CN1930291A - 经过修饰的启动子 - Google Patents
经过修饰的启动子 Download PDFInfo
- Publication number
- CN1930291A CN1930291A CNA2005800071171A CN200580007117A CN1930291A CN 1930291 A CN1930291 A CN 1930291A CN A2005800071171 A CNA2005800071171 A CN A2005800071171A CN 200580007117 A CN200580007117 A CN 200580007117A CN 1930291 A CN1930291 A CN 1930291A
- Authority
- CN
- China
- Prior art keywords
- asn
- glu
- ala
- asp
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供一种能够使编码蛋白质或多肽的基因的转录量提高的经过修饰的启动子DNA,以及使用其的蛋白质或多肽的有效的制造方法。通过对包含由SigA识别的启动子及其附近的碱基的碱基序列进行修饰以使得由SigA和SigE识别而形成的启动子DNA;含有该启动子DNA的表达载体、含有该表达载体的重组微生物、以及以培养该重组微生物为特征的蛋白质或多肽的制造方法。
Description
技术领域
本发明涉及经过修饰的启动子DNA、含有该DNA的表达载体、含有该表达载体的重组微生物以及利用该重组微生物的蛋白质或多肽的制造方法。
背景技术
利用微生物的有用物质的工业生产,以酒精饮料和味噌、酱油等的食品类为代表,还有氨基酸、有机酸、核酸相关物质、抗生素、糖、脂质和蛋白质等,其种类跨越多方面,而且关于其用途,也扩展到广泛的领域直到食品、医药和洗涤剂、化妆品等的日用品或各种化学合成品原料等。
在这样的利用微生物的有用物质的工业生产中,其生产率的提高是一个重要课题,作为其手段,可以进行利用突变等的遗传学手段的生产菌的育种。另一方面,由于微生物遗传学和生物技术的进步,特别是最近,使用基因重组技术等更更有效的有用物质的生产得到关注。
关于基因转录中需要的启动子的研究也进行了很多,作为在枯草杆菌(
Bacillus
subtilis)中强有力地转录编码异种蛋白质或多肽的基因的启动子区域,例如,可以利用来自芽孢杆菌sp(
Bacillus sp.)KSM-64株(FERM BP-2886)的碱性纤维素酶基因的启动子区域(例如,参考非专利文献1)、或者存在于来自芽孢杆菌sp KSM-S237株(FERMBP-7875)的碱性纤维素酶基因(例如,参考专利文献1)的上游部位的启动子区域等。
但是,在工业生产中需要降低生产成本,而即使在现在所使用的上述那样的启动子领域内生产率提高的效果也不能称为充分,需要更高的生产率。
[专利文献1]日本特开2000-210081号公报
[非专利文献2]Biosci.Biotech.Biochem.,59,2172,(1995)
发明内容
本发明提供一种对含有由SigA识别的启动子及其附近的碱基的碱基序列,进行修饰以使得被SigA和SigE识别而形成的启动子DNA。
而且,本发明提供含有该启动子DNA的表达载体、含有该表达载体的重组微生物、以及以培养该重组微生物为特征的蛋白质或多肽的制造方法。
此外,本发明提供一种启动子DNA的构建方法,其特征在于,对含有由SigA识别的启动子及其附近的碱基的碱基序列进行修饰,以使得被SigA和SigE识别。
附图说明
图1是表示使用SOE-PCR法的SigE识别启动子序列的导入方法的示意图;
图2是表示导入了SigE识别启动子序列的碱性纤维素酶生产用的质粒的构建方法的示意图。
具体实施方式
本发明涉及提供一种使编码蛋白质或多肽的基因的转录量提高的经过修饰的启动子DNA,以及一种使用其的蛋白质或多肽的有效制造方法。
在枯草杆菌中,作为RNA聚合酶复合体的亚单位而参与启动子序列的识别的σ因子鉴定有17个,以在营养增殖期中参与生殖所必需的基因的转录的主要σ因子(管家σ因子)SigA为代表,存在控制孢子形成过程的σ因子SigH、SigF、SigE、SigG、SigK,控制鞭毛形成和细胞壁溶解的σ因子SigD,控制某种氨基酸或糖的代谢的σ因子SigL,控制对环境变化的响应的σ因子SigB或称为ECFσ的σ因子等。结合在由除σ因子之外的5个(α、β、β’、δ、ω)亚单位构成的RNA聚合酶轴心复合体(core complex)上的各σ因子,被认为通过每个σ因子参与不同的启动子序列的识别,从而控制不同的基因的转录,由此,对存在于基因组上的4100多个基因、进行根据情况的基因的表达控制。
有报告称,尽管在营养增殖期内,主要是SigA与RNA聚合酶轴心复合体会合,诱导具有SigA识别的启动子的基因或操纵子的转录,但是进入孢子形成期而控制孢子形成过程的σ因子被活化时,引起与RNA聚合酶轴心复合体会合的σ因子的置换,与SigA会合的RNA聚合酶的量相对下降(J.Bacteriol.,179,4969,(1999))。因此,被认为在孢子形成期之后,来自通过SigA识别的启动子的转录量相对下降。
鉴于这种情况,本发明者们发现,对含有由枯草杆菌的σ因子SigA识别的启动子DNA片段,利用基因工程学的操作实施碱基的修饰,在保留该启动子的功能、重新构建由SigE识别的序列的情况下,可以提高编码在下游处连接的蛋白质或多肽的基因的转录量。
和使用天然启动子的情况相比,本发明的启动子DNA可以极大地提高编码在下游处连接的蛋白质或多肽的基因的转录量,可以有效地生产蛋白质或多肽。
在本发明中,氨基酸序列和碱基序列的同一性,通过Lipman-Pearson法(Science,227,1435,(1985))计算。具体地说,通过使用遗传信息处理软件Genetyx-Win(软件开发)的同源性解析(Searchhomology)程序,将参数Unit size to compare(ktup)设为2进行分析而算出。
通常,σ因子识别并结合存在于转录起始点的上游10碱基和35碱基附近的数个碱基序列,分别称为-10区域、-35区域。另外,两区域的序列、两区域间的距离,已知每个σ因子分别具有共通的特征,称为共有序列,可以认为作为启动子的本体。有报告称,SigA的共有序列,为由用TTGaca表示的-35区域、用在其14碱基之后连接的tgnTAtaat所表示的-10区域所形成(n为A或G或C或T。此外,大写字母表示保存性高,而小写字母表示保存性低。Bacillus Subtilis andIts Closest Relatives:From Genes to Cells,Edited by A.L.Sonenshein,American Society for Microbiology,pp289,(2002))。并且,有报告称,在上述序列号1的碱基号92~552、序列号2的碱基号133~589之间,存在多个SigA的共有序列(Biosci Biotechnol Biochem.64,2281,2000、Biosci Biotechnol Biochem.56,872,(1992))。
