突变体编码基因的启动子包括但不限于PbmoR、Ptac、PT7、 PLlacO1等；
进一步地，所述报告基因包括但不限于gfp、rfp、cfp、sfgfp、egfp、yfp、ecfp 等基因；
优选地，所述生物传感器为包含W21R突变体、E54V突变体或W21R/E54V 突变体编码基因及其启动子PbmoR、启动子Pbmo及gfp报告基因的重组质粒；进一步地，所述重组质粒可选用的表达载体包括但不限于本领域通用的表达载体，如pET、pUC19、pMAL等；
更优选地，所述生物传感器是将质粒pYH1上的野生型BmoR蛋白编码基因替换为W21R突变体、E54V突变体或W21R/E54V突变体编码基因所得，也即，将由PbmoR启动的W21R突变体、E54V突变体或W21R/E54V突变体编码基因连接至colE1复制起始位点、ampr和Pbmo驱动的gfp基因所得；
进一步地，所述正丁醇生产菌株包括但不限于大肠杆菌，酿酒酵母，枯草芽孢杆菌等；
进一步地，所述启动子PbmoR的核苷酸序列如序列表SEQ ID NO.9所示；
进一步地，所述启动子Pbmo的核苷酸序列如序列表SEQ ID NO.10所示；
进一步地，所述gfp报告基因的核苷酸序列如序列表SEQ ID NO.11所示。
本发明还提供上述生物传感器在正丁醇检测中的应用，特别是在含有正丁醇的环境、食品、医学、生物类样品，或正丁醇生产菌株筛选中的应用，通过将上述质粒导入生产菌株，如大肠杆菌，酿酒酵母，枯草芽孢杆菌等，或将上述质粒导入单独的宿主中(如大肠杆菌XL10-Gold等)后加入检测体系中，用以检测正丁醇的产生。
有益效果：
1、基于BmoR的生物传感器可用于筛选高产正丁醇菌株，但野生型BmoR 对正丁醇和乙醇、异丁醇均具有响应，无法区分，特异性差，本发明提供的W21R 突变体、E54V突变体和W21R/E54V突变体仅对正丁醇敏感，W21R/E54V突变体对乙醇无响应，解决了野生型BmoR蛋白无法区分正丁醇、乙醇和异丁醇的问题。
2、野生型BmoR的检测范围过窄，对正丁醇的检测范围均为0-40mM，在底物浓度达到40mM时响应已经达到了饱和，因此无法用于鉴别正丁醇产量高于40mM的菌株。而本发明提供的W21R/E54V突变体对正丁醇的检测范围达到0-100mM，提高了BmoR蛋白对正丁醇的响应饱和度，可用于正丁醇高水平生产菌株的筛选和应用。
附图说明：
图1为原理流程图；
首先通过易错PCR对野生型bmoR的N端前1000bp进行随机突变，得到 BmoR的随机突变库；在bmoR基因下游加入GFP荧光蛋白，可通过检测荧光强度来反映突变体BmoR对正丁醇/异丁醇/乙醇的响应情况。通过添加不同浓度的正丁醇，检测突变体BmoR对于醇分子的响应强度。
图2为BmoR突变体/野生型对10mM正丁醇和异丁醇的响应情况；
图3为BmoR W21R/E54V突变体/野生型对0-100mM正丁醇或异丁醇的响应情况；
图4为BmoR W21R/E54V突变体/野生型对0-800mM乙醇的响应情况；
图5为BmoR突变体W21R/E54V与乙醇，正丁醇及异丁醇的分子对接情况。
具体实施方式：
下面通过具体的实施方案叙述本发明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
本发明提供的生物传感器是基于W21R突变体、E54V突变体或W21R/E54V 突变体的生物传感器，所述传感器是包含W21R突变体、E54V突变体或 W21R/E54V突变体编码基因及其启动子、启动子Pbmo及报告基因的表达元件；启动子启动bmoR基因表达，BmoR蛋白与醇分子结合形成六聚体，进而启动下游启动子Pbmo，从而表达报告基因，产生荧光等信号。本领域技术人员可根据实际情况在现有技术中选择启动子启动BmoR突变体基因的表达，如采用PbmoR、 Ptac、PT7、PLlacO1等启动子。所述的报告基因也可以有多种选择，本领域常用的能够产生可视化检测信号、或可供检测的小分子物质的蛋白分子，如荧光蛋白、颜色蛋白等均可实现本发明所述生物传感器的响应，优选地，如gfp、rfp、cfp、 sfgfp、egfp、yfp、ecfp等。上述传感器中还包含复制起始位点等实现表达的必要元件，优选地，如colE1复制起始位点等。上述传感器中还可以包含抗性基因等标记，如ampr等，便于筛选。本领域技术人员还可根据实际需求对上述传感器增加其他元件，如将上述元件构建至现有技术中的表达载体上，如pET、pUC19、 pMAL等，获得可作为传感器的重组质粒。
下述实例中所用的材料、试剂等，如无特殊说明，均可从商业途径获取。
以下将通过具体实施例对本发明做进一步地解释说明。
实施例1 BmoR突变体W21R、E54V和W21R/E54V的筛选
构建转录因子BmoR的随机突变文库
(1)以带有野生型BmoR编码基因(SEQ ID NO.2所示)的质粒pYH1(构建见DOI：https://doi.org/10.1016/j.ymben.2019.08.015； https://doi.org/10.1186/s12934-019-1084-2)为模板，通过易错PCR(通过在PCR 体系中添加Mn2+，提高PCR体系中Mg2+浓度并调整dNTP的比例来引入随机突变。配制10×不均衡dNTPs混合液，其中dCTP，dTTP的浓度是dATP，dGTP 的四倍。PCR程序设置如下：94℃预变性2min，30个扩增循环中包括：95℃变性1min，55-68℃退火1min，按照每分钟1kb的扩增速度确定合适延伸时间，延伸温度为72℃)扩增得到bmoR突变体基因；通过凝胶电泳对PCR产物进行确认，并进行回收纯化；将纯化产物放置于37℃水浴锅中，DpnI(1μL/50 μL纯化产物)消化1-2h。取2μL bmoR突变基因片段(同时取bmoR野生型片段作对照)，3μL pYH1骨架(也可采用构建有PbmoR(或其他启动子)、Pbmo、gfp 荧光蛋白基因(或其他报告基因)的质粒作为骨架，与pYH1骨架效果等价)及 5μLGibson Assemble Mix，混合后放置于50℃水浴锅中连接1h。取5-10μL的连接产物转入50μL大肠杆菌XL10-Gold化转感受态细胞中，37℃过夜培养，得到BmoR N端随机突变库。
(2)挑每个单菌落，接种到5mL LB(100μg/mL Amp)液体培养基中，37℃， 220rpm培养8小时作为种子液。使用96深孔板中进行初筛。每个孔先用移液枪加入950μL新鲜的LB(100μg/mL Amp)培养基，然后向孔中分别加入正丁醇、异丁醇或乙醇，使其终浓度为10mM，最后分别吸取50μL种子液接种到每个孔中。封好封口膜后，将深孔板放置于30℃，220rpm的摇床培养16h；
(3)使用微孔板对荧光强度和OD600进行检测：吹打混匀菌液，吸取200μL 放入酶标仪中，30℃定量检测，设置参数为：470nm激发波长和510nm发射波长，增益值为50；得到的GFP和OD600值首先减去背景对照值，在此基础上计算每个孔的GFP/OD600作为相对荧光强度值。
(4)分析初筛结果后，对有效突变菌的质粒进行测序，获得有效突变体分别为：第21位氨酸由Trp突变为Arg的W21R突变体(氨基酸序列如序列表SEQ ID NO.3，核苷酸序列如SEQ ID NO.4)、第54位氨基酸由Glu突变为Val的E54V 突变体(氨基酸序列如序列表SEQ IDNO.5，核苷酸序列如SEQ ID NO.6)和第 21位氨酸由Trp突变为Arg同时第54位氨基酸由Glu突变为Val的W21R/E54V 突变体(氨基酸序列如序列表SEQ ID NO.7，核苷酸序列如SEQ IDNO.8)，上述突变体的GFP/OD600初筛结果如下表1和图2所示，可知相对于野生型BmoR，W21R/E54V突变体仅对正丁醇具有响应，对乙醇和异丁醇无响应；W21R突变体和E54V突变体仅对正丁醇具有响应，对异丁醇无响应。
表1 GFP/OD600
| BmoR蛋白 | 正丁醇 | 异丁醇 | 乙醇 |
| WT | 983 | 868 | 68.4 |
| W21R/E54V | 39.1 | 0.00 | 0.00 |
| W21R | 17.9 | 0.00 | —— |
| E54V | 20.3 | 0.00 | —— |
实施例2浓度梯度实验测定野生型和突变体的检测限变化
在初筛结果的基础上，对含有W21R/E54V突变体基因和含有野生型BmoR 基因的菌株分别进行浓度梯度的正丁醇或异丁醇的外源添加实验，测定响应曲线，计算Km，响应强度值等参数。
挑平板上的单克隆，接种到5mL LB(100μg/mL Amp)液体培养基中，37℃， 220rpm培养8h作为种子液。
外源添加实验在灭菌的2mL 96深孔板中进行。每个孔先用移液枪添加950 μL新鲜的LB(100μg/mL Amp)培养基中，然后在培养基中分别加入异丁醇、正丁醇、乙醇，使它们的终浓度分别为0、1、10、20、40、60、80、100mM(乙醇终浓度设置至800mM)，最后取50μL种子液接种到每个孔中，封好封口膜之后，将深孔板放于30℃，220rpm的摇床培养16h。
