CN113913357B - 一种生产碱性蛋白酶的底盘菌株及其构建方法与应用 - Google Patents
一种生产碱性蛋白酶的底盘菌株及其构建方法与应用 Download PDFInfo
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Abstract
本发明属于生物工程技术领域，涉及工业微生物的育种，特别涉及一种生产碱性蛋白酶的底盘菌株及其构建方法与用途。本发明提供了一种解淀粉芽孢杆菌基因工程菌株，该基因工程菌株不表达解淀粉芽孢杆菌基因组上的六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因，优选的还异源过表达碱性蛋白酶基因aprE。本发明从实验和生产中的实际情况出发，分析影响发酵性能的不良因素，通过对基因工程宿主的改造和基因精简，最终获得了一种能够高产碱性蛋白酶的底盘菌株，并且为构建蛋白酶高产底盘菌株提供了新的思路。
Description
技术领域
本发明属于生物工程技术领域，涉及工业微生物的育种，特别涉及一种生产碱性蛋白酶的底盘菌株及其构建方法与用途。
背景技术
在众多的工业用酶中，主要产自微生物的碱性蛋白酶是能够在碱性条件下水解蛋白质肽键的一类酶，其最适pH一般为9～11，在洗涤剂、食品加工、皮革制造、纺织制造等工业中被广泛应用和研究。
随着市场对碱性蛋白酶需求的不断增长，人们开始将注意力集中在碱性蛋白酶高产底盘菌株的构建方面。大肠杆菌(E.coli)和毕赤酵母(Pichia pastoris)作为首选宿主菌株被用于碱性蛋白酶的高效表达，但产量较低，稳定性较差，难以满足工业生产的需要。近年来，为获得性能优良的工业微生物细胞工厂，学者们在生产菌株的遗传改良方面开展了深入的研究。Zhang等人通过敲除影响泡沫形成的脂肽编码基因srfA，优化了未经驯化的野生菌株B.subtilis ATCC 6051a的生产性能；Illing等人通过删除芽孢形成关键控制基因sigE提高了酶的合成能力；模式菌B.subtilis WB800属于多重蛋白酶缺陷型菌株，这明显促进其它胞外分泌蛋白的积累。
对解淀粉芽孢杆菌基因组进行简化，在维持其基因稳定的同时，也能达到优化代谢网络，改善细胞的能量利用效率，提高细胞生理性能的预测性及可控性的目的，获得性能优秀的工业底盘。在对解淀粉芽孢杆菌CGMCC No.11218研究的过程中发现一系列未经驯化的不良特性，例如在发酵过程中产生粘性物质、形成泡沫以及合成各种次级代谢产物等。不仅会导致较高的染菌风险，不利于目的产物的分泌和纯化，还会增加工业操作成本，影响经济效益提升。但作为重要的工业菌株，解淀粉芽胞杆菌较强的蛋白分泌能力和蛋白酶耐受能力促使研究者们对其进行改造，不断优化解淀粉芽胞杆菌的表型，提升其蛋白酶生产能力。
发明内容
针对上述问题，本发明的目的是通过对基因工程宿主的改造和基因精简，提供能够高产碱性蛋白酶的解淀粉酶芽孢杆菌底盘菌株。
为了实现上述目的，本发明采取如下的技术方案：
第一方面，本发明提供了一种解淀粉芽孢杆菌基因工程菌株，该基因工程菌株不表达解淀粉芽孢杆菌基因组上的六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因；优选的，该基因工程菌株还异源过表达碱性蛋白酶基因aprE。
第二方面，本发明提供了上述解淀粉芽孢杆菌基因工程菌株的构建方法，包括：在解淀粉芽孢杆菌中，将六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因失活或敲除，获得底盘菌株；优选的，进一步在底盘菌株中引入碱性蛋白酶基因aprE。
第三方面，本发明提供上述解淀粉芽孢杆菌基因工程菌株在高产碱性蛋白酶中的用途。
第四方面，本发明提供一种高效生产碱性蛋白酶的方法，所述方法是在适宜条件下培养上述解淀粉芽孢杆菌基因工程菌株，并从其培养物中收集碱性蛋白酶。
本发明的有益效果如下：
本发明通过对宿主解淀粉芽孢杆菌的基因组的分析，对其存在的孤岛基因、冗余基因、假基因等非必需基因，以及控制次级代谢产物合成的基因进行合理化的精简，这大大减少了细胞代谢负担和能耗，以及营养资源浪费，进而影响细胞的生长与发酵性能。基因组岛是一类具有特定结构和功能的大片段DNA的总称，一般被认为是通过水平基因转移产生的，细菌可经过接合、转导、转化等方式获得基因组岛。本发明中通过基因组、转录组信息以及结合预测手段，预测得到噬菌体来源的基因组岛基因序列，从中选取3049-3052序列作为敲除的靶基因，其中3049基因合成功能未知蛋白，3050基因合成噬菌体蛋白，3051基因推测合成原噬菌体LambdaCh01门户蛋白，3052基因推测合成噬菌体衣壳蛋白，进而有针对性地简化解淀粉芽孢杆菌基因组，去除非必需基因干扰，以降低基因组的复杂度，提高解淀粉芽孢杆菌对底物和能量的利用效率，最终获得能够表达异源蛋白的理想宿主细胞。
本发明从实验和生产中的实际情况出发，通过分析影响发酵性能的不良因素，结合基因组/转录组数据以及SubtiWiki基因数据库等信息，选定了胞外多糖EPS合成，聚谷氨酸合成和噬菌体相关基因作为菌株改造的靶基因，以此来改善菌株的生产性能与潜力，通过对解淀粉芽孢杆菌基因组中上述物质的合成相关基因进行精简，最终获得了一种能够高产碱性蛋白酶的底盘菌株，并且为构建蛋白酶高产底盘菌株提供了新的思路。
本发明提供了一种能够高产碱性蛋白酶的解淀粉芽孢杆菌底盘菌株，在携带碱性蛋白酶表达盒的情况下，经48h摇瓶发酵，发酵液酶活力高达22523.7±531.8U/mL，以出发菌株为对比酶活提升了2倍左右；通过7L发酵罐放大培养，发酵液中碱性蛋白酶酶活力在72h达到峰值，为96783.17±1637.46U/mL。
附图说明
图1：温敏型敲除载体T2的构建过程；
图2A：pgs基因簇敲除验证；
图2B：噬菌体相关基因3049-3052敲除验证；
图2C：六个胞外蛋白酶敲除和对照验证图；
图3：各敲除菌株的生长曲线比较；
图4：各敲除菌株表达碱性蛋白酶的酶活比较；
图5：基因工程菌BAΔ6ΔepsΔpgsΔ3049-30521表达碱性蛋白酶的酶活测定。
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。
第一方面，本发明提供了一种解淀粉芽孢杆菌基因工程菌株，该基因工程菌株不表达解淀粉芽孢杆菌基因组上的六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因。
优选的，所述胞外蛋白酶基因aprE的核苷酸序列如SEQ ID NO：2所示，GenBank:GU825966.1(594bp至1742bp)。
优选的，所述胞外蛋白酶基因bpr的核苷酸序列如SEQ ID NO：3所示，GenBank:CP054415.1(全基因组1624354bp至1628643bp)。
优选的，所述胞外蛋白酶基因vpr的核苷酸序列如SEQ ID NO：4所示，GenBank:CP002634.1(全基因组3706130bp至3708541bp)。
优选的，所述胞外蛋白酶基因mpr的核苷酸序列如SEQ ID NO：5所示，GenBank:CP054415.1(全基因组886397bp至887311bp)。
优选的，所述胞外蛋白酶基因nprE的核苷酸序列如SEQ ID NO：6所示，GenBank:K02497.1(254bp至1819bp)。
优选的，所述胞外蛋白酶基因epr的核苷酸序列如SEQ ID NO：7所示，GenBank:CP054415.1(全基因组3751547bp至3753295bp)。
优选的，所述胞外多糖基因簇eps的核苷酸序列如SEQ ID NO：8所示，GenBank:CP018902.1(全基因组：2456935bp至2472647bp)。
优选的，所述聚谷氨酸基因簇pgs的核苷酸序列如SEQ ID NO：9所示，GenBank:CP018902.1(全基因组：2616648bp至2617796bp)。
根据本发明，作为底盘菌株为了进一步高产碱性蛋白酶，所述基因工程菌株还异源过表达碱性蛋白酶基因aprE。
优选的，所述碱性蛋白酶基因aprE的核苷酸序列如SEQ ID NO：10所示，GenBank：FJ940727.1。
根据本发明，不表达上述基因的方式可以采取本领域常规手段，例如，通过本领域常规手段使该基因失活或者将该基因敲除。
根据本发明，所述不表达是指该基因表达产物的量显著低于原有水平，例如，显著降低了至少50％、60％、70％、80％、90％、100％。
根据本发明一种优选的实施方式，所述解淀粉芽孢杆菌基因组上的六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因被敲除。敲除的方式可以采取本领域常规手段，例如，通过同源重组的方式，构建敲除载体并电转化入解淀粉芽孢杆菌中，经过单双交换将上述基因从基因组上敲除，优选的，所述敲除载体是包含Kana抗性基因的pWH-T2质粒。
根据本发明，异源过表达上述基因的方式可以采取本领域常规手段，例如，通过本领域常规手段使碱性蛋白酶基因aprE的表达盒插入在解淀粉芽孢杆菌的基因组中，或者通过本领域常规手段将包含有碱性蛋白酶基因aprE表达盒的表达载体转入解淀粉芽孢杆菌中。
根据本发明，所述过表达是指该基因表达产物的量显著高于原有水平。
根据本发明一种优选的实施方式，所述碱性蛋白酶基因aprE的异源过表达是通过将包含有碱性蛋白酶基因aprE表达盒的表达质粒pWB980电转入解淀粉芽孢杆菌中实现的。
根据本发明一种优选的实施方式，所述出发菌株为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)CGMCC No.11218(该菌种的生物保藏信息已公开于专利申请CN105087448A)。
第二方面，本发明提供了上述解淀粉芽孢杆菌基因工程菌株的构建方法，包括：在解淀粉芽孢杆菌中，将六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因失活或敲除，并且可选的在解淀粉芽孢杆菌中引入碱性蛋白酶基因aprE并使其过表达。
以上在第一方面中已详细介绍过的内容不再赘述。
根据本发明一种具体实施方式，所述构建方法包括：在出发菌株解淀粉芽孢杆菌中，敲除六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，得到菌株BAΔ6；进一步敲除胞外多糖基因簇eps，得到菌株BAΔ6Δeps；进一步敲除聚谷氨酸基因簇pgs，得到菌株BAΔ6ΔepsΔpgs；进一步敲除噬菌体相关基因3049-3052，得到菌株BAΔ6ΔepsΔpgsΔ3049-3052。
进一步地，将含有碱性蛋白酶基因aprE表达盒的表达载体转入菌株BAΔ6ΔepsΔpgsΔ3049-3052中，得到高产碱性蛋白酶的解淀粉酶芽孢杆菌BAΔ6ΔepsΔpgsΔ3049-30521。
根据本发明一种更为优选的实施方式，所述构建方法包括如下步骤：
(1)敲除目的基因、获得各敲除菌株
所述目的基因包括解淀粉芽孢杆菌的六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和噬菌体相关基因3049-3052，所述目的基因均按照如下步骤敲除，需要说明的是，上述基因敲除的顺序并不影响本发明目的的实现，只要最终能达到上述基因全部敲除的结果即可，根据本发明具体实施方式所采取的敲除顺序仅仅是为了获得各步骤敲除菌株以便于进行敲除效果的比较：
1)通过PCR扩增获得目的基因两侧同源序列UP端与DOWN端；
2)通过双酶切与琼脂糖核酸凝胶电泳获得线性的敲除质粒载体；
3)通过无缝克隆技术连接同源序列与线性载体获得敲除质粒；
4)通过甲基化诱导使敲除质粒被甲基化修饰；
5)将甲基化修饰的敲除质粒电转化入解淀粉芽孢杆菌感受态细胞中；
6)通过单、双交换筛选出敲除菌株，并通过测序验证序列；
7)通过摇瓶培养，在LB培养基中测定敲除菌OD600，并绘制生长曲线。
上述各步骤的具体操作方法根据本领域人员容易获得的技术手册、教科书或文献报道均可以实现。
(2)异源过表达碱性蛋白酶基因
将携带有碱性蛋白酶基因aprE表达盒的重组质粒电转化入以上敲除菌株BAΔ6ΔepsΔpgsΔ3049-3052中，作为对比，同时也电转化入以上敲除菌株BAΔ6、BAΔ6Δeps、BAΔ6ΔepsΔpg以及出发菌株中，得到产碱性蛋白酶的各基因工程菌株BA、BAΔ61、BAΔ6Δeps1、BAΔ6ΔepsΔpgs1、BAΔ6ΔepsΔpgsΔ3049-30521，上述菌株通过摇瓶发酵产碱性蛋白酶，国标法测定碱性蛋白酶酶活力。
根据本发明一种优选的实施方式，所述碱性蛋白酶基因aprE通过重组质粒Ply-2-SPamyE-aprE-pWB980转入菌株，其中启动子Ply-2与信号肽SPamyE的核苷酸序列如SEQ IDNO：11所示。所述重组质粒Ply-2-SPamyE-aprE-pWB980的构建可以参照专利申请CN112522173 A的实施例1，也可以采用其他本领域常规手段。
第三方面，本发明提供上述解淀粉芽孢杆菌基因工程菌株在高产碱性蛋白酶中的用途。
根据本发明一种优选的实施方式，所述基因工程菌株BAΔ6ΔepsΔpgsΔ3049-30521用于发酵生产碱性蛋白酶的用途，所述基因工程菌株的发酵液中碱性蛋白酶的酶活最高达到22523.7±531.8U/mL或96783.17±1637.46U/mL，以出发菌株为对比酶活提升了2倍左右。
第四方面，本发明提供一种高效生产碱性蛋白酶的方法，所述方法是在适宜条件下培养上述解淀粉芽孢杆菌基因工程菌株，并从其培养物中收集碱性蛋白酶。
根据本发明一种优选的实施方式，所述适宜条件是指培养温度37℃及采用如下发酵培养基组成：玉米粉64g/L，豆饼粉40g/L，磷酸氢二钠4g/L，磷酸二氢钾0.3g/L，高温淀粉酶0.7g/L。
以下将通过具体的实施例对本发明进行更详细描述。如无特别说明，以下实施例中：
种子培养基：酵母粉5g/L，蛋白胨10g/L，氯化钠10g/L；
发酵培养基：玉米粉64g/L，豆饼粉40g/L，磷酸氢二钠4g/L，磷酸二氢钾0.3g/L，高温淀粉酶0.7g/L。
解淀粉芽孢杆菌感受态制备培养基：
LBS培养基：酵母粉5g/L，蛋白胨10g/L，氯化钠5g/L，山梨醇9.1085g/L；
复苏培养基：酵母粉5g/L，蛋白胨10g/L，氯化钠5g/L，山梨醇9.1085g/L，甘露醇6.92246g/L。
碱性蛋白酶的酶活力测定方法参照GB/T 23527-2009附录B福林酚法进行，即1个酶活力单位(U/mL)定义为1mL酶液在40℃、pH 10.5条件下反应1min水解酪蛋白产生1μg酪氨酸所需要的酶量。每一样品设三次重复，结果取平均值。
实施例所涉及的菌株及质粒见于表1和表2：
表1
表2
注：其中6表示epr,vpr,aprE,bpr,mpr,nprE六种胞外蛋白酶基因，eps基因编码eps合成蛋白酶，pgs基因编码聚谷氨酸合成相关酶，3049-3052这段基因预测为编码噬菌体蛋白。pWH-T2质粒、pWB980质粒为商业性质粒。
实施例所涉及的引物信息如表3所示：
表3
实施例1：基因工程菌株的构建
(1)敲除目的基因
1)扩增目的基因同源序列
根据解淀粉芽孢杆菌CGMCC No.11218基因组数据设计并通过PCR进行扩增获得目的基因序列两端的同源臂序列UP端与DOWN端，分别以目的基因-UP-F、目的基因-UP-R和目的基因-DOWN-F、目的基因-DOWN-R两组引物(参见引物表)，以CGMCC No.11218基因组为模板进行PCR扩增；扩增反应体系如下：
| 引物F | 2μL |
| 引物R | 2μL |
| DNA模板 | 2μL |
| PrimerStar酶 | 25μL |
| ddH2O | 19μL |
扩增程序的设置为：预变性：95℃5min；变性：95℃30s；退火：56℃45s；延伸：72℃5s；反应30个循环；延伸：72℃，10min。
将PCR产物进行琼脂糖凝胶电泳，UP端和DOWN端电泳条带大小在1000bp至1500bp之间，再由小量DNA回收试剂盒回收PCR产物，即目的基因上下游同源臂片段。
2)表达载体的线性化
提取pWH-T2质粒，其提取过程参照试剂盒的操作手册。经XbaI和SmaI双酶切后进行琼脂糖凝胶电泳，再由DNA凝胶回收试剂盒回收产物，得到了线性化载体序列。
双酶切体系如下：
| ddH2O | 25μL |
| 质粒模板 | 15μL |
| Q.cut Buffer | 5μL |
| 限制性内切酶XbaI | 2.5μL |
| 限制性内切酶SmaI | 2.5μL |
混匀后，37℃水浴酶切2h，反应完成后将酶切产物进行琼脂糖凝胶电泳，4260bp，再由小量DNA回收试剂盒回收酶切产物：线性pWH-T2质粒。
3)敲除载体的构建
将酶切获得的线性载体片段和目的基因的上下游同源臂通过无缝克隆连接形成重组质粒pWH-T2-Δpgs。
无缝克隆酶反应体系如下：
| 无缝克隆酶 | 5μL |
| 线性载体 | xμL |
| 插入片段 | yμL |
混合均匀后，50℃水浴反应15min。
PS：首先使用NanoDrop 2000C超微量分光光度计测量线性载体，上下游同源臂UP和DOWN端的DNA浓度，根据公式：
pmols＝质量ng/(片段长度bp×0.65KDa)，
x：y＝1：2；x+y＝5，
此外，x、y的添加量应在0.01～0.25pmols之间，由此计算线性载体和插入片段的添加量。
4)敲除载体的甲基化修饰以及电转化入解淀粉芽胞杆菌感受态细胞
将构建的敲除载体通过化转转化入EC135.P.Bam.感受态细胞中，当培养液OD600值为0.2时，加入50mg/mL阿拉伯糖水溶液80μL进行甲基化诱导，于30℃摇床过夜培养。
通过使用电转仪将经甲基化修饰的敲除载体电转入解淀粉芽胞杆菌CGMCCNo.11218感受态细胞中，并使用敲除载体验证引物XbaI-F、SmaI-R进行菌落PCR进行验证。
5)单交换验证
挑选成功电转的单克隆在45℃下培养3-4代后，经稀释涂布，挑单菌落进行菌落PCR验证，由于单交换过程中上游或下游同源序列均有可能发生单交换，因此使用两组引物XbaI-F、目的基因-DJH-DOWN-R和目的基因-DJH-UP-F、SmaI-R进行验证。
6)双交换验证
挑选成功单交换的单克隆在37℃下培养6-9代，经稀释涂布，挑单菌落进行菌落PCR验证，所使用引物为目的基因-SJH-F、目的基因-SJH-R。
7)按照上述步骤1)至步骤6)的操作分别敲除目的基因：六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和噬菌体相关基因3049-3052，涉及到的引物参见以上引物表。除特别说明外，操作方法与条件基本相同。单双交换验证正确后的菌株分别命名为：BAΔ6、BAΔ6Δeps、BAΔ6ΔepsΔpgs、BAΔ6ΔepsΔpgsΔ3049-3052，上述基因工程菌株以及出发菌株(解淀粉芽孢杆菌CGMCC No.11218)均可以作为转入及表达碱性蛋白酶基因的宿主。