因此,作为含有本发明的由SigA识别的启动子及其附近的碱基的碱基序列,优选含有由序列号1所表示的碱基号92~552的碱基序列、由序列号2所表示的碱基序列的碱基号133~589的碱基序列,或者包含具有对该碱基序列具有80%以上、优选90%以上、更优选95%以上、特别优选98%以上的同一性的碱基序列、且是具有SigA的共有序列的碱基序列和/或具有相同的启动子功能的碱基序列,或者,具有由序列号1所表示的碱基序列、由序列号2所表示的碱基序列、或者对该碱基序列具有90%、优选95%以上、更优选98%以上的同一性的碱基序列、且是具有SigA的共有序列的和/或具有相同的启动子功能的。作为上述由含有序列号1所表示的碱基号92~552的碱基序列的碱基序列,优选在序列号1中含有碱基号的92~552的461~570bp的连续的碱基序列,更优选为461~520bp,特别优选461~480bp的连续的碱基序列。作为上述含有由序列号2所表示的碱基号133~589的碱基序列的碱基序列,优选在序列号2中含有碱基号133~589的457~610bp的连续的碱基序列,更优选为457~520bp,特别优选457~480bp以下的连续的碱基序列。
在此,由序列号1所表示的碱基序列是存在于来自芽孢杆菌sp.KSM-S237株(FERM BP-7875)的碱性纤维素酶基因的上游的,由序列号2所表示的碱基序列是存在于来自芽孢杆菌sp.KSM-64株(FERMBP-2886)的碱性纤维素酶基因的上游的,两者具有95.6%的同一性。
本发明的启动子DNA,对上述碱基序列实施碱基的修饰,进行构建以使得除了SigA,也可以由SigE识别。在此,所构建的启动子序列可以是单独的,也可以是多个。
另一方面,有报道称,由SigE识别的启动子序列为,由ATAHTT(H为A或C或T)所表示的-35区域、和、在其13或14碱基之后连接的CATAYAHT(Y为C或T)所表示的-10区域形成的碱基序列,优选为由ATATTT所表示的-35区域和在其13或14碱基之后连接的CATACAAT所表示的-10区域形成的碱基序列,更优选为由ATATTTCAAGTAGTAATAACATACAAT所形成的碱基序列(J.Mol.Biol.327,945,(2003)),优选该碱基序列为重新构建的。
在本发明中的最优选的启动子DNA,可以举出,由序列号7所表示的、将序列号1修饰的碱基序列,或者由序列号8所表示的、将序列号2修饰的碱基序列。
碱基的修饰,尽管可以通过***具有由上述SigE识别的启动子序列的DNA片段,或者缺失、置换或者***1个或多个碱基而进行,但是其中优选通过置换1个或多个碱基而进行的方法。即,例如在来源于在质粒载体上克隆的来自芽孢杆菌sp.KSM-S237株(FERMBP-7875)的碱性纤维素酶基因的上游的DNA片段(序列号1)或者来源于来自芽孢杆菌sp.KSM-64株(FERM BP-2886)的碱性纤维素酶基因的上游的DNA片段(序列号2)的任意部位处,通过Kunkel法(Proc.Natl.Acad.Sci.USA.,82,488,1985)等的部位特异的变异导入法而导入限制性内切酶的识别部位,另一方面,通过化学合成等制备在两端添加有该限制性内切酶识别部位的含有由SigE识别的启动子序列的DNA片段后,可以通过用酶结合由该限制性内切酶处理过的两个片段,从而构建***由SigE识别的启动子序列的启动子DNA。
此外,可以通过将来自于由序列号1或序列号2所表示的碱性纤维素酶基因的上游处的DNA片段的一部分通过SOE(重叠延伸拼接法,splicing by overlap extension)-PCR法(Gene,77,51,1989)等而进行碱基置换,从而构建该DNA片段。
下面,更具体地说明通过使用SOE-PCR法在由序列号1所表示的碱基序列所形成的DNA片段的一部分上施行碱基置换,重新构建由SigE识别的启动子(序列)的方法。
首先,通过第一次的PCR制备将实施了碱基置换的部位作为下游末端的上游侧DNA片段、以及将实施了碱基置换的部位作为上游末端的下游侧DNA片段,但是此时,例如,实施设计以使得,在用于上游侧DNA片段的下游侧以及下游侧DNA片段的上游侧的引物中,相互退火、且由SigE识别的启动子序列被重新构建(图1)。
随后,将在第一次的PCR中制备的2种DNA片段作为模板,通过使用上游侧DNA片段的上游侧引物和下游侧DNA片段的下游侧引物进行第二次的PCR,在上游侧DNA片段的下游末端和下游侧DNA片段的上游末端之间发生退火,PCR扩增的结果是可以得到2个DNA片段连接、且在其连接部分处重新构建有由SigE识别的启动子序列的DNA片段(图1)。
如此构建的启动子DNA,由于除了SigA、还能被SigE识别,所以即使在孢子形成期中,也可以进行编码在其下游处连接的蛋白质或多肽的基因的转录。
即,在通过枯草杆菌重组生产异种蛋白质或多肽时,通过在编码目标蛋白质或多肽的基因的上游处连接该启动子DNA,该基因除了在营养增殖期内由与SigA会合的RNA聚合酶转录,而且还在继营养增殖期之后的孢子形成期内由与SigE会合的RNA聚合酶转录,并且还在孢子形成期内维持该基因的转录。因此,导入有含有该启动子DNA的表达载体的重组枯草杆菌,与使用在重新构建由SigE识别的启动子序列之前的天然的启动子的情况相比较,可以生产大量的目标蛋白质或多肽。
目标蛋白质或多肽的基因,没有特别限制,包括洗涤剂、食品、纤维、饲料、化学品、医疗、诊断等各种工业用酶以及生物活性肽等。此外,尽管按照工业用酶的功能分类,包括氧化还原酶(Oxidoreductase)、转移酶(Transferase)、水解酶(Hydrolase)、裂解酶(Lyase)、异构酶(Isomerase)、合成酶(Ligase/Synthetase)等,但优选的可以举出,纤维素酶、α-淀粉酶、蛋白酶等水解酶的基因。具体地说,可以举出,在多糖水解酶的分类(Biochem.J.,280,309(1991))中属于族5的纤维素酶,也可以举出其中来自微生物、特别是来自芽孢杆菌属细菌的纤维素酶。作为更具体的例子,可以举出,由用序列号4或序列号6所表示的氨基酸序列所组成的、分别来自芽孢杆菌sp.KSM-S237株(FERM BP-7875)或芽孢杆菌sp.KSM-64株(FERMBP-2886)的纤维素酶,或者由与该氨基酸序列具有70%、优选80%、更优选90%以上、进一步优选95%以上、特别优选98%以上的同一性的氨基酸序列构成的纤维素酶。
而且,作为α-淀粉酶的具体例子,可以举出来自微生物的α-淀粉酶,特别优选来自芽孢杆菌属细菌的液化型淀粉酶。作为更具体的例子,可以举出由用序列号14所表示的氨基酸序列组成的来自芽孢杆菌属sp.KSM-K38株(FERM BP-6946)的碱性纤维素酶,或者由与该氨基酸序列具有70%、优选80%、更优选90%以上、进一步优选95%以上、特别优选98%以上的同一性的氨基酸序列所组成的纤维素酶。此外,作为蛋白酶的具体例子,可以举出来自微生物、特别是来自芽孢杆菌属细菌的丝氨酸蛋白酶和金属蛋白酶等。
另一方面,希望目标蛋白质或多肽的基因,除了本发明的启动子DNA,还适当地结合有与该基因的翻译、分泌有关的控制区域,即,核糖体结合部位(SD序列)和含有起始密码子的翻译起始区域和分泌用信号肽区域。希望适当地与目标蛋白质或多肽的结构基因结合有,例如,在日本特开2000-210081号公报和日本特开平4-190793号公报等中记载的来自芽孢杆菌属细菌、即KSM-S237株(FERM BP-7875)和KSM-64株(FERM BP-2886)的纤维素酶基因的翻译起始区域、分泌用信号肽区域,更具体地说为,由序列号3所表示的碱基序列的碱基号563~659的碱基序列或由用序列号5所表示的碱基序列所组成的纤维素酶基因的碱基号600~696的碱基序列,或者由相对于该碱基序列具有70%以上、优选80%以上、更优选90%以上、进一步优选95%以上、特别优选98%以上的同一性的碱基序列所构成的DNA片段,或者由上述任意碱基序列的一部分缺失的碱基序列组成的DNA片段。
可以通过将在含有上述目标蛋白质或多肽基因的DNA片段的上游处连接有本发明的启动子DNA并***到适合的载体中的表达载体,按照一般的转化法摄入到枯草杆菌中并构建重组微生物,而使目标蛋白质或多肽的生产率提高。此外,也可以由通过使用在本发明的启动子DNA上结合有与枯草杆菌基因组适当的相同区域的DNA片段、而直接编入枯草杆菌基因组中而构建的重组菌株,使目标蛋白质或多肽的生产率提高。