使用微孔板对荧光强度和OD600进行检测：吹打混匀菌液，吸取200μL放入酶标仪中，30℃定量检测，设置参数为：470nm激发波长和510nm发射波长，增益值为50；得到的GFP和OD600值首先减去背景对照值，在此基础上计算每个孔的GFP/OD600作为相对荧光强度值。
以GFP/OD600为纵坐标，分别以正丁醇、异丁醇和乙醇的终浓度为横坐标，用OriginPro 8.5或GraphPad Prism 8软件作图，对数据进行米氏拟合，根据拟合结果计算BmoR突变体对乙醇、正丁醇和异丁醇的Km，最大响应强度等参数(图 3)。
在10mM底物浓度下对随机突变库进行筛选，得到的突变体W21R/E54V 对正丁醇特异性响应，对异丁醇不响应；进一步在0-100mM梯度浓度条件下进行验证，结果表明，该突变体在不同正丁醇和异丁醇浓度的条件下，仍保持对正丁醇特异性响应，对异丁醇几乎不响应。通过OriginPro 8.5进行做图，野生型 BmoR在0-100mM底物浓度下，对正丁醇的Km为3.78，对异丁醇的Km为4.24； W21R/E54V突变体在0-100mM底物浓度下，对正丁醇的Km为5.58，对异丁醇的Km为44.65；Km作为亲和度的表征，Km值越大，亲和力越小；Km值越小，亲和力越大。
野生型BmoR能够响应0-40mM的正丁醇，当浓度高于40mM时，响应将达到饱和，因此野生型BmoR无法区分40mM以上的正丁醇；计算结果表明，突变体相较于野生型，Km值有明显的提升，证明BmoR突变体能够实现更高浓度正丁醇的特异性响应(0-100mM)；同时，W21R/E54V突变体的正丁醇Km值小于异丁醇Km值，进一步说明该突变体对正丁醇的亲和力大于对异丁醇的亲和力。与实验结果保持一致。
首先在0-200mM乙醇浓度下进行野生型BmoR和突变体W21R/E54V的响应分析。结果表明，该突变体在0-200mM乙醇的条件下，几乎没有任何响应。通过OriginPro 8.5进行做图，野生型BmoR在0-200mM底物浓度下，对乙醇的 Km为9.70；W21R/E54V突变体在0-200mM底物浓度下，对乙醇的Km为228。 Km作为亲和度的表征，Km值越大，亲和力越小；Km值越小，亲和力越大。进一步将乙醇浓度提升至800mM,该突变体对乙醇仍几乎没有响应，即对800mM 以内的乙醇不敏感(图4)。
实施例3模型分析
对W21R/E54V突变体进行测序，分析突变位点上氨基酸的变化，同时使用AUTODOCK及ChimeraX等软件对BmoR突变体进行建模，并将突变体分别与底物小分子乙醇、正丁醇和异丁醇进行对接，分析突变体与这两种醇的结合位点和氢键的形成情况。
以野生型BmoR蛋白质三维结构为模板，对W21R/E54V突变体进行同源建模，同源率为99.6％。进一步将突变体结构与底物分子(乙醇、正丁醇或异丁醇)进行分子对接。结果表明，在复合体中，该突变体与正丁醇共1个氢键相互作用(Glu261)，同时与乙醇和异丁醇都没有形成任何相互作用力，说明正丁醇能紧密与该突变体结合，与Km值分析结果保持一致，即对正丁醇特异性响应(图 5)。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。
SEQUENCE LISTING
<110> 北京理工大学
<120> 对乙醇不敏感对正丁醇特异性响应的BmoR蛋白突变体
<130> 1
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 669
<212> PRT
<213> 假单胞杆菌（Pseudomonas butanovora）
<400> 1
Met Ser Lys Met Gln Glu Phe Ala Arg Leu Glu Thr Val Ala Ser Met
1 5 10 15
Arg Arg Ala Val Trp Asp Gly Asn Glu Cys Gln Pro Gly Lys Val Ala
20 25 30
Asp Val Val Leu Arg Ser Trp Thr Arg Cys Arg Ala Glu Gly Val Val
35 40 45
Pro Asn Ala Arg Gln Glu Phe Asp Pro Ile Pro Arg Thr Ala Leu Asp
50 55 60
Glu Thr Val Glu Ala Lys Arg Ala Leu Ile Leu Ala Ala Glu Pro Val
65 70 75 80
Val Asp Ala Leu Met Glu Gln Met Asn Asp Ala Pro Arg Met Ile Ile
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Leu Asn Asp Glu Arg Gly Val Val Leu Leu Asn Gln Gly Asn Asp Thr
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Leu Leu Glu Asp Ala Arg Arg Arg Ala Val Arg Val Gly Val Cys Trp
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Asp Glu His Ala Arg Gly Thr Asn Ala Met Gly Thr Ala Leu Ala Glu
130 135 140
Arg Arg Pro Val Ala Ile His Gly Ala Glu His Tyr Leu Glu Ser Asn
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Thr Ile Phe Thr Cys Thr Ala Ala Pro Ile Tyr Asp Pro Phe Gly Glu
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Phe Thr Gly Ile Leu Asp Ile Ser Gly Tyr Ala Gly Asp Met Gly Pro
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Val Pro Ile Pro Phe Val Gln Met Ala Val Gln Phe Ile Glu Asn Gln
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Leu Phe Arg Gln Thr Phe Ala Asp Cys Ile Leu Leu His Phe His Val
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Arg Pro Asp Phe Val Gly Thr Met Arg Glu Gly Ile Ala Val Leu Ser
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Arg Glu Gly Thr Ile Val Ser Met Asn Arg Ala Gly Leu Lys Ile Ala
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Gly Leu Asn Leu Glu Ala Val Ala Asp His Arg Phe Asp Ser Val Phe
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Asp Leu Asn Phe Gly Ala Phe Leu Asp His Val Arg Gln Ser Ala Phe
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Gly Leu Val Arg Val Ser Leu Tyr Gly Gly Val Gln Val Tyr Ala Arg
290 295 300
Val Glu Pro Gly Leu Arg Val Pro Pro Arg Pro Ala Ala His Ala Arg
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Pro Pro Arg Pro Ala Pro Arg Pro Leu Asp Ser Leu Asp Thr Gly Asp
325 330 335
Ala Ala Val Arg Leu Ala Ile Asp Arg Ala Arg Arg Ala Ile Gly Arg
340 345 350
Asn Leu Ser Ile Leu Ile Gln Gly Glu Thr Gly Ala Gly Lys Glu Val