(2)引入碱性蛋白酶基因aprE
碱性蛋白酶基因AprE转入菌株BAΔ6ΔepsΔpgsΔ3049-3052。
1)由苏州金唯智生物科技有限公司合成pLY-2启动子与枯草芽孢杆菌amyE信号肽的核苷酸序列(如SEQ ID NO：11所示)；
2)利用PCR技术以克劳氏芽孢杆菌基因组为模板扩增碱性蛋白酶基因(序列如SEQID NO：10所示)，对其进行纯化回收，引物的选择参见表3，反应体系及反应条件如下：
3)利用从北京全式金生物技术有限公司购买的无缝克隆酶将pLY-2启动子与枯草芽孢杆菌amyE信号肽片段、碱性蛋白酶基因AprE回收片段及线性化载体pWB980连接；连接体系如下：
4)将步骤3)中的反应体系50℃反应15min后化转到枯草芽孢杆菌WB600中，方法如下所述；
①挑取新活化的枯草芽孢杆菌WB600单菌落于5mL LB液体培养基中，37℃，220r/min，过夜培养；
②取100μL培养液转接至5mL SPI培养基中，37℃，220r/min培养至对数生长末期OD600＝1.2(约3–4h)；
③取200μL生长至对数期末的培养液至2mL SPII培养基中，37℃，100r/min培养1.5h；
④在上述SPII培养基的菌体中加入20μL 10mmol/L EGTA，37℃，100r/min培养10min；
⑤加入连接产物，37℃，100r/min培养30min；
⑥调节转速至220r/min，继续培养1.5h，取菌液涂布于含有100μg/mL卡那霉素的LB筛选平板，37℃培养12h，筛选阳性转化子进行验证。
5)提取验证正确的转化子的质粒，电转至出发菌株CGMCC No.11218中，验证阳性转化子，记为BA；同时将质粒分别电转至基因工程菌株BAΔ6，BAΔ6Δeps，BAΔ6ΔepsΔpgs，BAΔ6ΔepsΔpgsΔ3049-3052中，获得产碱性蛋白酶的基因工程菌株分别命名为BAΔ61，BAΔ6Δeps1，BAΔ6ΔepsΔpgs1，BAΔ6ΔepsΔpgsΔ3049-30521，验证阳性转化子。
实施例2：基因工程菌株生产碱性蛋白酶的用途
摇瓶发酵：将基因工程菌株BA、BAΔ61，BAΔ6Δeps1，BAΔ6ΔepsΔpgs1，BAΔ6ΔepsΔpgsΔ3049-30521在LB平板上进行三区划线，37℃倒置培养过夜，挑取活化的单菌落于5mL LB培养基中，37℃,220r/min振荡培养12h，以2％的接种量转接于50mL LB液体培养基至OD600达0.8-1.0，再以2％的接种量转接于100mL发酵培养基的挡板瓶中，37℃，220r/min振荡培养48-60h。根据实验去求定点取样，4℃，12000r/min离心2min，取发酵液上清液，适当稀释后按照国标法测定碱性蛋白酶活力。
发酵罐发酵：菌种接种于装有液体LB培养基(50mL/250mL)的摇瓶中37℃振荡培养至OD600值至0.8-1.0，取菌液于脱脂乳板上三区划线，37℃静置培养18h，挑取平板上透明圈相对较大的单克隆至装有液体LB培养基(50mL/250mL)的摇瓶中，37℃振荡培养8h得种子液，以4％的接种量转接至装有发酵培养基(100mL/500mL)的摇瓶中，37℃振荡培养7-8h；取培养液以5％的接种量接入3L的发酵培养基中(7L发酵罐)，补料控制发酵(糖浓度低于15％时，补料30糊精，5％棉籽蛋白；发酵过程中控制pH为7.3，温度为37℃，残糖为15-20％，溶氧为30％-50％，发酵60-72h。
通过测量生长曲线可知，所有的敲除菌株在生长状态方面均没有较大的变化，如图3所示，可见敲除了eps基因簇、pgs基因簇以及3049-3052基因对菌株的生长没有任何的影响；而将含有碱性蛋白酶基因的质粒电转到敲除菌株中，通过国标法测酶活，通过酶活数据可知，基因工程菌株BAΔ6ΔepsΔpgsΔ3049-30521表达碱性蛋白酶的酶活最高为22523.7±531.8U/mL，如图4所示，以出发菌株为对比酶活提升了2倍左右；通过7L发酵罐放大培养，发酵液中碱性蛋白酶酶活力在72h达到峰值，为96783.17±1637.46U/mL，如图5所示。可见，本发明通过对解淀粉芽孢杆菌基因组上相关基因进行精简，最终获得了一种生长不受影响并能够高效异源表达碱性蛋白酶的基因工程菌株，并且为构建蛋白酶高产底盘菌株提供了创新思路。
虽然本发明已经以较佳实施例公开如上，但其并非用以限定本发明，任何本领域技术人员，在不脱离本发明的精神和原理的情况下，可以对这些实施例进行各种形式和细节的变化、修改、替换和变型，本发明的范围由权利要求及其等同物所限定。
SEQUENCE LISTING
<110> 天津科技大学
<120> 一种生产碱性蛋白酶的底盘菌株及其构建方法与应用
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 4319
<212> DNA
<213> Bacillus amyloliquefaciens CGMCC No.11218
<400> 1
taaagcgaaa aaagccaccg actaccatat cgatatgttc cggaagaata aggccgaatt 60
aattaaactg aagcgcgttc accgtgccga ggtggatatc gcatatttca cctacgagta 120
tttgagcgac ggtctaaacc cggaaaacga ggataatgtc gtaaggaact cggatgacgg 180
gacgcctcac gacggcattg aagatatcgc gaagatccac gaagagtttt ttgagctttg 240
cgattacgtc aacgaagaga aaaggaacgc gcggcttgca atcgcggctg cccggggcca 300
ctcaaaatcc ggaatgtttt cgaacgcctt gccgttacat caagcggctt atagaaagcg 360
gaaatatatt ctcgttattt ctgagacgga ttctctctcg aaaaagctca tcggatgggt 420
taacaagcaa atgaaattta acggcaagct ccgggaagac ttcgggccta tgatgcacga 480
gtcggcaagc cgcaatgaaa aggataacga agaggcgttc attacaacga cgaatatcct 540
tattgagtcg tcatcctccg gaaagcagct tcgcggtaaa cggcacggag ctgttcggcc 600
ggacctcgta ataattgacg acccgtcatc gacaaacaac gaaggaacca aggaagcgcg 660
ggaaaagctc gttcattggt ttaactcggt cgttgttccg atcgggtcca aggcgacggc 720
aatcgtgtta gtcgggacaa tggtttcggc gaccgggctt cttaatcacg ttctgaagcg 780
taaggatttc aaggcgagct ttcacggagc gatcataagt gagccgacgc atccggaatt 840
atgggacaaa tactgcgagg tctatgcgag agcagaaacg atcgaagagg ctgacgaatt 900
ctacgaagcg cataaagagg cgttggaaga cggcgtcgaa ctggcgtggc cttggcgctg 960
gacctatcgc gagctcatgc acgagaaagt aaacatggga acacgcgcat acaactcgga 1020
gttccgaaac cttgctttca gtgaagacga gcagtttttc tttccggata actacgctta 1080
ctatcactac taccatgaga acggaacagc atacgtcgtg tacaacgaat tgaaaatacc 1140
ggtcagcgat cttctgattg ttggggcgtg ggatatcgcg cagggtaaga acgcgcggtc 1200
ttgctataac tcggttatta cgctcggcaa acacgagccg accggctata ttttcgtact 1260
cgatgaatac gcgtcgaagg aaccggcgca catatacatt gacgtcatta ttcggaagat 1320
caaacaatac cggcatcgtt ccttcaacgt cgaaacgatt aacgcgcagc acgaatatta 1380
tcgtcagctt caggagcggg tccggcagga aggtctttat acgtgccgcg tgaacgatgt 1440
gaaatcgcat aagtcttcga aagatgagcg tattcaatcg atggaaccga tgctgcataa 1500
caaaacgctg attctgaacg ataggcacac gatgctgctc gatcagatgg cgcagtatcc 1560
tttcggcgat tacgttgatt cactcgacgc gcttcagcag gcgctcgaat ccgtattcag 1620
gccgaaaaca aggctggtga agaagccggg atggctataa gaaaggaggc gataaaatgt 1680
cgcgtatgaa agagcttgaa gcaaaactaa cgcttcagca acgtaaggca gcgcaagtcg 1740
ttgcgagtaa tgaagtcacg cccgaagacg ggaacaaacg aagtcaggac gcgctggccg 1800
aagaaatcgg cgtaagtcgt atgacgctct atcgatggcg gtttcaaaat ccgatattca 1860
tcgaatacat gaacctatta gcggacgaca tgctttccgg gcaccggtcc gaagtatacg 1920
ggcagctcct aaagctgata aaaggaccgc agccttcagt taaagctatc gacttgttca 1980
tgcgtcgtca tggcttactg accgatcgtc agataacgac caatgaaaca gacgacggcg 2040
cccgcagtaa cgaagatatc gagaaggaaa tcgaagacct acctgacatt atcggagagg 2100
aggaatagcg ttggggtttt tcaatttact aaagaaacac gaccagcctt cggatgagta 2160
cacggttaga aagacgtaca gcatcattcg ggaaggcgcg caatttcctc cggacgattc 2220
aatcaagagg ttggcgaaat ataaacgtat gcgtaagctc ttcgaaggaa atcaccgaga 2280
cgtatacgaa agggctacgg acattttaaa agattcgcca caagcgaaac agctcgaaaa 2340
gctctacatc gctataaatc tcgcggatat tttggtaacg aagcctgcgg atctgttggt 2400
aggagaaccg gttcactttg agagcggact ggacgacact agcgaagagc aaaaggcgct 2460
caacagatat gtagaagaaa acgatctcaa tcaactgtta cacgagagcg ccatgtcgaa 2520
cggatatcgc ggtgacgcgt ggattaaggt tcgatttggc taccgccaag actattccga 2580
acttatcgcg aggggcttag agattccaga agatgcgaaa atggagtccg ttgttgagca 2640
tgttaatgcg aattgcgttt tccctgagtt ttcagccgga aacgttaagc agatcaaagc 2700
ggtcaatata gcccaagtcg aatgggtaga gacggagcag acggaaattc ctttcctaaa 2760
cgtagagcgc catataccgg gacacatctt ctatacgcgc tacagactat atcagaatgg 2820
cgtcgatgtt tcaggtggcg ccccaatcag cgttttcaat ataggcgaag aggtgccgac 2880
gggccgcgaa gaagatcacg aagagacgtt ccttccgcac attccggttt ttcacattcc 2940
gtacaagtca atcgatgaca gctatttcgg tattggcgga ttggagaaga tcgaaacgac 3000
gctcgcggca attaacgatc gtctggtgca gatcgattac atcctatgga agcacagcga 3060
cccgactgcg tatgggccgg acttagaagg gtccggggat tcggttcagt ttggcggcaa 3120
gtacattccg gttacaaaag acgatacgac tccgggctat atggtttggc aggcgcagct 3180
tgacgcagcg ttcaaagaac ttgacgtcct attcagtaat gtctttcaaa tggccgagac 3240
gccgcagtgg ttattcggca cgacgatttc cggagataat tcaggcggca caggcacgtc 3300
tcacacagac ggcgcggcga tcaaagctcg cttcatgccg attctttcga aggtaaagcg 3360
gatacgagcg cactatgacc gggcggtaag agacgccctt tggacgtgta tgctactcga 3420
aaaagcagtc gggatcatca aggttgaaaa agcggtttat ccacgggcta tttggagcga 3480
cggactcccg aagaatgaaa aagagctcgc tgagattatg caaatccgca caggcggcaa 3540
gcctacgatt gatgttcgcg gggccattaa acagcaggac gatgtggacg acgaaaaagc 3600
tgacgaaatc attcgccgca ttgaagaaga cgaaaaaacg acgagcggct ttgttgacgg 3660
atcgattttc aacaaggaag aaccggaaag gagcccttcg gacgaggata aagaggaaat 3720
cacagaggag gacgatagtt aatgcctcgt tctcctgagc ctcaatacga ttatcagacg 3780
aatcgacttg ccgggtatta tcaggaagcg gtccgagaca tcttggcgga gcttgaacga 3840
attgacctcg ataattttcg aagggctaac gcgttggcga ccttgaaatc tattagcgag 3900
atcctcagcg atttagacga aaaatcttcc gcatgggtca aaaagaatgt tccgaaggcg 3960
gccaccgatg gtattgcgcg tgcactggtc gttctggacg tagcaaagac ggtggcggac 4020
gcggaaaagg tcgttgcttt taacgaggta aacgagtaca tggttgcggc tgctgtagcg 4080
gatacgcagt cggatttatt agcaattacg caaaacgtcg ataggaagac gcgggccgcc 4140
gtgaggaaag ccgtttcgga ttcgatgcgg cttaatatga ccaaagggac gaacggacgg 4200
cggagcataa cggatatggt gcgcaagtct cttcgagctt cggtgagtac cgggattatt 4260
gatgtgaggg gaaatcgatg gaagccggaa gtgtatgcgg atatggtcgt ccgcacaaa 4319
<210> 2
<211> 1149
<212> DNA
<213> Bacillus amyloliquefaciens CGMCC No.11218
<400> 2
gtgagaggca aaaaagtatg gatcagtttg ctgtttgctt tagcgttaat ctttacgatg 60
gcgttcggca gcacatcctc tgcccaggcg gcagggaaat caaacgggga aaagaaatat 120
attgtcgggt ttaaacagac aatgagcacg atgagcgccg ctaagaagaa agatgtcatt 180
tctgaaaaag gcgggaaagt gcaaaagcaa ttcaaatatg tagacgcagc ttcagctaca 240
ttaaacgaaa aagctgtaaa agaattgaaa aaagacccga gcgtcgctta cgttgaagaa 300
gatcacgtag cacatgcgta cgcgcagtcc gtgccttacg gcgtatcaca aattaaagcc 360
cctgctctgc actctcaagg ctacactgga tcaaatgtta aagtagcggt tatcgacagc 420
ggtatcgatt cttctcatcc tgatttaaag gtagcaggcg gagccagcat ggttccttct 480
gaaacaaatc ctttccaaga caacaactct cacggaactc acgttgccgg cacagttgcg 540
gctcttaata actcaatcgg tgtattaggc gttgcgccaa gcgcatcact ttacgctgta 600
aaagttctcg gtgctgacgg ttccggccaa tacagctgga tcattaacgg aatcgagtgg 660
gcgatcgcaa acaatatgga cgttattaac atgagcctcg gcggaccttc tggttctgct 720