使用本发明的启动子DNA的目标蛋白质或多肽的制造,可以通过,将上述重组微生物接种到含有同化性的碳源、氮源、其它必需成分的培养基中,按照常规的微生物培养方法培养,培养结束后,收集精制蛋白质或多肽而进行。
以下,使用实施例,对本发明的DNA片段的构建方法以及利用该DNA片段的纤维素酶的重组生产方法进行具体说明。
实施例
实施例1 SigE识别启动子(序列)向碱性纤维素酶基因的上游区域的构建
如图2所示地,进行了SigE识别启动子序列向碱性纤维素酶基因的上游区域的导入。即,在穿梭载体pHY300PLK的
BamHI限制性内切酶的切断位点处,将***有编码来自芽孢杆菌sp.(
Bacillus sp.)KSM-S237株(FERM BP-7875)的碱性纤维素酶基因(日本特开2000-210081号公报)的DNA片段(3.1kb)的重组质粒pHY-S237作为模板,用表1中所示的237UB1和EP1UPr的引物组制备碱性纤维素酶基因的上游区域0.4kb片段(A)。同样用表1中所示的EP1DNf和S237RV的引物组制备碱性纤维素酶基因的下游区域2.7kb片段(B)。随后,通过将所得的(A)(B)2片段混合后作为模板,进行使用表1中所示的237UB1和S237RV的引物组的SOE-PCR,按照(A)(B)的顺序使2片段结合而得到3.1kb的DNA片段(C)。对引物EP1UPr和EP1DNf分别实施碱基置换,如图2所示,在DNA片段(C)中在从碱性纤维素酶基因的翻译起始部位开始大约150bp的上游区域处重新构建SigE识别启动子(序列)。将得到的3.1kb的DNA片段(C)***穿梭载体pHY300PLK的
SmaI限制性内切酶切断点,构建重组质粒pHY-S237EP1。并将重组质粒pHY-S237作为模板,用表1中所示的237UB1和S237RV的引物组制备含有碱性纤维素酶基因的整个区域的3.1kb片段(D),并将其***穿梭载体pHY300PLK的SmaI限制性内切酶切断点,构建重组质粒pHY-S237W。
实施例2 碱性纤维素酶的分泌生产评价
将实施例1中所得的重组质粒pHY-S237EP1和作为对照的重组质粒pHY-S237W,通过原生质体转化法导入枯草杆菌168株中。将由此得到的菌株在10mL的LB培养基中37℃进行振荡培养一夜,并将该培养液0.05mL接种到50mL的2×L-麦芽糖培养基(2%胰胨、1%酵母提取物、1%NaCl、7.5%麦芽糖、7.5ppm硫酸锰4-5水合物和15ppm四环素)中,在30℃进行振荡培养3天。培养后,测定通过离心分离除去菌体的培养液上清液的碱性纤维素酶活性,从而求得通过培养在菌体外分泌生产的碱性纤维素酶的量。如表2所示,其结果发现,和对照的pHY-S237W(野生型)的情况相比,作为重组质粒使用pHY-S237EP1的情况有高的碱性纤维素酶的分泌生产。由此推测,在使用pHY-S237EP1的情况中,除了从由SigA识别的启动子开始的转录,还通过进行从重新构建的由SigE识别的启动子开始的转录,提高了纤维素酶分泌生产率。
表1
引物 | 碱基序列 | 序列号 |
237UB1 | TTGCGGATCCAACAGGCTTATATTTAGAGGAAATTTC | 9 |
EP1UPr | GTATGTTATTACTACTTGAAATATTCTACCCCCCTTCCTA | 10 |
EP1DNf | ATATTTCAAGTAGTAATAACATACAATACTTATAAGTTG | 11 |
S237RV | TCGCTACCCTTTTATTATCG | 12 |
表2
重组质粒 | 碱性纤维素酶的分泌生产量(相对值) |
pHY-S237W(野生型) | 100 |
pHY-S237EP1 | 165 |
实施例3 对导入有SigE识别启动子(序列)的碱性纤维素酶基因上游区域的碱性纤维素酶的生产的有效性的验证
将实施例1中构建的质粒pHY-S237EP1作为模板,用表3中所示的S237ppp-F2(BamHI)和S237ppp-R2(ALAA)的引物组进行PCR,扩增含有导入了SigE识别启动子(序列)的碱性纤维素酶基因的启动子区域和编码分泌信号序列的区域的0.6kb的DNA片段(E)。而且,将从芽孢杆菌sp.(
Bacillus sp.)KSM-K38株(FERM BP-6946)提取的基因组DNA作为模板,用表3中所示的K38matu-F2(ALAA)和SP64K38-R(XbaI)的引物组进行PCR,将编码具有由序列号14所表示的氨基酸序列的碱性纤维素酶(Appl.Environ.Microbiol.,67,1744,(2001))的1.5kb的DNA片段(F)进行扩增。随后将所得的(E)(F)2片段混合后作为模板,通过用表3中所示的S237ppp-F2(BamHI)和SP64K38-R(XbaI)的引物组进行SOE-PCR,得到在导入有SigE识别启动子(序列)的碱性纤维素酶基因的启动子区域和在其之后的编码分泌信号序列的区域的下游处连接有碱性纤维素酶基因的2.1kb的DNA片段(G)。将得到的2.2kb的DNA片段(G)***穿梭载体pHY300PLK(Yakult)的
BamHI-
XbaI限制性内切酶切断点,构建重组质粒pHY-K38(S237ps)EP1。此外,通过将上述用于0.6kb的DNA片段(E)的扩增的模板取代为实施例1中构建的质粒pHY-S237W,同样构建重组质粒pHY-K38(S237ps)W。
将重组质粒pHY-K38(S237ps)EP1和作为对照的pHY-K38(S237ps)W,通过原生质体转化法导入枯草杆菌168株中。将由此得到的菌株在与实施例2相同的条件下进行振荡培养5天。培养后,测定通过离心分离除去了菌体的培养液上清液的碱性纤维素酶活性,从而求得通过培养在菌体外分泌生产的碱性纤维素酶的量。其结果,如表4所示,和对照的pHY-K38(S237ps)W(野生型)的情况相比,使用pHY-K38(S237ps)EP1作为重组质粒的情况可以看到高的碱性纤维素酶的分泌生产。由此,显示出导入有SigE识别启动子(序列)的碱性纤维素酶基因上游区域在各种蛋白质或多肽的生产中是有效的。
表3
引物 | 碱基序列 | 序列号 |
S237ppp-F2(BamHI) | CCCGGATCCAACAGGCTTATATTTA | 15 |
S237ppp-R2(ALAA) | TTCAATCCATCTGCTGCAAGAGCTGCCGG | 16 |
K38matu-F2(ALAA) | GCTCTTGCAGCAGATGGATTGAACGGTACG | 17 |
SP64K38-R(XbaI) | TTGGTCTAGACCCCAAGCTTCAAAGTCGTA | 18 |
表4
重组质粒 | 碱性纤维素酶的分泌生产量(相对值) |
pHY-K38(S237ps)W(野生株) | 100 |
pHY-K38(S237ps)EP1 | 143 |
序列表
<110>花王株式会社(KAO CORPORATION)
<120>经过修饰的启动子
<130>KS0817
<150>JP 2004-062853
<151>2004.03.05
<160>18
<170>PatentIn Ver.3.1
<210>1
<211>572
<212>DNA
<213>芽孢杆菌sp.KSM-S237
<400>1
gatttgccga tgcaacaggc ttatatttag aggaaatttc tttttaaatt gaatacggaa 60
taaaatcagg taaacaggtc ctgattttat ttttttgagt tttttagaga actgaagatt 120
gaaataaaag tagaagacaa aggacataag aaaattgcat tagttttaat tatagaaaac 180