355 360 365
Phe Ala Lys His Leu His Ala Glu Ser Pro Arg Ser Lys Gly Pro Phe
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Val Ala Val Asn Cys Ala Ala Ile Pro Glu Gly Leu Ile Glu Ser Glu
385 390 395 400
Leu Phe Gly Tyr Glu Glu Gly Ala Phe Thr Gly Gly Arg Arg Lys Gly
405 410 415
Asn Ile Gly Lys Val Ala Gln Ala His Gly Gly Thr Leu Phe Leu Asp
420 425 430
Glu Ile Gly Asp Met Ala Pro Gly Leu Gln Thr Arg Leu Leu Arg Val
435 440 445
Leu Gln Asp Arg Ala Val Met Pro Leu Gly Gly Arg Glu Pro Met Pro
450 455 460
Val Asp Ile Ala Leu Val Cys Ala Thr His Arg Asn Leu Arg Ser Leu
465 470 475 480
Ile Ala Gln Gly Gln Phe Arg Glu Asp Leu Tyr Tyr Arg Leu Asn Gly
485 490 495
Leu Ala Ile Ser Leu Pro Pro Leu Arg Gln Arg Ser Asp Leu Ala Ala
500 505 510
Leu Val Asn His Ile Leu Phe Gln Cys Cys Gly Gly Glu Pro His Tyr
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Ser Val Ser Pro Glu Val Met Thr Leu Phe Lys Arg His Ala Trp Pro
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Gly Asn Leu Arg Gln Leu His Asn Val Leu Asp Ala Ala Leu Ala Met
545 550 555 560
Leu Asp Asp Gly His Val Ile Glu Pro His His Leu Pro Glu Asp Phe
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Lys Lys Leu Gln Asp Leu Ala Leu Asp Ala Ile Glu Gln Ala Ile Glu
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Gln Asn Glu Gly Asn Ile Ser Val Ala Ala Arg Gln Leu Gly Val Ser
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Arg Thr Thr Ile Tyr Arg Lys Leu Arg Gln Leu Ser Pro Thr Gly Cys
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<210> 2
<211> 2010
<212> DNA
<213> 假单胞杆菌（Pseudomonas butanovora）
<400> 2
atgtctaaaa tgcaggaatt cgctcgtctg gaaaccgttg cttctatgcg tcgtgctgtt 60
tgggacggta acgaatgcca gccgggtaaa gttgctgacg ttgttctgcg ttcttggacc 120
cgttgccgtg ctgaaggtgt tgttccgaac gctcgtcagg aattcgaccc gatcccgcgt 180
accgctctgg acgaaaccgt tgaagctaaa cgtgctctga tcctggctgc tgaaccggtt 240
gttgacgctc tgatggaaca gatgaacgac gctccgcgta tgatcatcct gaacgacgaa 300
cgtggtgttg ttctgctgaa ccagggtaac gacaccctgc tggaagacgc tcgtcgtcgt 360
gctgttcgtg ttggtgtttg ctgggacgaa cacgctcgtg gtaccaacgc tatgggtacc 420
gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480
accatcttca cctgcaccgc tgctccgatc tacgacccgt tcggtgaatt caccggtatc 540
ctggacatct ctggttacgc tggtgacatg ggtccggttc cgatcccgtt cgttcagatg 600
gctgttcagt tcatcgaaaa ccagctgttc cgtcagacct tcgctgactg catcctgctg 660
cacttccacg ttcgtccgga cttcgttggt accatgcgtg aaggtatcgc tgttctgtct 720
cgtgaaggta ccatcgtttc tatgaaccgt gctggtctga aaatcgctgg tctgaacctg 780
gaagctgttg ctgaccaccg tttcgactct gttttcgacc tgaactttgg cgcgttcctg 840
gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900
gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960
ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020
ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080
gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140
aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200
ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260
gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320
ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380
gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 1440
atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct 1500
ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560
tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt 1620
cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680
ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740
gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800
ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860
caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920
cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980
cactggtctc agtctcgtat cggtacctaa 2010
<210> 3
<211> 669
<212> PRT
<213> 人工序列
<400> 3
Met Ser Lys Met Gln Glu Phe Ala Arg Leu Glu Thr Val Ala Ser Met
1 5 10 15