gctttaaaag cggcagttga taaagccgtt gcatccggcg tcgtagtcgt tgcggcagcc 780
ggtaacgaag gcacttccgg cagctcaagc acagtgggct accctggtaa atacccttct 840
gtcattgcag taggcgctgt tgacagcagc aaccaaagag catctttctc aagcgtagga 900
cctgagcttg atgtcatggc acctggcgta tctatccaaa gcacgcttcc tggaaacaaa 960
tacggggcgt acaacggtac gtcaatggca tctccgcacg ttgccggagc ggctgctttg 1020
attctttcta agcacccgaa ctggacaaac actcaagtcc gcagcagttt agaaaacacc 1080
actacaaaac ttggtgattc tttctactat ggaaaagggc tgatcaacgt acaggcggca 1140
gctcagtaa 1149
<210> 3
<211> 4290
<212> DNA
<213> Bacillus amyloliquefaciens CGMCC No.11218
<400> 3
ttgaagaaaa aaacgagaaa aagatggaca ggctccgttt taagcgcgat tgtcgtcagt 60
tcactgctgt ttccgggtac ggccggagcg aacagcacac cgggagcggt ttctttcacc 120
aaagatcttt ctgcctctaa gagcattcag cataagattt ccgattcagt caaaaagcgt 180
ttggaaaaaa gtgataaagt cacgtttctc atcaaattta aagagaaagc caatacgaaa 240
aaagcagtta aagaagcaga aaaaaacgcc aagtctcaat cgctttccgt ggcaaaaacc 300
gaatatcaaa agcggtcagc cgtcatctct tctttaaaag tggcggctca tgatgcgcag 360
caaaatatga aaacatattt ggaaaaacaa aaaaagaaag gcaaggcgga tcatatccat 420
tcctattata ttgtcaacgg aatggcagtc actgcttcga aagaggtaat ggaaaaagcc 480
gcctcatttc ctgaagtaga aaaagttctt ccgaatgaaa agcgtcagct tactcaatca 540
aaagctcctt ttcaaatgaa gaaaaaacaa aaagagatca aagcaaaggg aggcattgaa 600
tggaacatca gtcaaattga cgcgccgaag gcatgggcct cagggtacga cggcaccggt 660
acggttgtcg cctccattga tacaggggtt cagtgggatc atccggcgct taaggaaaaa 720
taccgggggt acgatcctaa aaaccccgca gtgccgaacc atgaaatgaa ctggtatgat 780
gccgtggcta aaaaagacgc gccctacgat gaccttgaac atggcactca tgtaacaggc 840
accatgacgg gttcagaacc tgacggctca aatcaaatcg gtgtggcccc tggcgcgaaa 900
tggatcgcag taaaagcatt ttcggatgat ggaggaaccg atgccgacat tttggatgcc 960
ggtgaatggg tgcttgcgcc aaaagataaa aacggtactc ctcatcccga gatggcgccg 1020
gatgttgtca ataattcctg ggcaggcggc tcaggaatcg atgagtggta ccgcgatatg 1080
gtgaatgcat ggagagcagc agggattttt ccggaattct ctgcgggaaa tgtagacttg 1140
tttacaccgg gggggcccgg ttcaatcgca aatccggcca attatccgga agcatttgcg 1200
acaggtgcga ccgacagcca aaagaaactc gctgatttct cacttcaagg cccctcaccg 1260
tatcatgaaa caaaaccgga tatttcggca ccgggagtga atatccgttc ctctattcct 1320
gggggaacat atgaaggcgg ctggaacgga acttcaatgg ccggaccgca tgtggcagcg 1380
acggcggctt tgctacggca ggcgaacgct tctattacgg tcgatgaaat ggaagacgtt 1440
ttgacgcgca ccgccgaaaa gctgacagac tccgattttc cggaatcacc gaataacggt 1500
tacggccacg gcttagtcaa tgcttttgac gctgtttctg ctgtaacaga tggaatagga 1560
agcatagaag gcaaggtatc ctctgcgggt gaagatcaca atccgcccag ctggcatcat 1620
gaacccgtct cagaagctta caaaggtgca aacctcccgt taacggttac ggcggaagat 1680
gatgtaagtg tgacggaggt gttactttcc tatcagtttg ataaaggaga gtggaaaacg 1740
atcgccgccg ttcgaaaaag cggtgatgaa aaaaagggaa cataccaggc ggacattccg 1800
cacgtaacgg gcagcacggt aagttataaa tggacaataa aagatttcgg aggtcattcc 1860
gcagaatctg atacgtaccg ggctgacgta aaaccaagca ttacggcggg gtacaaagag 1920
gattttgaat cacagccggc cggctggttc agttacggga cacatgacca atgggaatgg 1980
ggaatcccga gttcagggcc aggcgctgca ttttccggag ataaagtata tgcgacgaac 2040
ctgtccggac cttatgccga ttcagccaat atgaatttgg tgatgcctcc gatccaggtt 2100
ccggattcag gaaggttgtt tttgcaattt aaaagctggc acaagctcga agaattcttt 2160
gattacgggt atgtgttcgt tcttccggaa ggaaaatcaa attgggagca ggcggctgtt 2220
tacaatggtg actccgccgg atggaacgat gaagaagcag atttatctgc gtataaaggc 2280
caaaacataa agcttatgtt taatatgcag tcagacgaag tactcaatga agacggatgg 2340
tatatcgatg atgtccggct ttcaagcagc tctctcggca aagcagccga aaaaaggaaa 2400
tccaatagac aaacaacgcc cggtcatctg aagaaaaaag ctgtcagtcc gaaagaggct 2460
aaaccggcgg tgaaatcacc cgagaaaacg gtgaaacgtg aaaccaatct cctgccgctc 2520
agagcgcaaa tcagtgtcgc cgagacgggg aaatccgtgt attccgaccc tgcaaccgga 2580
agctacagcc tgtctcataa agcgggaaac tacacgctga aagctgaggc ttacggattt 2640
aagccagctg tgaaacaagt ttccattcaa tcagacaaaa ccgcacaggc agactttacc 2700
ttggagcaaa tgccgtccgg tacgttaaaa ggaaccatca ccaatcagtc aacaggcgaa 2760
ccggtggaag gggctgtgct ttatgttgta gaggatgccg caattgagcc ggcggtgaca 2820
aatgataaag gggaatattc gcttcaggcg tacgagggct cctatacaat caaagcagcc 2880
gcaaaaggct tttacaacag tgaattttct gttgatatca aaggagatgc tgaaaaaaac 2940
gccaagctta agccgtatat cggctatgaa ggggaaattg cctatgataa cgggaccgaa 3000
gaagccgcgc tgtcatattt taaggcaggc agcaaatccg cggtcaagat gacgcttaaa 3060
gacggaaaag aacacggtat gctgacagga ggcctcttta aattttgggg cgcggactgg 3120
cctgatccgg gcggcaccga atttcaagcg gaagtttacg atgcatccgg acctgacggg 3180
gccccgggca gcaaaattgc cggcccgttc caagctgaag cgcttcgaaa cggagaatgg 3240
acgaaagtgg atctcagctc aaaaggaatc gcagtcggaa aggattttta tctagtgttc 3300
cgccagacga aacccaatcc ttattcaccg gccctgtcat ctgatgacgg cagtccgtat 3360
tcgaacagaa attgggaata tctcgacggc agctggtcaa aagccgataa atcagacgga 3420
aactttatga tccgggctct ggtcaactac gaagctattg tgcctgacat tacgtcacct 3480
gatgaccggt cgtttacaaa caagaagaca atcacggtaa aaggaacggc ctctccggga 3540
accgctgtcc gccttaccaa taacggtaaa accgccgctg aaacgaaagc cggtgcagac 3600
ggcaatttcc aggcggatgt cacactccgt aaagatgcca atcggctgac ggcggcatct 3660
gtgacagaca ggggatcaac agatgagtca cgccctgtca cggtcatatt ggaccaggat 3720
aagccggatg tgacgattga cagtccggca aacggagata aaacaaataa agaagcagtg 3780
accgtaaaag gaaaagctta tgacgcccat ttaaaagagg taaaggtgaa cggcaaaaaa 3840
gcagaagtga ataacggctc ttatcaagca agaatccttc tggaaaacgg cagcaatgag 3900
atcaaggtta cggcctcaga cgaagcgggt aacaaaacaa cgaaaaagac ggtaatcgat 3960
gtcaattaca acgctcccgt catttcgggg ctggttcccg gagcggataa agaattaaaa 4020
gccggagaat cagtgaaaat cgccttttca agcgggaaga aattagacgc aaccttcgtt 4080
atccgtctgc ctctgacaaa cgcgcgggcg gggagccaaa acgcaacgga gcttccgctg 4140
agagaaattt ctccgggaag atatgagagt tactggacgg cgacctcttc tatcaaagca 4200
agcggagcaa aaattgacgt tatcgtaaga gatgactacg gaaatgaaac gagacagacg 4260
gcaaaaggaa aactttatat taacgattaa 4290
<210> 4
<211> 2412
<212> DNA
<213> Bacillus amyloliquefaciens CGMCC No.11218
<400> 4
ttgaaaaaag gaatcatccg ttatctgctt ccggcttttg tcttatcctt taccttatcc 60
acaagttcac aggctgcgcc ggcttcaaaa ccgcaagctc ctgatctcga gaaggctgag 120
gtgttcggtg acattgatat gaccaccggc aaacaaacga cggtcatcgt ggagctgaaa 180
gaaaaatcac tggccgaagc aaaggaactc ggcaaaacac aaacaaaaaa caagctgaaa 240
agcgaacgcg caaaagtgaa aaataaagcg ctcaaaacga ttaaacacgg aaagatcaac 300
agggaatatg agcaagtgtt ttctggtttc tccatgaaac tcccggcaaa tgaaatcccg 360
aaactgctga gtgatcagga tgtcaaagcg gtttatccga atgtcaccta tcataccgat 420
cagctgaaag ataaagacat caccctttcc aaggatgccg tgtctccgca gatggatgac 480
agcgcgcctt atataggggc aaatgacgcg tggaagctcg gctatacggg caaaggcgtc 540
aaagttgcca ttattgatac aggcgtcgaa tacaaacacc ctgacttaaa gaaaaatttc 600
gggcaatata aaggatacga ttttgtggat aacgattacg atcctgaaga aacgccggcc 660
ggtgatccga gaggcgctgc gactgaccat ggaacccatg tcgcgggcac ggttgctgca 720
aacggaacga ttaaaggcgt cgcgcctgat gcaacgcttc tggcctaccg tgtgcttggt 780
ccgggcggaa gcgggacaac ggagaacgtc atcgccggca tcgaacgcgc cgtacaggac 840
ggagcgaatg tcatgaacct ttcgctcggc aattctgtga ataatccgga ctgggcgaca 900
agcactgcgc ttgactgggc aatgtcagaa ggcgttacgg ccgttacgtc aaacggaaac 960
agcgggccga acaattggac cgtcggctct ccgggaacat ccagagaagc catctccgtc 1020
ggagcgacac agctgccgct gaacgaatat gccgtctcct tcggttctta ttcctcagcg 1080
aaagtgatgg gctacaacaa agaagatgac ataaaagcgc tgaataaaaa agaagtagaa 1140
ctcgcagaag cgggtatcgg cgagcaaaaa gactttgaag gcaaagatct gaaaggaaaa 1200
atcgcggttg tcaaacgcgg cagcatcgcc tttgtggata aagccgacaa tgccaaaaaa 1260
gcgggcgcga tcggtatggt tgtgtataac aacgccgcag gagaaattga agccaacgta 1320
ccgggcatgt ccgtgccgac cattaagctg tcatcagaag acggcgaaaa gctcgtcagc 1380
caaataaaag cgggcggcac aaaagcgaca ttcaatttat ccgtggctaa atcgctcact 1440
gaacaaatgg cagacttctc ttcacgcggt ccggttatgg acacgtggat gataaaacct 1500
gacgtctccg ctcccggcgt aaacatcgtc agcaccattc cgacccatga tccggccaac 1560
ccgtacggct acggatcgaa gcagggaaca agcatggctt caccgcatgt agccggcgca 1620
gctgctgtca tcaagcaggc taaaccgaaa tggagcccgg agcaaataaa agccgcactc 1680
atgaacacgg cggaaacctt aacggatgcg gacggtgacg tatacccgca taatgcgcag 1740
ggcgccggaa gcatcagaat catgaaggcg atcaaagcag attctctcgt cgccccggga 1800
agttattctt acggaacgtt tatgaaagac aaaggcaatg aaacgaaaaa agaaacattt 1860
acgattgaaa accaatcgtc catcagaaag tcatatcagc tggaatactc tttcaacggc 1920
acaggcatta ccgtttcagg cacagaccgg gtcgtgattc ccgctcatca aacaggaaaa 1980
gtcaatgcga aggtaaaggt caatgccaaa aaagtaaaag caggcacatt tgagggaaca 2040
gtcacagtgc gtgaaggcgg aaaaacagtc gcaaaagtgc cgacgcttct gattgtaaaa 2100