gcctttttat aattatttat acctagaacg aaaatactgt ttcgaaagcg gtttactata 240
aaaccttata ttccggctct tttttaaaac agggggtaaa aattcactct agtattctaa 300
tttcaacatg ctataataaa tttgtaagac gcaatatgca tctctttttt tacgatatat 360
gtaagcggtt aaccttgtgc tatatgccga tttaggaagg ggggtagatt gagtcaagta 420
gtaataatat agataactta taagttgttg agaagcagga gagcatctgg gttactcaca 480
agttttttta aaactttaac gaaagcactt tcggtaatgc ttatgaattt agctatttga 540
ttcaattact ttaaaaatat ttaggaggta at 572
<210>2
<211>609
<212>DNA
<213>芽孢杆菌sp.KSM-64
<400>2
agtacttacc attttagagt caaaagatag aagccaagca ggatttgccg atgcaaccgg 60
cttatattta gagggaattt ctttttaaat tgaatacgga ataaaatcag gtaaacaggt 120
cctgatttta tttttttgaa tttttttgag aactaaagat tgaaatagaa gtagaagaca 180
acggacataa gaaaattgta ttagttttaa ttatagaaaa cgcttttcta taattattta 240
tacctagaac gaaaatactg tttcgaaagc ggtttactat aaaaccttat attccggctc 300
tttttttaaa cagggggtga aaattcactc tagtattcta atttcaacat gctataataa 360
atttgtaaga cgcaatatac atcttttttt tatgatattt gtaagcggtt aaccttgtgc 420
tatatgccga tttaggaagg gggtagattg agtcaagtag tcataattta gataacttat 480
aagttgttga gaagcaggag agaatctggg ttactcacaa gttttttaaa acattatcga 540
aagcactttc ggttatgctt atgaatttag ctatttgatt caattacttt aataatttta 600
ggaggtaat 609
<210>3
<211>3150
<212>DNA
<213>芽孢杆菌sp.KSM-S237
<220>
<221>CDS
<222>(573)..(3044)
<223>
<220>
<221>sig_peptide
<222>(573)..(659)
<223>
<220>
<221>mat_peptide
<222>(660)..(3044)
<223>
<400>3
gatttgccga tgcaacaggc ttatatttag aggaaatttc tttttaaatt gaatacggaa 60
taaaatcagg taaacaggtc ctgattttat ttttttgagt tttttagaga actgaagatt 120
gaaataaaag tagaagacaa aggacataag aaaattgcat tagttttaat tatagaaaac 180
gcctttttat aattatttat acctagaacg aaaatactgt ttcgaaagcg gtttactata 240
aaaccttata ttccggctct tttttaaaac agggggtaaa aattcactct agtattctaa 300
tttcaacatg ctataataaa tttgtaagac gcaatatgca tctctttttt tacgatatat 360
gtaagcggtt aaccttgtgc tatatgccga tttaggaagg ggggtagatt gagtcaagta 420
gtaataatat agataactta taagttgttg agaagcagga gagcatctgg gttactcaca 480
agttttttta aaactttaac gaaagcactt tcggtaatgc ttatgaattt agctatttga 540
ttcaattact ttaaaaatat ttaggaggta at atg atg tta aga aag aaa aca 593
Met Met Leu Arg Lys Lys Thr
-25
aag cag ttg att tct tcc att ctt att tta gtt tta ctt cta tct tta 641
Lys Gln Leu Ile Ser Ser Ile Leu Ile Leu Val Leu Leu Leu Ser Leu
-20 -15 -10
ttt ccg gca gct ctt gca gca gaa gga aac act cgt gaa gac aat ttt 689
Phe Pro Ala Ala Leu Ala Ala Glu Gly Asn Thr Arg Glu Asp Asn Phe
-5 -1 1 5 10
aaa cat tta tta ggt aat gac aat gtt aaa cgc cct tct gag gct ggc 737
Lys His Leu Leu Gly Asn Asp Asn Val Lys Arg Pro Ser Glu Ala Gly
15 20 25
gca tta caa tta caa gaa gtc gat gga caa atg aca tta gta gat caa 785
Ala Leu Gln Leu Gln Glu Val Asp Gly Gln Met Thr Leu Val Asp Gln
30 35 40
cat gga gaa aaa att caa tta cgt gga atg agt aca cac gga tta cag 833
His Gly Glu Lys Ile Gln Leu Arg Gly Met Ser Thr His Gly Leu Gln
45 50 55
tgg ttt cct gag atc ttg aat gat aac gca tac aaa gct ctt tct aac 881
Trp Phe Pro Glu Ile Leu Asn Asp Asn Ala Tyr Lys Ala Leu Ser Asn
60 65 70
gat tgg gat tcc aat atg att cgt ctt gct atg tat gta ggt gaa aat 929
Asp Trp Asp Ser Asn Met Ile Arg Leu Ala Met Tyr Val Gly Glu Asn
75 80 85 90
ggg tac gct aca aac cct gag tta atc aaa caa aga gtg att gat gga 977
Gly Tyr Ala Thr Asn Pro Glu Leu Ile Lys Gln Arg Val Ile Asp Gly
95 100 105