Arg Arg Ala Val Arg Asp Gly Asn Glu Cys Gln Pro Gly Lys Val Ala
20 25 30
Asp Val Val Leu Arg Ser Trp Thr Arg Cys Arg Ala Glu Gly Val Val
35 40 45
Pro Asn Ala Arg Gln Glu Phe Asp Pro Ile Pro Arg Thr Ala Leu Asp
50 55 60
Glu Thr Val Glu Ala Lys Arg Ala Leu Ile Leu Ala Ala Glu Pro Val
65 70 75 80
Val Asp Ala Leu Met Glu Gln Met Asn Asp Ala Pro Arg Met Ile Ile
85 90 95
Leu Asn Asp Glu Arg Gly Val Val Leu Leu Asn Gln Gly Asn Asp Thr
100 105 110
Leu Leu Glu Asp Ala Arg Arg Arg Ala Val Arg Val Gly Val Cys Trp
115 120 125
Asp Glu His Ala Arg Gly Thr Asn Ala Met Gly Thr Ala Leu Ala Glu
130 135 140
Arg Arg Pro Val Ala Ile His Gly Ala Glu His Tyr Leu Glu Ser Asn
145 150 155 160
Thr Ile Phe Thr Cys Thr Ala Ala Pro Ile Tyr Asp Pro Phe Gly Glu
165 170 175
Phe Thr Gly Ile Leu Asp Ile Ser Gly Tyr Ala Gly Asp Met Gly Pro
180 185 190
Val Pro Ile Pro Phe Val Gln Met Ala Val Gln Phe Ile Glu Asn Gln
195 200 205
Leu Phe Arg Gln Thr Phe Ala Asp Cys Ile Leu Leu His Phe His Val
210 215 220
Arg Pro Asp Phe Val Gly Thr Met Arg Glu Gly Ile Ala Val Leu Ser
225 230 235 240
Arg Glu Gly Thr Ile Val Ser Met Asn Arg Ala Gly Leu Lys Ile Ala
245 250 255
Gly Leu Asn Leu Glu Ala Val Ala Asp His Arg Phe Asp Ser Val Phe
260 265 270
Asp Leu Asn Phe Gly Ala Phe Leu Asp His Val Arg Gln Ser Ala Phe
275 280 285
Gly Leu Val Arg Val Ser Leu Tyr Gly Gly Val Gln Val Tyr Ala Arg
290 295 300
Val Glu Pro Gly Leu Arg Val Pro Pro Arg Pro Ala Ala His Ala Arg
305 310 315 320
Pro Pro Arg Pro Ala Pro Arg Pro Leu Asp Ser Leu Asp Thr Gly Asp
325 330 335
Ala Ala Val Arg Leu Ala Ile Asp Arg Ala Arg Arg Ala Ile Gly Arg
340 345 350
Asn Leu Ser Ile Leu Ile Gln Gly Glu Thr Gly Ala Gly Lys Glu Val
355 360 365
Phe Ala Lys His Leu His Ala Glu Ser Pro Arg Ser Lys Gly Pro Phe
370 375 380
Val Ala Val Asn Cys Ala Ala Ile Pro Glu Gly Leu Ile Glu Ser Glu
385 390 395 400
Leu Phe Gly Tyr Glu Glu Gly Ala Phe Thr Gly Gly Arg Arg Lys Gly
405 410 415
Asn Ile Gly Lys Val Ala Gln Ala His Gly Gly Thr Leu Phe Leu Asp
420 425 430
Glu Ile Gly Asp Met Ala Pro Gly Leu Gln Thr Arg Leu Leu Arg Val
435 440 445
Leu Gln Asp Arg Ala Val Met Pro Leu Gly Gly Arg Glu Pro Met Pro
450 455 460
Val Asp Ile Ala Leu Val Cys Ala Thr His Arg Asn Leu Arg Ser Leu
465 470 475 480
Ile Ala Gln Gly Gln Phe Arg Glu Asp Leu Tyr Tyr Arg Leu Asn Gly
485 490 495
Leu Ala Ile Ser Leu Pro Pro Leu Arg Gln Arg Ser Asp Leu Ala Ala
500 505 510
Leu Val Asn His Ile Leu Phe Gln Cys Cys Gly Gly Glu Pro His Tyr
515 520 525
Ser Val Ser Pro Glu Val Met Thr Leu Phe Lys Arg His Ala Trp Pro
530 535 540
Gly Asn Leu Arg Gln Leu His Asn Val Leu Asp Ala Ala Leu Ala Met
545 550 555 560
Leu Asp Asp Gly His Val Ile Glu Pro His His Leu Pro Glu Asp Phe
565 570 575
Val Met Glu Val Asp Ser Gly Leu Arg Pro Ile Glu Glu Asp Gly Ser
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Thr Ala Ala His Arg Ala Arg Gln Pro Ala Ser Gly Ser Gly Pro Ala
595 600 605
Lys Lys Leu Gln Asp Leu Ala Leu Asp Ala Ile Glu Gln Ala Ile Glu
610 615 620
Gln Asn Glu Gly Asn Ile Ser Val Ala Ala Arg Gln Leu Gly Val Ser
625 630 635 640
Arg Thr Thr Ile Tyr Arg Lys Leu Arg Gln Leu Ser Pro Thr Gly Cys
645 650 655
His Arg Pro Ala His Trp Ser Gln Ser Arg Ile Gly Thr
660 665
<210> 4
<211> 2010
<212> DNA
<213> 人工序列
<400> 4
atgtctaaaa tgcaggaatt cgctcgtctg gaaaccgttg cttctatgcg tcgtgctgtt 60
cgggacggta acgaatgcca gccgggtaaa gttgctgacg ttgttctgcg ttcttggacc 120
cgttgccgtg ctgaaggtgt tgttccgaac gctcgtcagg aattcgaccc gatcccgcgt 180
accgctctgg acgaaaccgt tgaagctaaa cgtgctctga tcctggctgc tgaaccggtt 240
gttgacgctc tgatggaaca gatgaacgac gctccgcgta tgatcatcct gaacgacgaa 300
cgtggtgttg ttctgctgaa ccagggtaac gacaccctgc tggaagacgc tcgtcgtcgt 360
gctgttcgtg ttggtgtttg ctgggacgaa cacgctcgtg gtaccaacgc tatgggtacc 420
gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480
accatcttca cctgcaccgc tgctccgatc tacgacccgt tcggtgaatt caccggtatc 540