gaaccggact atccgcgcgt gacttcgatc gacgtacagg acggcacagc gcaaggttcc 2160
tatcaaattg aaacctatct gccggcgggc gccgaagagc tagccttcct tgtgtataac 2220
agcaatcttg actttgtcgg ccaagccggc atttataaaa aacaagataa aggctatcaa 2280
tatttcgact ggaacggcaa agtcaatggt gacaccgcac tgccggcagg agaatattat 2340
atgctggcct atgccgcgaa caaagggaaa tcaagccagg tgctgactga aaaaccattt 2400
atcattgaat aa 2412
<210> 5
<211> 915
<212> DNA
<213> Bacillus amyloliquefaciens CGMCC No.11218
<400> 5
ttattgattt gaccaatatt gaatgttgtc gaaaacacta tttgtcaccc ttgttcccag 60
attgtaagtc gacccgccgt ttgtatgaac ggcaatggcg gtctggccgg tatcgctgta 120
atcgtgataa acgggcgatc cgctctggca gccgaacgta tctgtcgtgt aagtgagttt 180
atatgtctcg gccgaacgta tcggcttgct gtcagaccac atggaaccgg acactttgtc 240
gcacggatat cccgtcacag ttgaagacag cccggccgga ctggaactgt tggtcgtccg 300
gtagccgtac cagccgacag tattcccggg cgatccgtct actttgattg ccccgtaatc 360
atagttggta tcaccgtttt ctgtccatcc tttgacactg tagaatgtct tgcctgaata 420
tgatccgtac ggatacgatg agccgttgcg gccgggagct gccgtgattt tcgatgccca 480
tccatttttc tgatcataca cacaatgtcc ggctgtgacg atcgtgtcgg gattgacaaa 540
aaaaccggtg catccgtaag tgcttgtgct gccgctgtat gtaacggtca gctgtgctgt 600
cgcgcgataa ggaaatgatg ttgtcggaga aattctgatt cgttcgtctg ccactataat 660
actggaaggc tttaatgact tgatgtcggc tatgccgttg ttttgtgcag gctgtctttt 720
ggtgaggcac gatccttcat aagcgggaga tgtttgtgtt gtttttacag gctgattcgg 780
acttgcgtca aactcctcac cgctgcttga aacagatgtc tgcggactgc cttgggcttc 840
tgctgaaaca gcaaataatg aaacggacag aaagaatgcg gtaaagctca gcaatcgttt 900
tgaaactgtc ttcat 915
<210> 6
<211> 1566
<212> DNA
<213> Bacillus amyloliquefaciens CGMCC No.11218
<400> 6
gtgggtttag gtaagaaatt gtctgttgct gtcgccgctt cctttatgag tttaaccatc 60
agtctgccgg gtgttcaggc cgctgagaat cctcagctta aagaaaacct gacgaatttt 120
gtaccgaagc attctttggt gcaatcagaa ttgccttctg tcagtgacaa agctatcaag 180
caatacttga aacaaaacgg caaagtcttt aaaggcaatc cttctgaaag attgaagctg 240
attgaccaaa cgaccgatga tctcggctac aagcacttcc gttatgtgcc tgtcgtaaac 300
ggtgtgcctg tgaaagactc tcaagtcatt attcacgtcg ataaatccaa caacgtctat 360
gcgattaacg gtgaattaaa caacgatgtt tccgccaaaa cggcaaacag caaaaaatta 420
tctgcaaatc aggcgctgga tcatgcttat aaagcgatcg gcaaatcacc tgaagccgtt 480
tctaacggaa ccgttgcaaa caaaaacaaa gccgagctga aagcagcagc cacaaaagac 540
ggcaaatacc gcctcgccta tgatgtaacc atccgctaca tcgaaccgga acctgcaaac 600
tgggaagtaa ccgttgatgc ggaaacagga aaaatcctga aaaagcaaaa caaagtggag 660
catgccgcca caaccggaac aggtacgact cttaaaggaa aaacggtctc attaaatatt 720
tcttctgaaa gcggcaaata tgtgctgcgc gatctttcta aacctaccgg aacacaaatt 780
attacgtacg atctgcaaaa ccgcgagtat aacctgccgg gcacactcgt atccagcacc 840
acaaaccagt ttacaacttc ttctcagcgc gctgccgttg atgcgcatta caacctcggc 900
aaagtgtatg attatttcta tcagaagttt aatcgcaaca gctacgacaa taaaggcggc 960
aagatcgtat cctccgttca ttacggcagc agatacaata acgcagcctg gatcggcgac 1020
caaatgattt acggtgacgg cgacggttca ttcttctcac ctctttccgg ttcaatggac 1080
gtaaccgctc atgaaatgac acatggcgtt acacaggaaa cagccaacct gaactacgaa 1140
aatcagccgg gcgctttaaa cgaatccttc tctgatgtat tcgggtactt caacgatact 1200
gaggactggg atatcggtga agatattacg gtcagccagc cggctctccg cagcttatcc 1260
aatccgacaa aatacggaca gcctgataat ttcaaaaatt acaaaaacct tccgaacact 1320
gatgccggcg actacggcgg cgtgcataca aacagcggaa tcccgaacaa agccgcttac 1380
aatacgatta caaaaatcgg cgtgaacaaa gcggagcaga tttactatcg tgctctgacg 1440
gtatacctca ctccgtcatc aacttttaaa gatgcaaaag ccgctttgat tcaatctgcg 1500
cgggaccttt acggctctca agatgctgca agcgtagaag ctgcctggaa tgcagtcgga 1560
ttgtaa 1566
<210> 7
<211> 1749
<212> DNA
<213> Bacillus amyloliquefaciens CGMCC No.11218
<400> 7
atgcatcaca tcctaaaacc gataacggcc gctgccttgt gcgtcagcct gttttccgcc 60
gcggcgccag cgcacgccga gacgcacgac cgggacgtta tcgtagtgta caaaaatcaa 120
aacggcaagg aatccgccat agacagcgga gcggatgtgg aacagaccta tcagcatctc 180
ccggcagtcg ccgtgtcggc cgattcgcaa acggtgaaag acctgaagca agatcctgac 240
atcttatatg tagaagacaa tgtacccttt caggcagcag gcggctctga tgtacacctg 300
ttatcagcgg ctccaagcag cagttacgtg ctgccgcaat gggatataga accgactcag 360
gtcaagcagg cctggaaaga aggcttgacg ggaaaaaaag tgaaagtggc tgtcattgac 420
agcgggattt atcctcatga tgacctttcc attgcgggag gctattccgc agtcagctat 480
acatcctcat acaaagatga caacggacac ggtacgcatg tcgccggaat cattgcggcc 540
aagcatgacg gatacggcat tgatggcatc gcgccggatg tccggttata tgcggttaaa 600
gctttggatc gaaaaggtgc gggagatctt aaaagccttc ttaaagcgat cgactggtca 660
atcgccaata aaatggacat catcaatatg agtctcggaa caaacgcaga cagcaaaatt 720
cttcacgacg ccgtcgacaa agcatacaaa aaaggaatcg tgatcgtagc cgccgcagga 780
aatgacggca acaaaaaacc cgtcaattat ccgggcgcct acagcagcgt aacggcagtc 840
tcagcgtcaa ccgaaacaaa cgggcttgcg gcgttttcca cgaccggaaa acaaattgaa 900
ttcgccgcac ccggcaccaa tataacaagc acttacctga atcaaatgta tgcaacggcg 960
gacgggacct ctcaagccgc gccgcacgtc accggcatgt tcgcgctgct caagcagaaa 1020
tatccggagg aaacaaatac gcagctccga cagcagatgc agcaaaacgt caaagacctg 1080
ggcgctcccg gacgtgacag ccaattcggt tacgggctgg tgcaatatta tacgaaccaa 1140
aagagttttg ccgaacgggc cgtcataaaa gcggaaaaaa cgaaaaagca ggcagatatc 1200
aatcaagcaa aaacgtcggt cagcaaactt cccaagtcaa agggaaaaac cgcacttgag 1260
acgcgaatca ataaagttca agccgcaaga aatgtgacgg atgcaagaga caaagtccgg 1320
actgccgaaa aacaaaagaa aaaaaacgcc gtcaatgccg cccaaagcgc catccgcaaa 1380
ttagcggccg gctcagagaa aaaagggctg caaaagcggc ttgatgctgt caactcaggc 1440
cttttgaaaa cagccgaagc cagcgtcaag caggcggaaa aaaagacctc tgagaccaac 1500
gccgccaaag caaaaaaagc agtcagcgag ctccagccgg gaaaagaaaa aaccgccctg 1560
gaaaaacggc ttgacaggat aaaagataag atcaaccgga agcaagcaag agacaaagtg 1620
aaaacggccg aaaaaaccaa aacgaaaaag gcaaaatcag cggcacaaac ggcggtcagc 1680
cggctgaagc cgtctgccga aaagaccggt ctgcaaaaac gtgtgcgcgc gatccgggtg 1740
aaacattaa 1749
<210> 8
<211> 15714
<212> DNA
<213> Bacillus amyloliquefaciens CGMCC No.11218
<400> 8
atgaatgaga atatgagttt taaagaatta tttgacatta ttaagcgcag atttttactg 60
atttttatca tgacagcagt agtgacgctg gtgacgggat acatccaatt tcgagtgatt 120
tcgcccgttt atcaggcgtc aacccaggtg ctcattcatg aaacaagcgg tgaaaaaaaa 180
tcaaatctca gcgacatcca gctgaatctc cattacaaca acacgtttca aacgataatg 240
aaaagcccgg ttgtgattga gaaagtgaag cggaagctcc atctttccga gacggcatcc 300
gctttgaaag caaagatcac gacaagcagc gaaaccgatt cagaaatcat caccgcagcg 360
gtgcaggatg agaatccgaa acaggccgcc gcaatagcga acacgctgat gaaaacattt 420
aaaaaagaag tccgcgacag gatgaatata aaaggcgtca tcgttttgtc tgaggcaaaa 480
gcatcggaaa gcccgatggt caagccttcg cgcatccgga atatcatgat ggcattcggt 540
gcggccctta tggccggtgt gacgcttgcg ttttttctcc actttcttga tgaaaccgtt 600
aaaagcgagc ggcagctcag cgaaaaaaca gacttgcctg ttttaggggt tgtgtatgat 660
atcaaaaatc agaagacacg gtctgatgaa aaacatttcg gggagtgagg catttgggat 720
tcagaaaaaa gaagtcaaga cggggattgg ctcaaatatc cgttttacac cacaaatcat 780
tggtagctga acaataccgc accattcgga caaatattga gttttcctct gtacagaccc 840
atttgcgctc tattctcgtc acttcttccg ttccgggaga aggaaaatca ttcagcgccg 900
ccaaccttgc cgcggtattt gcgcagcagg aaaaaaaggt gctgctcgtc gatgcggatt 960
tacgaaaacc gacgatacat gagacctatc agcttgaaaa tgtacaaggc cttacgaatg 1020
tgttagtcgg aaacgcttcc ctcggcgaaa cggtgcaaaa aacactgata gataatcttt 1080
atgtcttaac gagcggtccg acgccgccga atccggctga gcttttgtcc tctaaagcga 1140
tgggagagct gattcaggag atgtacagcc gatacagtct ggtcattttc gattcacctc 1200
cgcttttggc ggtggcggac ggacaggttt tagcgaacca gacggatgga agcgttctcg 1260
tcgttttgag cggaaaaaca aaaatggata ccgtccaaaa ggcgaaagac gcgcttcagc 1320
agtcgaaggc gaagcttttg ggcgcgcttt tgaataaaaa gaaaatcaaa aaaacagaac 1380
actactcgta ttgagcggtg aaggtgatgt ctccttacct ttatcaacgg aggcactcgg 1440
ctctgctgtg ggcagcgatt ttcgctgcct tagatgctgg tcgtcggtgg cggggtgtga 1500
ggacgggtta aatgcccaat ttatcttaaa atgccgcgtt tttatcggct tagctgttat 1560
gcctgttttt ttgcgaaaac cgtatcagct aaaccgataa atgaggaggg gaaagattga 1620
cttaccggag aagactttca atgatttttg cattggatac gtatctcgtt ttactttccg 1680
ttgttatagg atatcaattt tttgaggatt cttatcactt ttatgactcc ggagcgctcc 1740
tgctgaccgc ggtgagcatg ctgatcagcc accatgtatg cgcttttatg tttcaccagt 1800
ataagcaggt atggacgtat acgggaatag gggagctgct tgccctgctg aaggggatca 1860
ctctgtccgc agcggtgacg gccgccgttc aatatggggt gttccacacg attttgttcc 1920
ggcttttagc cgtcagctgg atggttcagc tattgtttat cggaggcagc cggatgattt 1980
cgcgcgtgct gaaagaaacg atcggcaaga agcaaaatga ctcttcacgg gcgctgatta 2040
tcggcgctgg cgcggggggg gacgctgctc gcccgtcagc ttacgcagaa aaacgatctc 2100