att gag tta gcg att gaa aat gac atg tat gtt att gtt gac tgg cat 1025
Ile Glu Leu Ala Ile Glu Asn Asp Met Tyr Val Ile Val Asp Trp His
110 115 120
gtt cat gcg cca ggt gat cct aga gat cct gtt tat gca ggt gct aaa 1073
Val His Ala Pro Gly Asp Pro Arg Asp Pro Val Tyr Ala Gly Ala Lys
125 130 135
gat ttc ttt aga gaa att gca gct tta tac cct aat aat cca cac att 1121
Asp Phe Phe Arg Glu Ile Ala Ala Leu Tyr Pro Asn Asn Pro His Ile
140 145 150
att tat gag tta gcg aat gag ccg agt agt aat aat aat ggt gga gca 1169
Ile Tyr Glu Leu Ala Asn Glu Pro Ser Ser Asn Asn Asn Gly Gly Ala
155 160 165 170
ggg att ccg aat aac gaa gaa ggt tgg aaa gcg gta aaa gaa tat gct 1217
Gly Ile Pro Asn Asn Glu Glu Gly Trp Lys Ala Val Lys Glu Tyr Ala
175 180 185
gat cca att gta gaa atg tta cgt aaa agc ggt aat gca gat gac aac 1265
Asp Pro Ile Val Glu Met Leu Arg Lys Ser Gly Asn Ala Asp Asp Asn
190 195 200
att atc att gtt ggt agt cca aac tgg agt cag cgt ccg gac tta gca 1313
Ile Ile Ile Val Gly Ser Pro Asn Trp Ser Gln Arg Pro Asp Leu Ala
205 210 215
gct gat aat cca att gat gat cac cat aca atg tat act gtt cac ttc 1361
Ala Asp Asn Pro Ile Asp Asp His His Thr Met Tyr Thr Val His Phe
220 225 230
tac act ggt tca cat gct gct tca act gaa agc tat ccg tct gaa act 1409
Tyr Thr Gly Ser His Ala Ala Ser Thr Glu Ser Tyr Pro Ser Glu Thr
235 240 245 250
cct aac tct gaa aga gga aac gta atg agt aac act cgt tat gcg tta 1457
Pro Asn Ser Glu Arg Gly Asn Val Met Ser Asn Thr Arg Tyr Ala Leu
255 260 265
gaa aac gga gta gcg gta ttt gca aca gag tgg gga acg agt caa gct 1505
Glu Asn Gly Val Ala Val Phe Ala Thr Glu Trp Gly Thr Ser Gln Ala
270 275 280
agt gga gac ggt ggt cct tac ttt gat gaa gca gat gta tgg att gaa 1553
Ser Gly Asp Gly Gly Pro Tyr Phe Asp Glu Ala Asp Val Trp Ile Glu
285 290 295
ttt tta aat gaa aac aac att agc tgg gct aac tgg tct tta acg aat 1601
Phe Leu Asn Glu Asn Asn Ile Ser Trp Ala Asn Trp Ser Leu Thr Asn
300 305 310
aaa aat gaa gta tct ggt gca ttt aca cca ttc gag tta ggt aag tct 1649
Lys Asn Glu Val Ser Gly Ala Phe Thr Pro Phe Glu Leu Gly Lys Ser
315 320 325 330
aac gca acc aat ctt gac cca ggt cca gat cat gtg tgg gca cca gaa 1697
Asn Ala Thr Asn Leu Asp Pro Gly Pro Asp His Val Trp Ala Pro Glu
335 340 345
gaa tta agt ctt tct gga gaa tat gta cgt gct cgt att aaa ggt gtg 1745
Glu Leu Ser Leu Ser Gly Glu Tyr Val Arg Ala Arg Ile Lys Gly Val
350 355 360
aac tat gag cca atc gac cgt aca aaa tac acg aaa gta ctt tgg gac 1793
Asn Tyr Glu Pro Ile Asp Arg Thr Lys Tyr Thr Lys Val Leu Trp Asp
365 370 375
ttt aat gat gga acg aag caa gga ttt gga gtg aat tcg gat tct cca 1841
Phe Asn Asp Gly Thr Lys Gln Gly Phe Gly Val Asn Ser Asp Ser Pro
380 385 390
aat aaa gaa ctt att gca gtt gat aat gaa aac aac act ttg aaa gtt 1889
Asn Lys Glu Leu Ile Ala Val Asp Asn Glu Asn Asn Thr Leu Lys Val
395 400 405 410
tcg gga tta gat gta agt aac gat gtt tca gat ggc aac ttc tgg gct 1937
Ser Gly Leu Asp Val Ser Asn Asp Val Ser Asp Gly Asn Phe Trp Ala
415 420 425
aat gct cgt ctt tct gcc aac ggt tgg gga aaa agt gtt gat att tta 1985
Asn Ala Arg Leu Ser Ala Asn Gly Trp Gly Lys Ser Val Asp Ile Leu
430 435 440
ggt gct gag aag ctt aca atg gat gtt att gtt gat gaa cca acg acg 2033
Gly Ala Glu Lys Leu Thr Met Asp Val Ile Val Asp Glu Pro Thr Thr
445 450 455
gta gct att gcg gcg att cca caa agt agt aaa agt gga tgg gca aat 2081
Val Ala Ile Ala Ala Ile Pro Gln Ser Ser Lys Ser Gly Trp Ala Asn
460 465 470