ctggacatct ctggttacgc tggtgacatg ggtccggttc cgatcccgtt cgttcagatg 600
gctgttcagt tcatcgaaaa ccagctgttc cgtcagacct tcgctgactg catcctgctg 660
cacttccacg ttcgtccgga cttcgttggt accatgcgtg aaggtatcgc tgttctgtct 720
cgtgaaggta ccatcgtttc tatgaaccgt gctggtctga aaatcgctgg tctgaacctg 780
gaagctgttg ctgaccaccg tttcgactct gttttcgacc tgaactttgg cgcgttcctg 840
gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900
gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960
ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020
ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080
gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140
aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200
ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260
gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320
ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380
gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 1440
atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct 1500
ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560
tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt 1620
cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680
ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740
gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800
ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860
caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920
cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980
cactggtctc agtctcgtat cggtacctaa 2010
<210> 5
<211> 669
<212> PRT
<213> 人工序列
<400> 5
Met Ser Lys Met Gln Glu Phe Ala Arg Leu Glu Thr Val Ala Ser Met
1 5 10 15
Arg Arg Ala Val Trp Asp Gly Asn Glu Cys Gln Pro Gly Lys Val Ala
20 25 30
Asp Val Val Leu Arg Ser Trp Thr Arg Cys Arg Ala Glu Gly Val Val
35 40 45
Pro Asn Ala Arg Gln Val Phe Asp Pro Ile Pro Arg Thr Ala Leu Asp
50 55 60
Glu Thr Val Glu Ala Lys Arg Ala Leu Ile Leu Ala Ala Glu Pro Val
65 70 75 80
Val Asp Ala Leu Met Glu Gln Met Asn Asp Ala Pro Arg Met Ile Ile
85 90 95
Leu Asn Asp Glu Arg Gly Val Val Leu Leu Asn Gln Gly Asn Asp Thr
100 105 110
Leu Leu Glu Asp Ala Arg Arg Arg Ala Val Arg Val Gly Val Cys Trp
115 120 125
Asp Glu His Ala Arg Gly Thr Asn Ala Met Gly Thr Ala Leu Ala Glu
130 135 140
Arg Arg Pro Val Ala Ile His Gly Ala Glu His Tyr Leu Glu Ser Asn
145 150 155 160
Thr Ile Phe Thr Cys Thr Ala Ala Pro Ile Tyr Asp Pro Phe Gly Glu
165 170 175
Phe Thr Gly Ile Leu Asp Ile Ser Gly Tyr Ala Gly Asp Met Gly Pro
180 185 190
Val Pro Ile Pro Phe Val Gln Met Ala Val Gln Phe Ile Glu Asn Gln
195 200 205
Leu Phe Arg Gln Thr Phe Ala Asp Cys Ile Leu Leu His Phe His Val
210 215 220
Arg Pro Asp Phe Val Gly Thr Met Arg Glu Gly Ile Ala Val Leu Ser
225 230 235 240
Arg Glu Gly Thr Ile Val Ser Met Asn Arg Ala Gly Leu Lys Ile Ala
245 250 255
Gly Leu Asn Leu Glu Ala Val Ala Asp His Arg Phe Asp Ser Val Phe
260 265 270
Asp Leu Asn Phe Gly Ala Phe Leu Asp His Val Arg Gln Ser Ala Phe
275 280 285
Gly Leu Val Arg Val Ser Leu Tyr Gly Gly Val Gln Val Tyr Ala Arg
290 295 300
Val Glu Pro Gly Leu Arg Val Pro Pro Arg Pro Ala Ala His Ala Arg
305 310 315 320
Pro Pro Arg Pro Ala Pro Arg Pro Leu Asp Ser Leu Asp Thr Gly Asp
325 330 335
Ala Ala Val Arg Leu Ala Ile Asp Arg Ala Arg Arg Ala Ile Gly Arg
340 345 350
Asn Leu Ser Ile Leu Ile Gln Gly Glu Thr Gly Ala Gly Lys Glu Val
355 360 365
Phe Ala Lys His Leu His Ala Glu Ser Pro Arg Ser Lys Gly Pro Phe
370 375 380
Val Ala Val Asn Cys Ala Ala Ile Pro Glu Gly Leu Ile Glu Ser Glu
385 390 395 400
Leu Phe Gly Tyr Glu Glu Gly Ala Phe Thr Gly Gly Arg Arg Lys Gly
405 410 415
Asn Ile Gly Lys Val Ala Gln Ala His Gly Gly Thr Leu Phe Leu Asp
420 425 430
Glu Ile Gly Asp Met Ala Pro Gly Leu Gln Thr Arg Leu Leu Arg Val
435 440 445
Leu Gln Asp Arg Ala Val Met Pro Leu Gly Gly Arg Glu Pro Met Pro
450 455 460
Val Asp Ile Ala Leu Val Cys Ala Thr His Arg Asn Leu Arg Ser Leu
465 470 475 480
Ile Ala Gln Gly Gln Phe Arg Glu Asp Leu Tyr Tyr Arg Leu Asn Gly