ggaatcatgc ctgtggcttt tattgatgat gaccagacga agcataagct cgagattatg 2160
gggctgcccg tcatcggcgg aaaagaaagc attttgccgg cggtgcagag gctgagaatt 2220
caccatatca tcatcgccat tccgtctctg cgaacccatg agcttcagac tttatacaaa 2280
gaatgtgtgc agacgggcgc ccatattaaa atcatgccgc aatttgatga gatcctgctc 2340
ggaacgcagg ctgccggaca catcagagat gtaaacgccg aagatttgct cggcagaaaa 2400
ccggtcactc tggatacgag caaaatttct gacagcataa agggaaaaac gattctggtc 2460
acgggtgccg gcggctctat cggttctgag atctgccgcc agatcagctc gtttcgtccg 2520
cgtgaaatcg tccttctcgg ccacggggaa aacagcattt attccgtgca tggcgaactg 2580
tcagcacgct ttgggaaaga ggtgcttttt cacgcggaga tcgccgatat tcaggataga 2640
gataaaatct ttaccttgat gaaaaaatac gagccgcacg tcgtctatca tgcggctgcc 2700
cataaacatg tgccgttaat ggaacataac ccggaggaag ctgtcaaaaa caacatcctc 2760
ggcacaaaaa atgtcgccga ggccgccgat atgtgcggaa cggaaacgtt cgtgctgatt 2820
tcttctgata aagcggtcaa tccggccaat gttatggggg cgacgaagcg gtttgcggaa 2880
atggtcatca tgaacctcgg aaaaatcagc cgcaccaaat tcgccgccgt ccgtttcgga 2940
aatgtgctcg gaagccgggg cagcgtcatt ccgattttca aaaagcagat tgcaaaaggc 3000
ggacccgtca ccgtcacgca tccggcgatg acaagatatt ttatgacgat ccccgaagcg 3060
tcaagactcg tcattcaggc gggggcgctt gcaaaagggc ggcagatttt cgttctggat 3120
atgggagaac ccgtcaaaat cgtcgatctg gcaaaaaacc tgattcattt atcaggctat 3180
acgacagaac agattcctat tgaattctcc ggcatccggc cgggagaaaa gatgtatgaa 3240
gaactgctga atcataatga agtacatacg gaacagattt ttccgaaaat ccatatcgga 3300
aaagcggtag acggggattg ggccgtgctt atccgcttta tggaagaatt cagccgtctg 3360
cctgaggaag agctgagaaa acggctgttt gaggcaatcg aatcagtaca tgaagaagcg 3420
gccgcgggcg tgtgatggga ggtcagaaag atgacgaaaa aagtattatt ctgcgccact 3480
gtcgattatc attttaaagc ctttcatctt ccgtattttc aatggtttca ggatatgggc 3540
tgggaggtgc acgtcgcggc aggcggtgat atgaatctgc cgttcgtgaa tgaaaagttt 3600
tccgttccga tccggcgctc accgtttcat cccgaaaacc tttctgttta cagacggctg 3660
aaacggctca ttcaggataa cggctatgac atgattcact gccatacgcc ggtcggcggc 3720
gtgctcgccc gccttgccgc gagacaagca cgccaaaaag ggacaaaggt gctctacacc 3780
gcgcatggtt ttcacttctg tgagggagcg cctctgaaaa attggcttct ttattacccg 3840
atcgaaaaat ttctttcttc ttatacggat tgcctgatta ccattaatga agaggattac 3900
gaacgggcca agcaaatgaa aaaaaccgat tgcgacgcga aaaaaataca cggcatcggc 3960
gttgatacgg acaggtttcg gcctgtaagc cgggcagaga gtgaacgtct gagagaaaaa 4020
cacggcttca ccgccggaga atttatcctc gtttatccag ctgaattaaa cagcaataaa 4080
aaccagggta tcctcattga ggctgcggca ttgctgaaga acagcatccc tgagctgaag 4140
cttgtatttg cgggagaagg cgcgatggaa gagtcgtaca gaaaaaaggc tgaatcactc 4200
ggtgtatccg atattgtgcg gttttacggt ttttgccggg acattcatga actcattcag 4260
cttgctgatc tgtctgtcgc ctcaagtatc cgggaagggc tcggcatgaa tgtccttgag 4320
ggaatggctg ccgaaaagcc cgccgttgcc gcagataaca gggggcaccg tgagatcatc 4380
gaagacggcg tgaacggttt tctcgtgccg gccggagaca gcgcggcgtt tgccgatcgg 4440
attggaaagc tgtaccgctc ccccggcctg cgaaaagcaa tggggcaaaa agggcgccgt 4500
acggcggaat gcttttcaga aacacgcacg gtaaaggaaa tggagcatat ttacgccggt 4560
tatatggata aaaaggagaa aagcgtatga atgcaggagc acaaccgaaa atttccgtca 4620
tcatgggaat ttataattgc gaacagacat tggcggagag tattgaatcc atcctgagcc 4680
aatcttataa gaattgggag ctgattatgt gtgatgacgc gtcaactgac ggaacgtatc 4740
agattgcgcg ccgttacgcg gatcattaca gcgaccggat tacgctgatt caaaatagga 4800
caaatcaacg gcttgccgct tctttgaacc gctgtctgac atacgcgtcg ggggattata 4860
tcgccagaca ggacggagac gatatctccg cccctaggag actggaaaaa caggccgctt 4920
tcttaaacaa gcacgcccac tatcaagtcg taggcacggg tatgctcgta tttgacgaat 4980
tcggcgtgag gggcgcccgg cttttgccgc cggtgccgaa accggaaatc atggcgaaag 5040
gcacaccgtt ctgccacggc accattatga tgcgggccga agcgtacaaa gcgctcggcg 5100
gctaccggtc agtcaggcgg acgcggcgga tggaggatat cgatttgtgg ctgcgctttt 5160
ttgaagaggg cttcagaggg tacaatctcc aagaaacgct gtataaagtg agagaggaca 5220
gcgatgcgtt taaacggagg tcgttcacgt attccattga taatgctgtg ctcgtttttc 5280
aggcgtgcag acggctgaag ctgccgtttt cccattacgt gtatatcgca aagccgctca 5340
tccgcgccat tacgccgccc gcggtcatga accgttatca taaaaacagg gacatccgcc 5400
aaaaagaagg gcttgccgaa catgactgac acacctgtgc gggtgctgca catcttcagc 5460
gggatgaaca gaggcggcgc agaaacgatg atgatgaatt tgtaccggaa gatggacaga 5520
acgaaggtgc agtttgattt tttaacccat aggaacgatc cgtgcgctta tgatgaggaa 5580
attcttactc tcggcggacg gcttttttat gtgccgagca tcggcagcac aaatccgata 5640
acatttgtga aacaggtcaa acgggtgatt caggaaaaag gcccgtttgc cgccgtgcac 5700
gcccataccg attttcaatc gggatttatc gcgctggctg cgcgtttagc cggtgtcccg 5760
gtgaggatct gccattctca cagcacgtcg tggcgggggc ggtcgtcgcg gcttgccggg 5820
atgcagctat ttgtattcag acggatgatc acggcaaacg ccaccgcgct gtgcgcctgc 5880
ggggaagagg cgggacgttt cttattcggt aaagaaaaag atgtgcatct tttgccgaac 5940
ggcattgatc tcgatttatt tgccggcggg cgtgcggaca cagaatcgga aaaaaggaag 6000
cggggcattg ccggcggccg tttggcaatc ggacatatcg gccgctttac agaggagaaa 6060
aaccaccaat ttctgctccg gctggccgct gatatgaaag agcggggcat cggctttcag 6120
ctgattctcg ccggagacgg tccgcttcgc accgatatgg aaaaactggc ggagaagctg 6180
gggcttgatg atgatgtgcg ttttatcggc gtagaggagc gcgttcatga tctgctgaaa 6240
acgctggatg tatttgtcat gccgtctcta tatgaggggc tgcctgtcac gctcgttgaa 6300
gcgcaggctt ccggggtgcc gtgcgtgata tcagacggca taacagaaga agccgatgcc 6360
gggctcggac tggtcagcag gctcagcctc aaagagccgc ccgaacaatg ggcatccgcg 6420
attttacggg cagcggagac gccggtgcct gacaaggccc gcattaaaga aaccctcgga 6480
cggctgggat atgatgcggg agaaaatgcg ggggcgatta tgaagcttta caatatgaat 6540
tgtgaaaagg aacagtaacg aatgattgta tatgtagtca atatgggaat tgtttatatt 6600
tggtcatggt tcgcgaaaat gtgcggcggg cgtgatcaat cgctttcgac cgggtaccgc 6660
ccgaatcagc tcgtcatgct ggttccgctg ctatcgctgg tattggtctc cgggttacgc 6720
tacagagtcg gaacggattt tcagacgtac gcgctgatgt acaaactggc gggcaattac 6780
ggcagtatat gggagatttt cgggttcgga gcgaaaaaag ccgctgttga tccggggttt 6840
accgctctca tctggctcat gaattttatc acgaaagatc cgcaaatcat gtatttcacc 6900
gttgcggtcg tgacgtgcag ctttatttta aaagggctcg ccgagttcgg ccgtccgttt 6960
gagctgagtg tgtttttgtt tttaggcacg tatcattatt acgcttcttt taacggcatc 7020
aggcagtata tggtggcggc cgtgctgttt tgggccgtcc gctacgtgat cagcggaagc 7080
tggaagcgct attttttgat cgtgctgttg tgctcgctct ttcattcctc ggccctgatc 7140
atgattccgg tttattttat cgtcagaaga agagcctggt cgcccgcgat tttcgggctg 7200
tcggccttgt ttcttctcat gacgtttctg tatcagaagt tcatttcgct gtttgtcgtc 7260
gtgctggaga acagctcgta cggccattat gagaaatggc tgatgaccaa tacgaacggg 7320
atgaacgtcg taaaaatcgc ggtgctgctt ctgccgctgt ttttggcttt ctgctaccgg 7380
gagcggctgc ggaagctctg gcctggcatt gatgtcatcg tgaatttctg tctgctcggc 7440
ctgctgttcg gtctgctggc gacaaaggat gttatttttg cccggtttaa tatatatttc 7500
ggcctgtacc aaatgatcct gatcccttat tttgtgagga tttttgatga aaaatcgaat 7560
gccctgattt atatcgccat tattgtctgt tattttcttt acagctattt gcttatgccg 7620
gtcgattcgt ctgtgcttcc gtacagaacg attttttccc ggtaatggat actggaggtt 7680
tatatggaaa cacccgcggt cagtttatta attgctgtgt ataatacgga aacctattta 7740
gaaaaatgtt tagattcatt gctcaatcag tcgctcgcca atatcgaaat tatcgccgtc 7800
aatgacgggt caacggatca aagcccggcc attcttgaaa cctatcaaaa gcgggatgag 7860
agaatacggg tcattcatca gaaaaaccgg gggctcggcg cggtccgcaa caagggcatt 7920
gaggcggcgc gcggcgaatt tatcgctttt attgatgcgg acgactgggt tgaaccggat 7980
tattgcctgc gcatgtatga aaaagcgaag gctgatcagg ctgatctcgt catttgcgaa 8040
tatgcggctg aatttgcgga tacggggaaa actgtcgtat caacgatcgc ttcagcgtat 8100
gagggccgtc cgaaacaact ctacctgaaa gatttatttg agggaagagt cagcggattt 8160
tcatggaaca agctgtacaa cagaaccatg atcgaacatc atcggctgcg ctttccgctt 8220
cgtgatgagc tggaacacgt ggaggatcag tacttcagcc tccgggcgca tgtattcgcg 8280
gatgcggtct cgtacgtgga tcagccgctc tatcattatc ggattcactt gacttccatt 8340
gtgcaaagct atcagaaaaa gctgtttgac tcaggtctgg ttctctatcg gctgaacgag 8400
gcttttttgc gtgaaaacgg ctgcttacag gaataccggc aggagctgga cttttttatc 8460
gtccagcacg gaacagtctg cctgcttaat gaatggaagc ggaataacgg cggacgtttt 8520
tctgagaaat ggcagaatat cagccggata tgcgctgatc ccgtgttccg cctgagtctt 8580
tccaaaacag gaacggcgcc gtttgacgct aaacgctcat gccttctgct tttggcgaag 8640
ctgaagctga ttccgttcgt atcgctggca tcagccgcgt atcagcgggc aatcgagtac 8700
aaaatgaaaa tcagagggtg aagacatgtc gatacaatct ttaaaaatca atctcgcgga 8760
atggcttttg cttaaggtga aatacccgtc tcaatttcat ttcggaacgg cagcggacgg 8820
ggcggggcat acagcggcaa gaaaaaagat cattctgacg ctgcttccgt cccacgataa 8880
tctcggcgat cacgccatcg cctacgcaag caaaacgttt cttgaacggg agtatcccga 8940
ttttgacatc attgaggtgg atatgaagga tatatacaga tccgcaaaag cgctgatcaa 9000