cca gag cgt gct gtt cga gtg aac gcg gaa gat ttt gtc cag caa acg 2129
Pro Glu Arg Ala Val Arg Val Asn Ala Glu Asp Phe Val Gln Gln Thr
475 480 485 490
gac ggt aag tat aaa gct gga tta aca att aca gga gaa gat gct cct 2177
Asp Gly Lys Tyr Lys Ala Gly Leu Thr Ile Thr Gly Glu Asp Ala Pro
495 500 505
aac cta aaa aat atc gct ttt cat gaa gaa gat aac aat atg aac aac 2225
Asn Leu Lys Asn Ile Ala Phe His Glu Glu Asp Asn Asn Met Asn Asn
510 515 520
atc att ctg ttc gtg gga act gat gca gct gac gtt att tac tta gat 2273
Ile Ile Leu Phe Val Gly Thr Asp Ala Ala Asp Val Ile Tyr Leu Asp
525 530 535
aac att aaa gta att gga aca gaa gtt gaa att cca gtt gtt cat gat 2321
Asn Ile Lys Val Ile Gly Thr Glu Val Glu Ile Pro Val Val His Asp
540 545 550
cca aaa gga gaa gct gtt ctt cct tct gtt ttt gaa gac ggt aca cgt 2369
Pro Lys Gly Glu Ala Val Leu Pro Ser Val Phe Glu Asp Gly Thr Arg
555 560 565 570
caa ggt tgg gac tgg gct gga gag tct ggt gtg aaa aca gct tta aca 2417
Gln Gly Trp Asp Trp Ala Gly Glu Ser Gly Val Lys Thr Ala Leu Thr
575 580 585
att gaa gaa gca aac ggt tct aac gcg tta tca tgg gaa ttt gga tat 2465
Ile Glu Glu Ala Asn Gly Ser Asn Ala Leu Ser Trp Glu Phe Gly Tyr
590 595 600
cca gaa gta aaa cct agt gat aac tgg gca aca gct cca cgt tta gat 2513
Pro Glu Val Lys Pro Ser Asp Asn Trp Ala Thr Ala Pro Arg Leu Asp
605 610 615
ttc tgg aaa tct gac ttg gtt cgc ggt gag aat gat tat gta gct ttt 2561
Phe Trp Lys Ser Asp Leu Val Arg Gly Glu Asn Asp Tyr Val Ala Phe
620 625 630
gat ttc tat cta gat cca gtt cgt gca aca gaa ggc gca atg aat atc 2609
Asp Phe Tyr Leu Asp Pro Val Arg Ala Thr Glu Gly Ala Met Asn Ile
635 640 645 650
aat tta gta ttc cag cca cct act aac ggg tat tgg gta caa gca cca 2657
Asn Leu Val Phe Gln Pro Pro Thr Asn Gly Tyr Trp Val Gln Ala Pro
655 660 665
aaa acg tat acg att aac ttt gat gaa tta gag gaa gcg aat caa gta 2705
Lys Thr Tyr Thr Ile Asn Phe Asp Glu Leu Glu Glu Ala Asn Gln Val
670 675 680
aat ggt tta tat cac tat gaa gtg aaa att aac gta aga gat att aca 2753
Asn Gly Leu Tyr His Tyr Glu Val Lys Ile Asn Val Arg Asp Ile Thr
685 690 695
aac att caa gat gac acg tta cta cgt aac atg atg atc att ttt gca 2801
Asn Ile Gln Asp Asp Thr Leu Leu Arg Asn Met Met Ile Ile Phe Ala
700 705 710
gat gta gaa agt gac ttt gca ggg aga gtc ttt gta gat aat gtt cgt 2849
Asp Val Glu Ser Asp Phe Ala Gly Arg Val Phe Val Asp Asn Val Arg
715 720 725 730
ttt gag ggg gct gct act act gag ccg gtt gaa cca gag cca gtt gat 2897
Phe Glu Gly Ala Ala Thr Thr Glu Pro Val Glu Pro Glu Pro Val Asp
735 740 745
cct ggc gaa gag acg cca cct gtc gat gag aag gaa gcg aaa aaa gaa 2945
Pro Gly Glu Glu Thr Pro Pro Val Asp Glu Lys Glu Ala Lys Lys Glu
750 755 760
caa aaa gaa gca gag aaa gaa gag aaa gaa gca gta aaa gaa gaa aag 2993
Gin Lys Glu Ala Glu Lys Glu Glu Lys Glu Ala Val Lys Glu Glu Lys
765 770 775
aaa gaa gct aaa gaa gaa aag aaa gca gtc aaa aat gag gct aag aaa 3041
Lys Glu Ala Lys Glu Glu Lys Lys Ala Val Lys Asn Glu Ala Lys Lys
780 785 790
aaa taatctatta aactagttat agggttatct aaaggtctga tgtagatctt 3094
Lys
795
ttagataacc tttttcttgc ataactggac acagagttgt tattaaagaa agtaag 3150
<210>4
<211>795
<212>PRT
<213>芽孢杆菌sp.KSM-S237
<400>4
Ala Glu Gly Asn Thr Arg Glu Asp Asn Phe Lys His Leu Leu Gly Asn
1 5 10 15
Asp Asn Val Lys Arg Pro Ser Glu Ala Gly Ala Leu Gln Leu Gln Glu
20 25 30
Val Asp Gly Gln Met Thr Leu Val Asp Gln His Gly Glu Lys Ile Gln
35 40 45