485 490 495
Leu Ala Ile Ser Leu Pro Pro Leu Arg Gln Arg Ser Asp Leu Ala Ala
500 505 510
Leu Val Asn His Ile Leu Phe Gln Cys Cys Gly Gly Glu Pro His Tyr
515 520 525
Ser Val Ser Pro Glu Val Met Thr Leu Phe Lys Arg His Ala Trp Pro
530 535 540
Gly Asn Leu Arg Gln Leu His Asn Val Leu Asp Ala Ala Leu Ala Met
545 550 555 560
Leu Asp Asp Gly His Val Ile Glu Pro His His Leu Pro Glu Asp Phe
565 570 575
Val Met Glu Val Asp Ser Gly Leu Arg Pro Ile Glu Glu Asp Gly Ser
580 585 590
Thr Ala Ala His Arg Ala Arg Gln Pro Ala Ser Gly Ser Gly Pro Ala
595 600 605
Lys Lys Leu Gln Asp Leu Ala Leu Asp Ala Ile Glu Gln Ala Ile Glu
610 615 620
Gln Asn Glu Gly Asn Ile Ser Val Ala Ala Arg Gln Leu Gly Val Ser
625 630 635 640
Arg Thr Thr Ile Tyr Arg Lys Leu Arg Gln Leu Ser Pro Thr Gly Cys
645 650 655
His Arg Pro Ala His Trp Ser Gln Ser Arg Ile Gly Thr
660 665
<210> 6
<211> 2010
<212> DNA
<213> 人工序列
<400> 6
atgtctaaaa tgcaggaatt cgctcgtctg gaaaccgttg cttctatgcg tcgtgctgtt 60
tgggacggta acgaatgcca gccgggtaaa gttgctgacg ttgttctgcg ttcttggacc 120
cgttgccgtg ctgaaggtgt tgttccgaac gctcgtcagg tattcgaccc gatcccgcgt 180
accgctctgg acgaaaccgt tgaagctaaa cgtgctctga tcctggctgc tgaaccggtt 240
gttgacgctc tgatggaaca gatgaacgac gctccgcgta tgatcatcct gaacgacgaa 300
cgtggtgttg ttctgctgaa ccagggtaac gacaccctgc tggaagacgc tcgtcgtcgt 360
gctgttcgtg ttggtgtttg ctgggacgaa cacgctcgtg gtaccaacgc tatgggtacc 420
gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480
accatcttca cctgcaccgc tgctccgatc tacgacccgt tcggtgaatt caccggtatc 540
ctggacatct ctggttacgc tggtgacatg ggtccggttc cgatcccgtt cgttcagatg 600
gctgttcagt tcatcgaaaa ccagctgttc cgtcagacct tcgctgactg catcctgctg 660
cacttccacg ttcgtccgga cttcgttggt accatgcgtg aaggtatcgc tgttctgtct 720
cgtgaaggta ccatcgtttc tatgaaccgt gctggtctga aaatcgctgg tctgaacctg 780
gaagctgttg ctgaccaccg tttcgactct gttttcgacc tgaactttgg cgcgttcctg 840
gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900
gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960
ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020
ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080
gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140
aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200
ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260
gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320
ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380
gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 1440
atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct 1500
ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560
tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt 1620
cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680
ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740
gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800
ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860
caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920
cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980
cactggtctc agtctcgtat cggtacctaa 2010
<210> 7
<211> 669
<212> PRT
<213> 人工序列
<400> 7
Met Ser Lys Met Gln Glu Phe Ala Arg Leu Glu Thr Val Ala Ser Met
1 5 10 15
Arg Arg Ala Val Arg Asp Gly Asn Glu Cys Gln Pro Gly Lys Val Ala
20 25 30
Asp Val Val Leu Arg Ser Trp Thr Arg Cys Arg Ala Glu Gly Val Val
35 40 45
Pro Asn Ala Arg Gln Val Phe Asp Pro Ile Pro Arg Thr Ala Leu Asp
50 55 60
Glu Thr Val Glu Ala Lys Arg Ala Leu Ile Leu Ala Ala Glu Pro Val
65 70 75 80
Val Asp Ala Leu Met Glu Gln Met Asn Asp Ala Pro Arg Met Ile Ile
85 90 95
Leu Asn Asp Glu Arg Gly Val Val Leu Leu Asn Gln Gly Asn Asp Thr
100 105 110
Leu Leu Glu Asp Ala Arg Arg Arg Ala Val Arg Val Gly Val Cys Trp
115 120 125
Asp Glu His Ala Arg Gly Thr Asn Ala Met Gly Thr Ala Leu Ala Glu
130 135 140
Arg Arg Pro Val Ala Ile His Gly Ala Glu His Tyr Leu Glu Ser Asn
145 150 155 160