aaaacgccac cccgatgata tggtattcat catcggcggc gggaacatgg gtgatttgta 9060
ccgttatgag gaatggacgc gccgttttat tataaaaaca ttccatcagt atcggatcgt 9120
tcagcttccg gcgacggctc acttttctga ttcaaaaaag gggcggaagg aactgaaacg 9180
cgcccaaaaa gtgtataatg cgcatcccga ccttttgtta atggcaagag acgagacgac 9240
gtatcaatgg atgaagcgtc attttcccgg aaaaacagtg ctcaaacagc ctgacatggt 9300
cttatatttg gataaaagcg aacggcgcct cccgcgggaa gggatctacc tctgcttacg 9360
tgaggaccgg gagagcgcgc tgaccgctga ggaaagaacg atggtaaaag aagctttggc 9420
tgaagaatac ggagagctgc attcctttac cacgacggtc ggccggagag tcagccgcca 9480
tacacgggaa gaagagctgg aggcgctgtg gaatacactg aaaggcgcgg aagccgtcgt 9540
aactgacagg cttcacggga tgattttttg tgcgctgacg aaaacgcctt gtgtcgtcat 9600
tcgttctttt gaccataaag tgatggaagg ctttcaatgg ctgaaacata taccgaacat 9660
gacattgctt gaacgtccgg agcctgaagc cgtgacggcc gctgtaaacc ggctgctgag 9720
cggcaagtat gaggaaggcg gttctatgcg cagtgtttat tttgccgggc tgcgctcgaa 9780
aatcagcggt gatgcccaat gaacgcgccg ctggtaagcg taatcgtccc gatgtataag 9840
acagagcctt ttattaaagc atgcgcggta tctttaacaa aacagatgct gagagacatc 9900
gaaattattt tcgtcaatga cggctcaccg gatcagtccg gccggatggc cgaacaattc 9960
gccgcagaag atgcgagaat ccgggtgatt catcaagaaa acggcggtct cagctcggcc 10020
cgcaatgccg gaattaaagc cgcccgcggc cgttatatcg gctttgtgga cggcgatgac 10080
tatgtgacgg aaacgatgtt cgagcggctt tatgaagaag cggagaagaa ccggctggat 10140
atcgccggat gcggctatta taaggaaacg ccgtctaagg aaagagcgta tatgccgccg 10200
tccattccgc cgggccgcgt cttcacagcg gctgagatga ctgatctgct gacctgcgcg 10260
cacgaacatc ggtttatttg gtacgtatgg cgttatatat accggagaga agtattgcaa 10320
gggctgctgt ttcatgaaga tatccgattt gccgaggact ctccgtttaa cttggcagcg 10380
ttccgccatg cagcacgtgt caaagtgata gatgaagggc tttatattta ccgggaaaac 10440
cccacaagcc ttacagaaac tccgtttaag ccacacttag atgacgaact gcaaaagcaa 10500
tatgaagcga aaatggcttt ttatgaggcg aacgggctga cggacgcctg ccaaagcgat 10560
atcaatacgt atttgtgtaa gcatcagatc ccgatgctga tcgccaatgc atgcgccgcc 10620
ccgcagccgt cacatgagat tacggcgcac atcggcagaa ttttaacgta tgacatggta 10680
aaaacggcgg tgcgcagcac gccgtgccgg cataaacagc tgctggccgg ggaacgtgtg 10740
gtgctcggtc tgtgcaaaat gcgccttccg ctgcttctgc atgcattttt cgatcggaaa 10800
acgaaagaga aaggcagtgc ggaaggagca tgaaatttgc cattaatttc ggcgccaatg 10860
tgacggcgtt tttactatcg gtgtttttat cagtatggat gacccccttt attgtcaaaa 10920
cattaggtgt agaggctttt gggtttgtcc atctgacaca gaatatcatt aactatttct 10980
ctattattac cgttgcgctg agctctgtcg ttgtgaggtt tttctccgtc gccgcccaca 11040
gggggaacag ggatgaggcc aatgcgtatg taagcaatta tttagccgcg tctgtcgtga 11100
tttcgctatt gctcgctgtg ccgctcgccg ggacggcttt ttttattgac cgcatcatga 11160
atgttccggc cgggctgtta acggatgtga ggctctctat cgtgatcggc agtgtgctgt 11220
ttatgctgac gttttttatg gcaggtttcg cgacggggcc gttttttgcc aacaagcttt 11280
atatcacaag ctccattcag gccgtgcaaa tgctggtcag ggtgctgtgt gtgctcgctc 11340
tgtttacgtt ccttcccccg aagatctggc agatccagct ctcggcttta gccggtgccg 11400
tctgcgcggc cgtgctgacg tttttctttt ttaagaaatt gattccgtgg ttttctttca 11460
ggcggaaagc cttgtcatta cagacgagca aagtgctgtt ttcggccggt gcatggagct 11520
ctgtcaacca gatcggcgtg ctgctgtttc tgcagattga cctgatgacg gcaaaccttg 11580
tgctggggcc gtcggaagcg ggcatttatg cggcgattat ccaatttccg cttctgctta 11640
gaagtcttgc cgggacgctc gcttcgctgt ttgcgccggt tttgacttcc tgctattcaa 11700
aaggcgatat ggaaggtctc ctgtcttacg ccaataaggc cgtgcggctc aacgggcttc 11760
tgctcgcgct tcccgccgcc ttattaggag ggcttgccga accgtttctg tcgatttggc 11820
tgggcccgtc gtttgtgcag acggcgccta ttttatacat tcacgccgcg tatttggcgg 11880
tcagtctctc tgtcatgccg ctattttacg tatggacggc ttttaataaa caaaaaacgc 11940
ccgccgtcgt tactttatgc ttaggcggac tgaatgttct attggccgtt gttctcagcg 12000
ggccggctca tctcggcctg tacggcatca ccattgccgg agctgtctct ctcattttga 12060
aaaatgccgt cttcacgccg ctgtatgttt cgcatattac cggctttcag aaaaccgcgt 12120
tttacaaagg catgttcggt ccgttggcgg cggctgtatt tgcctgggct gtctgccggg 12180
gaatccagct gttttcaccg cttgacagct ggaccggcct gattgccgca gggcttgccg 12240
tatgcgtgag ctatgccgca ttcgcttttt tcttcatttg tacgaaggaa gaaaggcggc 12300
ttgctttaca gaaatgccga aaagtgaaag gagctgttca gatttgaagg cgaaacgtgt 12360
atttgatata acggccgcga ttttgttatt gtgcggcgca agtgtcattc ttctcttcgc 12420
catggccgcc gtcagattcg ccatcgggtc gccggtgttc tttaaacaga cgagacccgg 12480
gtataacggg cgtccgttta ctttgtataa actcaggacg atgacggatg cgcgtgacga 12540
aaacggtgcg ccgcttccgg atcatctgcg tctgaccaga gcgggaaagc tgatcagaaa 12600
actgagcatc gatgagcttc cccagctttt taatgtgatc aaaggcgata tcagtctggt 12660
cggcccccgc cctttgctga tggattatct gccgctttac acggcggagc aggcgcggcg 12720
gcatgaggtg aaaccgggca ttacgggctg ggcgcagatt aatgggagaa atgcgatttc 12780
ctgggaagaa aaattcaagc tcgacgtgtg gtatgtcgat aaccgcacat tccttctcga 12840
cttgaaaata ctgctgttaa ctgtgaaaaa ggttctcgtg tcagagggga tacatcaatc 12900
cggccatgcg acggcaaagc gcttcacagg cagcggagat atgtcatcgt gaaaaaggtc 12960
gtgctgatcg gaaacggcgg acacggaaaa gttgtaaagg aaatcgtgaa ggctcgttcc 13020
gatatggagc ttgcgggcat attggacgac ggattcagcg gttttaccgt tcgggacggt 13080
ttgtatacgg gccgaacgaa agacgtgcac atgcttcgga aactcgtccc gggagccgta 13140
ttcacgatat gcatcggtca caatggtgta agaaagcagc tcgctgaaac gcttgggctt 13200
gaacatgatg attacaccgc tctcattcac cccggggcaa tcgtcagtga tacggcttcc 13260
gtcggacacg gaacggttgt gatggcgggc gccgtcattc aggcgggcgc tgacatcggc 13320
gcgcattgca tcatcaatac aggtgcggtt gccgatcatg acaatgcaat cggagattac 13380
gtccatcttt ccccgcgcgc tgcgcttgcc ggcggagtga aagtcgggga aggagcgcac 13440
atcggaatcg gcgcgtctgt cataccgcgg acagacatcg gtccttggtc ggttatcggc 13500
gcgggggctg ccgtcatcag ccggattccc gatcatgtga cggcggtcgg cgttccggcc 13560
cgcgtcatct cctctattca taatgaaaaa ggatgatttc atgcaaacaa acaaacggat 13620
ttatttgtct ccgcctcata tgagcggaaa agaacaggaa tacatcgcgg aagccttccg 13680
ttccaattgg attgcgccgc tcggtccgct cgtcaattcg tttgaagcgc ggctcgcgga 13740
atatgcgggg gtgaaaagcg cggcggccgt aagctcggga acggcggcca tccatcttgc 13800
cctgcggctt gcgggtgtga aaaaaggaga cgctgtattt tgtccttcct ttacgtttgt 13860
cgccacggcg aatccgattg tgtatgagca ggcagagccc gttttcattg attcggaatg 13920
ggagacgtgg aacatgtcgc ccgccgcgct tgagcgggcg ctccgggatg caaaacggtg 13980
cggcaggctc ccgaaagcgg tgatcgccgt caatctttac ggccaaagcg caaaaatgga 14040
tgaattgtta cgcctttgcg acgcatacgg agtctgtctg attgaagacg cagccgagtc 14100
cttaggatca acatataaag gcagacagag cggcacgttc ggccgtttcg gcatttattc 14160
ctttaacggc aacaaaatca ttacaacatc gggaggcggc atgctcgtgt ctgacgatga 14220
agcagctatt gaaaaagcga agtttcttgc gtcacaggcg agagatgccg ccgttcatta 14280
ccagcacagt gagctcggtt ataactacag gctcagcaac attctcgcag gcgtcggtat 14340
atcgcagctg gaagtgctcg aagaacgcgt ccgggcgagg agagaaatct ttcacaggta 14400
taaggaagcg ctggaaacgt atccgggcat ccgcatgatg ccggagcttg aaggaacggt 14460
ttcaaaccga tggctgacgg ctctgacgct tgataacggc gtgacgcctg aggaagccgt 14520
cgcctgtctt gccgaacaaa atattgaggc ccgtccgctg tggaaaccgc tgcatacgca 14580
gccgcttttc tcatcttccg ttttttatcc tcacagcgat catgaacggg tctctgagaa 14640
ccttttcagc cggggcattt gtctgccgtc cgggtctgat ttgtctattg aagagcagca 14700
gcgtgtcatt gacgcactag cacaactgtt tgaaacgaaa ggggagaaaa catggacagc 14760
cgccatgtca tgagccgatt aaaagagacg ctgacaggtc tgctcagcgt cataccgccc 14820
cagtctgaca tcatttatgc cgactatccc ctctatggaa atgtagggga tttattgatt 14880
atgaaaggga cggaggcgtt tttttaagcg cacggcatcc gcgtgaaaca gcgctggaat 14940
cctgacaatt ttccgttcgg gcgcagggcg gataaaaaga cgatcatcgt ctgtcaggga 15000
ggcgggaatt tcggggatct ctatccgtac tatcagacgt tcagagaaaa aatcgtcaaa 15060
gcctttcccg aaaacaggat cgtcattctg ccccaatcca tctattatca ggatgaaaca 15120
cggctgcgaa agacagcggc gctttttgcg gagcacaagg atctgcacct gtttacgaga 15180
gatcatgtgt cacacgagac tgcgaaacgc tttttttcgg cgaatcacat cggacttctg 15240
cctgatatgg cccatcagct gtatccgatc aagacggcag cggctccgtc acgcggcagg 15300
cttcatttta tccgcactga cggggaaatc aatccgaaac tgcaaaacaa tcattccgtt 15360
aaaagctgcg actggcagca tgtgctgtca gcaagcgacc gccggggaat cgcttttttc 15420
cagacgctga atgtcctgaa taaaaaagcg ggaaacctgc tgccgattgc gcggttttgg 15480
aaattctatt cggatttcct gacgaaaaaa gcggtccggt ttttcagcaa gtacgaatcg 15540
gcggaaacgt caaggcttca cggccacatc ctgtcctcgc ttcttgggaa accgaatact 15600
gtcattgata attcatacgg gaaaaacgcc aactattatc acacctggac acacgaagcg 15660
ccggacgtcc gtctgatcga gactgcaggc acgaaggaaa accttccgct ttga 15714
<210> 9
<211> 10471
<212> DNA
<213> Bacillus amyloliquefaciens CGMCC No.11218
<400> 9