Leu Arg Gly Met Ser Thr His Gly Leu Gln Trp Phe Pro Glu Ile Leu
50 55 60
Asn Asp Asn Ala Tyr Lys Ala Leu Ser Asn Asp Trp Asp Ser Asn Met
65 70 75 80
Ile Arg Leu Ala Met Tyr Val Gly Glu Asn Gly Tyr Ala Thr Asn Pro
85 90 95
Glu Leu Ile Lys Gln Arg Val Ile Asp Gly Ile Glu Leu Ala Ile Glu
100 105 110
Asn Asp Met Tyr Val Ile Val Asp Trp His Val His Ala Pro Gly Asp
115 120 125
Pro Arg Asp Pro Val Tyr Ala Gly Ala Lys Asp Phe Phe Arg Glu Ile
130 135 140
Ala Ala Leu Tyr Pro Asn Asn Pro His Ile Ile Tyr Glu Leu Ala Asn
145 150 155 160
Glu Pro Ser Ser Asn Asn Asn Gly Gly Ala Gly Ile Pro Asn Asn Glu
165 170 175
Glu Gly Trp Lys Ala Val Lys Glu Tyr Ala Asp Pro Ile Val Glu Met
180 185 190
Leu Arg Lys Ser Gly Asn Ala Asp Asp Asn Ile Ile Ile Val Gly Ser
195 200 205
Pro Asn Trp Ser Gln Arg Pro Asp Leu Ala Ala Asp Asn Pro Ile Asp
210 215 220
Asp His His Thr Met Tyr Thr Val His Phe Tyr Thr Gly Ser His Ala
225 230 235 240
Ala Ser Thr Glu Ser Tyr Pro Ser Glu Thr Pro Asn Ser Glu Arg Gly
245 250 255
Asn Val Met Ser Asn Thr Arg Tyr Ala Leu Glu Asn Gly Val Ala Val
260 265 270
Phe Ala Thr Glu Trp Gly Thr Ser Gln Ala Ser Gly Asp Gly Gly Pro
275 280 285
Tyr Phe Asp Glu Ala Asp Val Trp Ile Glu Phe Leu Asn Glu Asn Asn
290 295 300
Ile Ser Trp Ala Asn Trp Ser Leu Thr Asn Lys Asn Glu Val Ser Gly
305 310 315 320
Ala Phe Thr Pro Phe Glu Leu Gly Lys Ser Asn Ala Thr Asn Leu Asp
325 330 335
Pro Gly Pro Asp His Val Trp Ala Pro Glu Glu Leu Ser Leu Ser Gly
340 345 350
Glu Tyr Val Arg Ala Arg Ile Lys Gly Val Asn Tyr Glu Pro Ile Asp
355 360 365
Arg Thr Lys Tyr Thr Lys Val Leu Trp Asp Phe Asn Asp Gly Thr Lys
370 375 380
Gln Gly Phe Gly Val Asn Ser Asp Ser Pro Asn Lys Glu Leu Ile Ala
385 390 395 400
Val Asp Asn Glu Asn Asn Thr Leu Lys Val Ser Gly Leu Asp Val Ser
405 410 415
Asn Asp Val Ser Asp Gly Asn Phe Trp Ala Asn Ala Arg Leu Ser Ala
420 425 430
Asn Gly Trp Gly Lys Ser Val Asp Ile Leu Gly Ala Glu Lys Leu Thr
435 440 445
Met Asp Val Ile Val Asp Glu Pro Thr Thr Val Ala Ile Ala Ala Ile
450 455 460
Pro Gln Ser Ser Lys Ser Gly Trp Ala Asn Pro Glu Arg Ala Val Arg
465 470 475 480
Val Asn Ala Glu Asp Phe Val Gln Gln Thr Asp Gly Lys Tyr Lys Ala
485 490 495
Gly Leu Thr Ile Thr Gly Glu Asp Ala Pro Asn Leu Lys Asn Ile Ala
500 505 510
Phe His Glu Glu Asp Asn Asn Met Asn Asn Ile Ile Leu Phe Val Gly
515 520 525
Thr Asp Ala Ala Asp Val Ile Tyr Leu Asp Asn Ile Lys Val Ile Gly
530 535 540
Thr Glu Val Glu Ile Pro Val Val His Asp Pro Lys Gly Glu Ala Val
545 550 555 560
Leu Pro Ser Val Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp Ala
565 570 575
Gly Glu Ser Gly Val Lys Thr Ala Leu Thr Ile Glu Glu Ala Asn Gly
580 585 590
Ser Asn Ala Leu Ser Trp Glu Phe Gly Tyr Pro Glu Val Lys Pro Ser
595 600 605
Asp Asn Trp Ala Thr Ala Pro Arg Leu Asp Phe Trp Lys Ser Asp Leu
610 615 620
Val Arg Gly Glu Asn Asp Tyr Val Ala Phe Asp Phe Tyr Leu Asp Pro
625 630 635 640
Val Arg Ala Thr Glu Gly Ala Met Asn Ile Asn Leu Val Phe Gln Pro
645 650 655
Pro Thr Asn Gly Tyr Trp Val Gln Ala Pro Lys Thr Tyr Thr Ile Asn
660 665 670
Phe Asp Glu Leu Glu Glu Ala Asn Gln Val Asn Gly Leu Tyr His Tyr
675 680 685
Glu Val Lys Ile Asn Val Arg Asp Ile Thr Asn Ile Gln Asp Asp Thr
690 695 700
Leu Leu Arg Asn Met Met Ile Ile Phe Ala Asp Val Glu Ser Asp Phe
705 710 715 720
Ala Gly Arg Val Phe Val Asp Asn Val Arg Phe Glu Gly Ala Ala Thr
725 730 735
Thr Glu Pro Val Glu Pro Glu Pro Val Asp Pro Gly Glu Glu Thr Pro