Thr Ile Phe Thr Cys Thr Ala Ala Pro Ile Tyr Asp Pro Phe Gly Glu
165 170 175
Phe Thr Gly Ile Leu Asp Ile Ser Gly Tyr Ala Gly Asp Met Gly Pro
180 185 190
Val Pro Ile Pro Phe Val Gln Met Ala Val Gln Phe Ile Glu Asn Gln
195 200 205
Leu Phe Arg Gln Thr Phe Ala Asp Cys Ile Leu Leu His Phe His Val
210 215 220
Arg Pro Asp Phe Val Gly Thr Met Arg Glu Gly Ile Ala Val Leu Ser
225 230 235 240
Arg Glu Gly Thr Ile Val Ser Met Asn Arg Ala Gly Leu Lys Ile Ala
245 250 255
Gly Leu Asn Leu Glu Ala Val Ala Asp His Arg Phe Asp Ser Val Phe
260 265 270
Asp Leu Asn Phe Gly Ala Phe Leu Asp His Val Arg Gln Ser Ala Phe
275 280 285
Gly Leu Val Arg Val Ser Leu Tyr Gly Gly Val Gln Val Tyr Ala Arg
290 295 300
Val Glu Pro Gly Leu Arg Val Pro Pro Arg Pro Ala Ala His Ala Arg
305 310 315 320
Pro Pro Arg Pro Ala Pro Arg Pro Leu Asp Ser Leu Asp Thr Gly Asp
325 330 335
Ala Ala Val Arg Leu Ala Ile Asp Arg Ala Arg Arg Ala Ile Gly Arg
340 345 350
Asn Leu Ser Ile Leu Ile Gln Gly Glu Thr Gly Ala Gly Lys Glu Val
355 360 365
Phe Ala Lys His Leu His Ala Glu Ser Pro Arg Ser Lys Gly Pro Phe
370 375 380
Val Ala Val Asn Cys Ala Ala Ile Pro Glu Gly Leu Ile Glu Ser Glu
385 390 395 400
Leu Phe Gly Tyr Glu Glu Gly Ala Phe Thr Gly Gly Arg Arg Lys Gly
405 410 415
Asn Ile Gly Lys Val Ala Gln Ala His Gly Gly Thr Leu Phe Leu Asp
420 425 430
Glu Ile Gly Asp Met Ala Pro Gly Leu Gln Thr Arg Leu Leu Arg Val
435 440 445
Leu Gln Asp Arg Ala Val Met Pro Leu Gly Gly Arg Glu Pro Met Pro
450 455 460
Val Asp Ile Ala Leu Val Cys Ala Thr His Arg Asn Leu Arg Ser Leu
465 470 475 480
Ile Ala Gln Gly Gln Phe Arg Glu Asp Leu Tyr Tyr Arg Leu Asn Gly
485 490 495
Leu Ala Ile Ser Leu Pro Pro Leu Arg Gln Arg Ser Asp Leu Ala Ala
500 505 510
Leu Val Asn His Ile Leu Phe Gln Cys Cys Gly Gly Glu Pro His Tyr
515 520 525
Ser Val Ser Pro Glu Val Met Thr Leu Phe Lys Arg His Ala Trp Pro
530 535 540
Gly Asn Leu Arg Gln Leu His Asn Val Leu Asp Ala Ala Leu Ala Met
545 550 555 560
Leu Asp Asp Gly His Val Ile Glu Pro His His Leu Pro Glu Asp Phe
565 570 575
Val Met Glu Val Asp Ser Gly Leu Arg Pro Ile Glu Glu Asp Gly Ser
580 585 590
Thr Ala Ala His Arg Ala Arg Gln Pro Ala Ser Gly Ser Gly Pro Ala
595 600 605
Lys Lys Leu Gln Asp Leu Ala Leu Asp Ala Ile Glu Gln Ala Ile Glu
610 615 620
Gln Asn Glu Gly Asn Ile Ser Val Ala Ala Arg Gln Leu Gly Val Ser
625 630 635 640
Arg Thr Thr Ile Tyr Arg Lys Leu Arg Gln Leu Ser Pro Thr Gly Cys
645 650 655
His Arg Pro Ala His Trp Ser Gln Ser Arg Ile Gly Thr
660 665
<210> 8
<211> 2010
<212> DNA
<213> 人工序列
<400> 8
atgtctaaaa tgcaggaatt cgctcgtctg gaaaccgttg cttctatgcg tcgtgctgtt 60
cgggacggta acgaatgcca gccgggtaaa gttgctgacg ttgttctgcg ttcttggacc 120
cgttgccgtg ctgaaggtgt tgttccgaac gctcgtcagg tattcgaccc gatcccgcgt 180
accgctctgg acgaaaccgt tgaagctaaa cgtgctctga tcctggctgc tgaaccggtt 240
gttgacgctc tgatggaaca gatgaacgac gctccgcgta tgatcatcct gaacgacgaa 300
cgtggtgttg ttctgctgaa ccagggtaac gacaccctgc tggaagacgc tcgtcgtcgt 360
gctgttcgtg ttggtgtttg ctgggacgaa cacgctcgtg gtaccaacgc tatgggtacc 420
gctctggctg aacgtcgtcc ggttgctatc cacggtgctg aacactacct ggaatctaac 480
accatcttca cctgcaccgc tgctccgatc tacgacccgt tcggtgaatt caccggtatc 540
ctggacatct ctggttacgc tggtgacatg ggtccggttc cgatcccgtt cgttcagatg 600
gctgttcagt tcatcgaaaa ccagctgttc cgtcagacct tcgctgactg catcctgctg 660
cacttccacg ttcgtccgga cttcgttggt accatgcgtg aaggtatcgc tgttctgtct 720
cgtgaaggta ccatcgtttc tatgaaccgt gctggtctga aaatcgctgg tctgaacctg 780
gaagctgttg ctgaccaccg tttcgactct gttttcgacc tgaactttgg cgcgttcctg 840
gaccacgttc gtcagtctgc tttcggtctg gttcgtgttt ctctgtacgg tggtgttcag 900
gtttacgctc gtgttgaacc gggtctgcgt gttccgccgc gtccggctgc tcacgctcgt 960
ccgccgcgtc cggctccgcg tccgctggac tctctggaca ccggtgacgc tgctgttcgt 1020
ctggctatcg accgtgctcg tcgtgctatc ggtcgtaacc tgtctatcct gatccagggt 1080
gaaaccggtg ctggtaaaga agttttcgct aaacacctgc acgctgaatc tccgcgttct 1140
aaaggtccgt tcgttgctgt taactgcgct gctatcccgg aaggtctgat cgaatctgaa 1200