atgaaattga tagccattga cttagacggc acattactga actcggaaag tgtgatttct 60
ccggaaaaca gagcggcttt acaaagagcg gatgaatcgg gcattcttgt ggccatctgt 120
acgggcagag ccacttttga cgttaaggcg ctcttaaaag atttggatat tccgatcatt 180
gcggcaaacg gcgggacagt tcacgacaaa ggataccgcc tcatcagccg gttcttaatg 240
gatcagcagg ccggtaagga tatcgccgct tatttaacgg gaaacgatat ttactttgag 300
gtttatacgg atgatcatct cttgtctccg tttgacggcg agtccaaact gcaggcggaa 360
ctggacattc tgaaaagcgc caatcctgag gaagatattc agacattgtg ggagggcgca 420
aagacgcagt tcaaacaatt cggcattaaa cccgtgcagg atatacaggc gatttttgac 480
ggagacgaac atatttacaa gctcctttgt ttctcttttg atatggaaaa actgaaaaac 540
gccaaagaag aaattacaca tcacccgaaa ttatcccaga cttcttccgg aaaacacatt 600
atcgaaatcc tccctgccgg ttccggaaag ggacgggctc tgagggagct tgcttcactg 660
tacggaatag aaaaacagga tatttacgcc atcggcgaca gcccgaatga cttctccatg 720
tttgaagcgg ccggaaaccg catcgccatg ggcaatgcga ttgatgaact gaaagaaaaa 780
agcacatata ttacaaaaag caatgatgaa aacggtgttg cctatttcat caaccggctt 840
ctcgacaatg aacaataaaa aatctgtccg gcaaaagcca ggtcttccac gacctggctt 900
ttcatgtttc caaaaaggag tttcatgatg tcgaatgcaa tcgctattat tggattttca 960
gcttttctct tcgcacttgg atacggaata ttccatctca tatgcaaagc agtaaaaaaa 1020
gaaaaacgtt tttcaaaaaa gctgttttgg ccccttctca tcggcgggtt tatcctgttc 1080
ttaatcggag gctcctcggc agagccggac acagcggccg tgaaagcgga ggagaaatac 1140
agcacccttg atacggctaa tcaaaaattg acgaaggaac atcaggcgct tgagaaaaaa 1200
tatgaaagca tcagcgccgc ggccgaaaag gaaaaatccg aggccgaagc aggcaatgaa 1260
gaaaaactca gcaaactcag caaagaaata aatgagctga aaaaaacaaa caaatcactc 1320
aaacaagaca atgagaaatt aaaagattca caaaagaaac tcgaaaaggc cgctgaaacg 1380
cttcagtcag aaaataaaac attaaagcgg caaaaggaag aaacaaaaac agccggaaac 1440
gctgaatcgg cgcaaaacac agcctcttcc tccggcgggc atgcagaaac gaaggcagcc 1500
gacacctcac agggatgcaa cattaaaggc agccgaaacg gcatttatca cacgccggga 1560
agtacatact acgatcggac gacagatcca gtagaaatgt tctgttctgt agaggaagcg 1620
gaagcggcgg gattcagagc gccgaaacgg taaatagaaa aagacgtcag agatactctc 1680
tgacgtcttt ttttacggaa tccggatgct gcccgcatac ttgtccttcc aataagcgct 1740
cgtattcata tttgttatgg tcacgcctgc ggacagagtg acatgaataa cttgtccgtt 1800
cccgatataa atggccgggt tcaggctttc cgctttaaaa aagaccagat cgcccggctt 1860
caaatcctgt ttactgaccg gagcgcccgc ctgcatctgc ttctccgcgt aacgcggcag 1920
gctgattcct ttcgcttttt gatagacgta ttgaacaaat cccgccgtat caaagccggt 1980
ttccggtgaa gcgccgcctt tcagataaga aattccgata taatcagctg ctgttgacac 2040
aacgggatta tctttggaaa tggccagatt gctgaatcgg cgcacccctg tataatgttt 2100
tttccaataa tcttccgaca agtaggaaat ggtgactttt gcagagcggc tcgcttggat 2160
gaatcttccc tctcccgcat aaatgcccgc atgggaaatt ccttttttat acgtatcact 2220
gaaataaatc acgtccccgg gctttatgct gctcagtttt attttctcac cgaccagcca 2280
ctgctgttcc gcagatctcg gcaggtaaat gtgaagcttg ttttcaaaaa catattggac 2340
aagccctgaa cagtcgaatc cctcttcagg tgtgcttccg ccgaatacat acggaatgtc 2400
gagataacgg gcggcctcct gaacgatcgg attatcagcc agctccggat cttttgtaat 2460
ccgctttgcg ccaagatata aattcatcca ataattgctt ttttgaaagc ttgtgacggt 2520
tacgccttct gacggagaac tgtgtatcat ctctccattt ccgatataaa ggccgtcttg 2580
gacgggcgcg gtttgtaatg aatccgactt tttgaaaaaa acaatatccc ccggtaaaag 2640
ctcattttct ttgacggcag ctccgacttt ccattgatca ctgaccgttc tcggcagatg 2700
gatgttctgc ctgctgaaaa gatactgtat caatccggac ggatcaaaac cgtccttagg 2760
ctcgtcgcct ccgtaaacat atttatttcc gacaagtttt tgcgcgtctg aaaccatcag 2820
ggcagaaact tgagaatccg cttcagcaat ttccgttctc gttactgcag gtgccacaag 2880
acaaatcata aggccggcaa ggatgtactt cctccagctt gtgagcatgt tttctgttca 2940
ctctccttat ttttactagt tcttacatcg gtaactttaa gtaaatgttt aggagagcat 3000
aaaaaaagaa tgcccgatac gggcattctt tactggattc ggtacaaaaa atatgccggc 3060
aagcggaagc atgcgcttat tttgcgcccg ctgtgtcctc ctgctgaatt ttttgcattt 3120
ctgtgtcaat tttttgtttt tcctgatctg tgagctgatc attgaattta aaagctgaca 3180
taaacataaa taccataatg attgcggcaa acggccatcc tgctttaatg attttcattt 3240
gtttttcact ccgctttctt ttttaaaata tcgctgtgat ccacatcaaa cgttaacttt 3300
ccgtccttga cgctccactt gaaattcgat tctttcgtca gttcacgtat gattgtttta 3360
tggttaatgc tgcctttttt aaccggcgca ggcgttgctt cgtgaatgtt catcggtgtg 3420
acttcaaagc gtcccgtgcc gtttttctga aggtgatatt gaacaagcgc cgtgtctctc 3480
gttcttgtcc atccctggtc aaatacgaag ttgcccaggc tgtagaaaat aacggtgccg 3540
tggtacattt cgatcggttc aagcacgtgc ggatggtgtc cgacgataat atccgctccg 3600
gcatctgaca ttgctcttgc caattggcgc tgtctgtcat tcggatcatt atcgtattcc 3660
tgtccccagt gtgtttgaac aatgacgagg tctgcattct ttttggcttt agagatcatt 3720
ggaataaaca gctcaggatc ggccggaagc acacccggcg cattccgttt agctgcaaag 3780
ccttttccgg aaacatctgt gaatcctaac gttgcgattt tgactccgtt tacatcttga 3840
tatgaaatgt tctcctcggc ttctttaagc gtcaggcctg caccgacaag ttcaaggttc 3900
tgctttgaaa attcttcaac cgtatcccgc attccttgag ggccgtaatc catcgcatgg 3960
ttgttggctg ccgtcaaaac ggtgaaattc atgtccttca atacttttac ggcatctttg 4020
tttgactgaa ggtggatatt tttttctgct tctttgtagt tctttttata tgttaccggg 4080
ttttcaaagt tccccgacac ataatcggat gcttcgaaga atggctttgc gtagcggaag 4140
acgctttccg ttccgttatg ctttgtcact ttctccacat tccggcccat cataatatct 4200
ccgacgaatg aggctgacac aaggtcatcg gcatttcttt tcacactcgg cgtctccgcc 4260
ttccctgtca ggatgcagac gaacatcagg caaaagacga tcggcagtgc gataaaaacg 4320
tgaagattag tcttcttttt ctgctgtttg gtcagcttca gcagtttttc ttgaaaggtt 4380
aattgttttt tcatcgtttg acacacctta cattagatta agtagtaagc aaacataatg 4440
gcaaaagttg cgccgcttaa gaacagcgtg ctggcaaatg taatcgttaa tccctgtttc 4500
tgaatggtgt tggcgatcag acccggtaca atgataccga ttccgcggaa ttctgatatc 4560
tcaaacggta cgatcgggta aagaaaatca agtgcgattt ttaagcagat tcccgtaatc 4620
agcattgctg cgaattttct gcgtccgtac aaaatcataa atctggataa accgtatttg 4680
acgatgacgt atgtaagaag actcactaac agcacgagta aaataaagac cggctggtta 4740
aaaacgagtc ctaaataacc cggtacgact aaaccggcag gtacgatacc tgttttttcc 4800
gcaaaaatta aactgattaa cacgcctaaa ataagtgaga tatataaatc tgatccgaac 4860
atttatcctt ttcctcctag ctaacgagct gtttcacctt atactcttgg attttttcaa 4920
ttaatggttc cgctgaaccg tggatattgc cgacgccata tatgacacgg ttttgcattc 4980
ttgttttcag cacgtccatg atttcgtctg ttgatttata ttccagatcg tgcagtgtat 5040
cagcaggaat cttgccttct tcataagctc tgacgatcgg ttctgttgtc tcgccgatga 5100
gaatgagttc cttcgtttta atgtacggaa gcacgtcgtt ggcaaactga atcgttctgt 5160
ccacgcggtc ggcacggcag ttcatgataa caatcggttc atctgtcggg tagccgattt 5220
cctttacacg tttccatata tttaaagtag aagatgcatc atttgcggca aagccgttta 5280
cgaaatgacc gggctccttc gtgctgagca gcgggagaat tctcatggcg ccaggatcag 5340
gcggcgcatt cagcattcct ttaaacgctg tttcttcatc tataccaaga gcttgggcaa 5400
caccgagcgc aagggaagcg ttatcaggaa agaccatata ttcgaatttc cgcaggtatt 5460
catcagtaat tttagagtta tccgcaataa taacttcggt attccgctct gcggcttttt 5520
ctttaaagaa atctgtgtat tcactgtctg caatcacaag atggccgtta tatggaatcg 5580
tcgcggtaaa tgcttcagca atttcatcaa gcgtcggccc cataacgtcc atatggtctt 5640
caagcacatt cacgatgacg ccgatgttgg cttgaagcag ttcttcctga aagataatct 5700
gataatccgg gtttaccgcc atacattcac tgacaatggc attggcgccc cgctctacgg 5760
tttccttcat tacctctttt tgctcgccga tattcgggcc ctgcggtttc cgtttgatcg 5820
gtttctcctc aggtgtatcc caatagatca tccttgcatc tgttcccgtc gtttttccga 5880
ctgttttgta gcccgcctcc atcagtattc cggtcgtcag cctcgttacg gttgacttgc 5940
cgcggatacc attaatattg acgcgtactg gcagagcatc gatattcttc tgatgccgcc 6000
ttttttcaat gattccgatg attagtacgg cagcacaggc tataatgagt aaccacattg 6060
atttttgaca tctccttctg tattgataaa ttcattcatc agtatatagc ttcactatga 6120
attctcaatc ggcattatgt aacataatct cctcgcctgc actaagccaa acttctctat 6180
tagcggcgct attcttccta ttatcgaatt gctttttttg ggaataggca ggacttcgaa 6240
cttactttaa tttttttaaa gaaaaatgaa tgcgtttaca ttttcctacg gatattcgtt 6300
tggactaaat ggactatttg cgttccggaa gaaaaagatg caatcataat ccatcttttc 6360
atagcccgtt atatcaacat tttccgcact gcgcatacga ttcgggactc tccgtccgct 6420
ggatgcagac tgcctttttt gttaaggata aggtcataaa gtcattcatt ttcccccata 6480
tcctgctgtc agtcctattc caaaataact atcggaatga cttgcacatg acagaaacag 6540
acgggtccgg agtgtttaca ggaaatacac aatttatatg cggaaagtct atttgctttt 6600
gttccgtata caggacattt atcactcttt tgaaataatt ttaataaatc tttatattcg 6660
ttcaagaaat gttcatccat atttgtgaag ttcttggtaa aacatcaagc atcatcaata 6720
aattcctcta ttttataaaa tgcataaaaa aggaatagcc gctgaaaact acagataacg 6780
cggaaaggag gcggtctgat gtctgcggat atacaaaaca aacggcttcc cggtgatttt 6840
gaatcggctg tacgaaaaat gcaggaactg atgtgtgttt gcggagattt taaaatacga 6900
aggatcaaac tgcaggatcg tcaaaccgct tttttttata ttgaacaaat gatagaagaa 6960