ctgttcggtt acgaagaagg tgctttcacc ggtggtcgtc gtaaaggtaa catcggtaaa 1260
gttgctcagg ctcacggtgg taccctgttc ctggacgaaa tcggtgacat ggctccgggt 1320
ctgcagaccc gtctgctgcg tgttctgcag gaccgtgctg ttatgccgct gggtggtcgt 1380
gaaccgatgc cggttgacat agcgctggtc tgcgcaaccc accgtaacct gcgttctctg 1440
atcgctcagg gtcagttccg tgaagacctg tactaccgtc tgaacggtct ggctatctct 1500
ctgccgccgc tgcgtcagcg ttctgacctg gctgctctgg ttaaccacat cctgttccag 1560
tgctgcggtg gtgaaccaca ttactctgta agcccggaag ttatgaccct gttcaaacgt 1620
cacgcttggc cgggtaacct gcgtcagctg cacaacgttc tggacgctgc tctggctatg 1680
ctggacgacg gtcacgttat cgaaccgcac cacctgccgg aagacttcgt tatggaagtt 1740
gactctggtc tgcgtccgat cgaagaagac ggttctaccg ctgctcaccg tgctcgtcag 1800
ccggcttctg gttctggtcc ggctaaaaaa ctgcaggacc tggctctgga cgctatcgaa 1860
caggctatcg aacagaacga aggtaacatc tctgttgctg cgcgtcagct gggtgtaagc 1920
cgtaccacca tctaccgtaa actgcgtcag ctgtctccga ccggttgcca ccgtccggct 1980
cactggtctc agtctcgtat cggtacctaa 2010
<210> 9
<211> 138
<212> DNA
<213> 假单胞杆菌（Pseudomonas butanovora）
<400> 9
gaccttgagg tgaccttgag cgggcagata ccaccaaaat ttcccacgtg ctattatggt 60
tttgctaaag ctctcgacag cgaggagaga ctcgcgaaga taagcaattc gcccgacaga 120
ggtgaatgag gagacggt 138
<210> 10
<211> 524
<212> DNA
<213> 假单胞杆菌（Pseudomonas butanovora）
<400> 10
ccccccaacg acgtccgtca gagcccggtt cgagtggctt ctatatgccg atcatcggtg 60
gctctattgt ggcggtcagt gacaccggtc gccttcaccc ccacagatag taggtgctgc 120
ggctgctcat gctcctgtcg cggtagcgcg ctgttacgcg accgcccccg gacctcggcg 180
gacagcgcgg aagattggaa acagcccgag cgtgcgtgcc tcgggctgca tccttgccac 240
acccaaccgg attcgtcgga ccgctcgaca ttcgcgttcg ctcccgcggc gccgcgggtg 300
taccgttgcg ttacagatgt acccttcttt aacgtgtaac acacgcctgg agcggccaag 360
agccccgcac cttgcggcgc gtcttcccca ggggcccacc ggttgcggcc ttttgctgcg 420
accgtccatg ctggcacgac acttgctgaa agcgttagag cggaatcggt ccgatggagc 480
attcgaagcc gctaccgaca gcagaacaca caaaggagga agtg 524
<210> 11
<211> 717
<212> DNA
<213> 人工序列
<400> 11
atgcgtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60
gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120
aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180
gtcactactt tcggttatgg tgttcaatgc tttgcgagat acccagatca tatgaaacag 240
catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc 300
aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360
aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa 420
ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga 480
atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540
cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600
ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660
cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaataa 717
Claims (11)
1.一种BmoR突变体蛋白，其特征在于，是在序列表SEQ ID NO.1所示的野生型BmoR蛋白的基础上发生W21R和E54V突变获得的，所述突变体蛋白的氨基酸序列如SEQ ID NO.7所示。
2.权利要求1所述BmoR突变体在检测含有正丁醇的样品，或在筛选正丁醇生产菌株中的应用。
3.权利要求1所述BmoR突变体在构建检测正丁醇的生物传感器中的应用。
4.权利要求1所述BmoR突变体的编码基因。
5.如权利要求4所述的编码基因，其特征在于，如序列表SEQ ID NO.8所示。
6.包含权利要求4所述编码基因的重组质粒或重组菌株。
7.一种生物传感器，其特征在于，所述的传感器是包含权利要求4所述突变体编码基因、由BmoR突变体激活的启动子Pbmo、由Pbmo驱动的报告基因和表达BmoR突变体的启动子的表达元件。
8.如权利要求7所述的生物传感器，其特征在于，所述报告基因包括但不限于gfp、rfp、cfp、sfgfp、egfp、yfp、ecfp基因。
9.如权利要求7所述的生物传感器，其特征在于，表达BmoR突变体的启动子为PbmoR。
10.如权利要求7所述的生物传感器，其特征在于，所述传感器是将由PbmoR启动的突变体编码基因连接至colE1复制起始位点、ampr和Pbmo启动的gfp基因所得；
启动子Pbmo的核苷酸序列如序列表SEQ ID NO.10所示。
11.权利要求7所述生物传感器在检测含有正丁醇的环境、食品、医学、生物类样品，及筛选生产正丁醇的菌株中的应用。
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| CN110615832A (zh) * | 2019-09-27 | 2019-12-27 | 北京理工大学 | 一种高效筛选异丁醇高产菌株的BmoR突变体 |
| CN110885777A (zh) * | 2019-08-19 | 2020-03-17 | 山东汇冠康博生物科技有限公司 | 一种利用BmoR生物传感器筛选异丁醇高产菌株的方法 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110885777A (zh) * | 2019-08-19 | 2020-03-17 | 山东汇冠康博生物科技有限公司 | 一种利用BmoR生物传感器筛选异丁醇高产菌株的方法 |
| CN110615832A (zh) * | 2019-09-27 | 2019-12-27 | 北京理工大学 | 一种高效筛选异丁醇高产菌株的BmoR突变体 |
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