cagaagctta atgagtttct gttaagcgac agcttgcaga acggctcact tgaggaagcg 7020
atggatgctg tcagcagtgc ggaaacggat gagatcagaa aaattatcga cgggattttg 7080
gccggccagg ttgtttttct cagcgatgcg tcaagtaagg cgaagctgtt ctctgtcggg 7140
cagaacccgc tgcgttccat tatggaacca gaatctgaaa gcatcgtccg cggggcccat 7200
gacgggtttg tggaaagttt ggaaacgaac attcatcagc tgcggtttca catacaggac 7260
cgccacctct ccgttcacta tatggatatc ggggaacggg ccaaatcaaa ggttgcagtc 7320
atttttgttg agaatatcgc caatccggag attgtggagg aagtgaagcg gcgcctgtcg 7380
tacctgcgta tcgacagtgt gcttgcgccc ggaattatcg aggagtcgat agaagataat 7440
tcgttttcca tattcccgca gatcgttcat acggaacggc cggataaagc aaaagcaagc 7500
gtgatggaag gctgtgtcct catcatgatg gatgggagcc cgtcagcctt aattgcgccg 7560
atcacgttct tttcattttt tcaatcgccg gatgactaca gcacaaggtg gatttccgcc 7620
agttttttga gggtgctgag atttttcgcc tttatgatcg cgattacgct gcccgctttt 7680
tatatcgcgc tgattgcgtt ccattttgaa gtcattccca acgatctgat tatcaccatg 7740
aaaaattcga ttgtagatat tcccttccct ccgctgatag aagccatgat tatggagatt 7800
acgatcgaac tgatccgcga agccggtatc aggctcccga aaccgatcag tcagacgatc 7860
ggcattgtcg gcggtctcgt gatcggtgac gcagtcgttc aggcgggact catttcgaat 7920
atgatgatta tcgtcgtagc cgttacggct gtttcctctt ttgtgcttcc ttcatatgaa 7980
atgagcacgt ccattcgaac gctcagattt ccgcttatgt ttctggcatc atcgttcggt 8040
tttatcggta tcgggctcgg cttcgggctt atcctgatga acctgtgcaa gcttgaatct 8100
ctcggggttc cttatctatc atctctgacg ccgtttcatt tcagtgactt aaaggatgcg 8160
tttatccggc ttccggcttg gttcattaaa agaaggccgt tttatttgag gccgcaagac 8220
tcaaagcaga ttaaacatgt tcgggggtgg agaaaaaatg aagactaaaa aaatcagccg 8280
gcttcaagca gtatttttca ttaatcagac gcagatcagc atcggcattt tggcgctgcc 8340
ccacttgctg atgatgaaat cgggaagcag cgggtggatt tccattctgc tcaccgggct 8400
gcttgtccag cttacattca gtctgtatat gatattattt caccggtttc cgaagatgaa 8460
tctgtacgaa atgctggagg aggtgttcgg aacattcgcc ggcaaagggc tgcaagtgct 8520
gtatatttta tattttacgt ttctctgcac gctcattctt tctgtattta cacggatctt 8580
ggatatctgg gtgctccctc tgactccgaa ttgggtcatc tgcttaatgc tgatactcgg 8640
ttctgtctat atcgcccaag ccccgctgaa tgctatcgcc aggttcagct ttatcttaac 8700
gccattttta ctgctgatct ctctgccgct tttatacacg ctgggaaaag ctgatgtgac 8760
aaacattatg ccgatttttc acggaaccgc caaagacatg ctgaccgggg tgcagcagac 8820
actggtcagc attaccggct ttcaggcagc gcttgtaatc gggcccctta tcaactgcac 8880
cgtcagccag cgctacaaaa taatgacgat ttcaaacgtc tgtattgtcc tttactatct 8940
gtacatcacc cttacttgct ttgtcacaat cagccccgaa gaaataaaga ttatcccgga 9000
gcctgtgctg tatttgttaa aaaccgtttc attcagaatt atcgaacgga cggatctgct 9060
ctttttgacg ttttggttcg tgacagtctt ttcaacattg gccaacgtgt tttatttggc 9120
aagcaccgga atggccaagc tgttcaacaa aaaagaccac agccggtttg tttatctgct 9180
ggccctgctc gtctttcttc catctatttg ggccataagc acggataaaa cgatacagca 9240
gctggatcag gttttccgtc tttccgatat tgtctttgtg tatttgtttc cggcgtttat 9300
gacattgatt gtccttgtac gcaaaaaggg tgtccgccat gcgcattaaa aaagcgcttc 9360
ttatcagtct catgtgtgca agccttacgc ttacgggctg ctgggaccaa aatttattta 9420
aagatatttc tctcgtgctg accgcaagcc tcgacgtgga tcaagacggt aaaaataaaa 9480
tcggcatcac ctatcggaag gttcaatctt cccaggcgga acaggaaaca ggcggctcct 9540
tcattacgac ggtgctcagc gcttcagggc acaccatccg tgaagcgcgg ttccaattgg 9600
ataagcttgt cgatcagcgc attgacacgt ccaagcaaag agtcttacta atcggtgaga 9660
aaatggctga aaaccgcttt ttccatatct cgatgtattt tacagggatc cgaaaagtcc 9720
gcttctcgct catccggcgg ttgtaaagga cggcgaggcg attgaagtgc tgaagctgat 9780
cgtccggcag aaattgatcg tcagcaagta tttaacgaat ttattaacaa gcgcttatac 9840
ggaatcaaat atcagcatgg aaaatttgca atccatccgt tcaagcatgc tgacagctaa 9900
tgaggatttc gcccttcctt atttatcgat taacggacag accataaagg tagagggttc 9960
agccctgttc agcggtcagc ggaaagtgac tgaggtgctc ggtcaggatt cgacccttct 10020
gactctttta agcgggcaat ttaataaatc ggccaatatt aatctgaagc tgtgggacaa 10080
atacaaaatg gatattgaaa attacatttc tctcggcatc acggatacct cgagaaagat 10140
tgtattttta gatacagatc ctcagcattt gaaagtgaaa attcgtttaa acctgaatgc 10200
acgtgtcttg gagtatccga aggacaaact gagttcttat acggctatca gccaaattaa 10260
caaacaggct tcagctaatc tgaatgaaca ggccagatcg gtgatcaggc atattcaaga 10320
gcataactcg gatattttcg gcatcggccg gagataccaa acccattatt atgagcgggg 10380
caaacatctc aattggcgtg agatctaccc ttccattgag attgttcctg atatcaaagt 10440
caatatctca caaatcggta tcgtgaatta a 10471
<210> 10
<211> 1171
<212> DNA
<213> Bacillus alcalophilus strain TCCC11004
<400> 10
atgaggaggg aaccgaatga agaaaccgtt ggggaaaatt gtcgcaagca ccgcactact 60
catttctgtt gcttttagtt catcgatcgc atcggctgct gaagaagcaa aagaaaaata 120
tttaattggc tttaatgagc aggaagctgt cagtgagttt gtagaacaag tagaggcaaa 180
tgacgaggtc gccattctct ctgaggaaga ggaagtcgaa attgaattgc ttcatgaatt 240
tgaaacgatt cctgttttat ccgttgagtt aagcccagaa gatgtggacg cgcttgaact 300
cgatccagcg atttcttata ttgaagagga tgcagaagta acgacaatgg cgcaatcagt 360
gccatgggga attagccgtg tgcaagcccc agctgcccat aaccgtggat tgacaggttc 420
tggtgtaaaa gttgctgtcc tcgatacagg tatttccact catccagact taaatattcg 480
tggtggcgct agctttgtac caggggaacc atccactcaa gatgggaatg ggcatggcac 540
acatgtggcc gggacgattg ctgctttaaa caattcgatt ggcgttcttg gcgtagcgcc 600
gagcgcggaa ctatacgctg ttaaagtatt aggggcgagc ggttcaggtt cggtcagctc 660
gattgcccaa ggattggaat gggcagggaa caatggcatg cacgttgcta atttgagttt 720
aggaagccct tcgccaagtg ccacacttga gcaagctgtt aatagcgcga cttctagagg 780
cgttcttgtt gtagcggcat ctgggaattc aggtgcaggc tcaatcagct atccggcccg 840
ttatgcgaac gcaatggcag tcggagctac tgaccaaaac aacaaccgcg ccagcttttc 900
acagtatggc gcagggcttg acattgtcgc accaggtgta aacgtgcaga gcacataccc 960
aggttcaacg tatgccagct taaacggtac atcgatggct actcctcatg ttgcaggtgc 1020
agcagccctt gttaaacaaa agaacccatc ttggtccaat gtacaaatcc gcaatcatct 1080
aaagaatacg gcaacgagct taggaagcac gaacttgtat ggaagcggac ttgtcaatgc 1140
agaagcggca acacgctaat caataataaa a 1171
<210> 11
<211> 725
<212> DNA
<213> 人工序列
<400> 11
cattatgttt gaatttccgt ttaaagaatg ggctgcaagc cttgtgtttt tgttcatcat 60
tatcttatat tactgcatca gggctgcggc atccggaatg ctcatgccga gaatagacac 120
caaagaagaa ctgcaaaaac gggtgaagca gcagcgaata gaatcaattg cggtcgcctt 180
tgcggtagtg gtgcttacga tgtacgacag ggggattccc catacattct tcgcttggct 240
gaaaatgatt cttcttttta tcgtctgcgg cggcgttctg tttctgcttc ggtatgtgat 300
tgtgaagctg gcttacagaa gagcggtaaa agaagaaata aaaaagaaat catctttttt 360
gtttggaaag cgagggaagc gttcacagtt tcgggcagct ttttttatag gaacattgat 420
ttgtattcac tctgccaagt tgttttgata gagtgattgt gataatttta aatgtaagcg 480
ttaacaaaat tctccagtct tcacatcggt ttgaaaggag gaagcggaag aatgaagtaa 540
gagggatttt tgactccgaa gtaagtcttc aaaaaatcaa ataaggagtg tcaagaatgt 600
ttgcaaaacg attcaaaacc tctttactgc cgttattcgc tggattttta ttgctgtttc 660
atttggttct ggcaggaccg gcggctgcga gtgctgaaac ggcgaacaaa tcgaatgagc 720
ttaca 725
Claims (8)
1.一种解淀粉芽孢杆菌基因工程菌株，其特征在于：所述基因工程菌株不表达解淀粉芽孢杆菌基因组上的六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因；所述基因工程菌株还异源过表达碱性蛋白酶基因aprE；
所述解淀粉芽孢杆菌的出发菌株为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)CGMCC No.11218；
所述胞外蛋白酶基因aprE的核苷酸序列如SEQ ID NO：2所示；和/或
所述胞外蛋白酶基因bpr的核苷酸序列如SEQ ID NO：3所示；和/或
所述胞外蛋白酶基因vpr的核苷酸序列如SEQ ID NO：4所示；和/或
所述胞外蛋白酶基因mpr的核苷酸序列如SEQ ID NO：5所示；和/或
所述胞外蛋白酶基因nprE的核苷酸序列如SEQ ID NO：6所示；和/或
所述胞外蛋白酶基因epr的核苷酸序列如SEQ ID NO：7所示；和/或
所述胞外多糖基因簇eps的核苷酸序列如SEQ ID NO：8所示；和/或
所述聚谷氨酸基因簇pgs的核苷酸序列如SEQ ID NO：9所示；和/或
所述碱性蛋白酶基因aprE的核苷酸序列如SEQ ID NO：10所示。
2.如权利要求1所述的基因工程菌株，其特征在于：所述基因工程菌株基因组上的六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因被敲除。
3.如权利要求1所述的基因工程菌株，其特征在于：所述碱性蛋白酶基因aprE通过重组质粒Ply-2-SPamyE-aprE-pWB980转入菌株，其中，启动子Ply-2与信号肽SPamyE的核苷酸序列如SEQ ID NO：11所示。
4.权利要求1-3任一所述基因工程菌株的构建方法，其特征在于：所述方法包括：在出发菌株解淀粉芽孢杆菌中，将六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，胞外多糖基因簇eps，聚谷氨酸基因簇pgs和核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因失活或敲除，并且在解淀粉芽孢杆菌中引入碱性蛋白酶基因aprE并使其过表达。
5.如权利要求4所述的构建方法，其特征在于：所述构建方法进一步包括：在出发菌株解淀粉芽孢杆菌中，敲除六种胞外蛋白酶基因aprE，bpr，vpr，mpr，nprE，epr，得到第一敲除菌株；进一步敲除胞外多糖基因簇eps，得到第二敲除菌株；进一步敲除聚谷氨酸基因簇pgs，得到第三敲除菌株；进一步敲除核苷酸序列如SEQ ID NO：1所示的噬菌体相关基因，得到第四敲除菌株；进一步将含有碱性蛋白酶基因aprE表达盒的表达载体转入第四敲除菌株中，得到所述基因工程菌株；
通过包含Kana抗性基因的pWH-T2质粒与上述基因同源重组的方式敲除上述基因；
碱性蛋白酶基因aprE通过重组质粒Ply-2-SPamyE-aprE-pWB980转入菌株，其中，启动子Ply-2与信号肽SPamyE的核苷酸序列如SEQ ID NO：11所示。
6.权利要求1-3任一所述基因工程菌株在发酵生产碱性蛋白酶中的用途。
7.一种高效生产碱性蛋白酶的方法，其特征在于：所述方法是在适宜条件下培养权利要求1-3任一所述基因工程菌株，并从其培养物中收集碱性蛋白酶。
8.如权利要求7所述的方法，其特征在于：所述适宜条件是指培养温度37℃及采用如下发酵培养基组成：玉米粉64g/L，豆饼粉40g/L，磷酸氢二钠4g/L，磷酸二氢钾0.3g/L，高温淀粉酶0.7g/L。
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