740 745 750
Pro Val Asp Glu Lys Glu Ala Lys Lys Glu Gln Lys Glu Ala Glu Lys
755 760 765
Glu Glu Lys Glu Ala Val Lys Glu Glu Lys Lys Glu Ala Lys Glu Glu
770 775 780
Lys Lys Ala Val Lys Asn Glu Ala Lys Lys Lys
785 790 795
<210>5
<211>3332
<212>DNA
<213>芽孢杆菌sp.KSM-64
<220>
<221>CDS
<222>(610)..(3075)
<223>
<220>
<221>sig_peptide
<222>(610)..(696)
<223>
<220>
<221>mat_peptide
<222>(697)..(3075)
<223>
<400>5
agtacttacc attttagagt caaaagatag aagccaagca ggatttgccg atgcaaccgg 60
cttatattta gagggaattt ctttttaaat tgaatacgga ataaaatcag gtaaacaggt 120
cctgatttta tttttttgaa tttttttgag aactaaagat tgaaatagaa gtagaagaca 180
acggacataa gaaaattgta ttagttttaa ttatagaaaa cgcttttcta taattattta 240
tacctagaac gaaaatactg tttcgaaagc ggtttactat aaaaccttat attccggctc 300
tttttttaaa cagggggtga aaattcactc tagtattcta atttcaacat gctataataa 360
atttgtaaga cgcaatatac atcttttttt tatgatattt gtaagcggtt aaccttgtgc 420
tatatgccga tttaggaagg gggtagattg agtcaagtag tcataattta gataacttat 480
aagttgttga gaagcaggag agaatctggg ttactcacaa gttttttaaa acattatcga 540
aagcactttc ggttatgctt atgaatttag ctatttgatt caattacttt aataatttta 600
ggaggtaat atg atg tta aga aag aaa aca aag cag ttg att tct tcc att 651
Met Met Leu Arg Lys Lys Thr Lys Gln Leu Ile Ser Ser Ile
-25 -20
ctt att tta gtt tta ctt cta tct tta ttt ccg aca gct ctt gca gca 699
Leu Ile Leu Val Leu Leu Leu Ser Leu Phe Pro Thr Ala Leu Ala Ala
-15 -10 -5 -1 1
gaa gga aac act cgt gaa gac aat ttt aaa cat tta tta ggt aat gac 747
Glu Gly Asn Thr Arg Glu Asp Asn Phe Lys His Leu Leu Gly Asn Asp
5 10 15
aat gtt aaa cgc cct tct gag gct ggc gca tta caa tta caa gaa gtc 795
Asn Val Lys Arg Pro Ser Glu Ala Gly Ala Leu Gln Leu Gln Glu Val
20 25 30
gat gga caa atg aca tta gta gat caa cat gga gaa aaa att caa tta 843
Asp Gly Gln Met Thr Leu Val Asp Gln His Gly Glu Lys Ile Gln Leu
35 40 45
cgt gga atg agt aca cac gga tta caa tgg ttt cct gag atc ttg aat 891
Arg Gly Met Ser Thr His Gly Leu Gln Trp Phe Pro Glu Ile Leu Asn
50 55 60 65
gat aac gca tac aaa gct ctt gct aac gat tgg gaa tca aat atg att 939
Asp Asn Ala Tyr Lys Ala Leu Ala Asn Asp Trp Glu Ser Asn Met Ile
70 75 80
cgt cta gct atg tat gtc ggt gaa aat ggc tat gct tca aat cca gag 987
Arg Leu Ala Met Tyr Val Gly Glu Asn Gly Tyr Ala Ser Asn Pro Glu
85 90 95
tta att aaa agc aga gtc att aaa gga ata gat ctt gct att gaa aat 1035
Leu Ile Lys Ser Arg Val Ile Lys Gly Ile Asp Leu Ala Ile Glu Asn
100 105 110
gac atg tat gtc atc gtt gat tgg cat gta cat gca cct ggt gat cct 1083
Asp Met Tyr Val Ile Val Asp Trp His Val His Ala Pro Gly Asp Pro
115 120 125
aga gat ccc gtt tac gct gga gca gaa gat ttc ttt aga gat att gca 1131
Arg Asp Pro Val Tyr Ala Gly Ala Glu Asp Phe Phe Arg Asp Ile Ala
130 135 140 145
gca tta tat cct aac aat cca cac att att tat gag tta gcg aat gag 1179
Ala Leu Tyr Pro Asn Asn Pro His Ile Ile Tyr Glu Leu Ala Asn Glu
150 155 160
cca agt agt aac aat aat ggt gga gct ggg att cca aat aat gaa gaa 1227
Pro Ser Ser Asn Asn Asn Gly Gly Ala Gly Ile Pro Asn Asn Glu Glu
165 170 175
ggt tgg aat gcg gta aaa gaa tac gct gat cca att gta gaa atg tta 1275
Gly Trp Asn Ala Val Lys Glu Tyr Ala Asp Pro Ile Val Glu Met Leu
180 185 190
cgt gat agc ggg aac gca gat gac aat att atc att gtg ggt agt cca 1323
Arg Asp Ser Gly Asn Ala Asp Asp Asn Ile Ile Ile Val Gly Ser Pro
195 200 205
aac tgg agt cag cgt cct gac tta gca gct gat aat cca att gat gat 1371
Asn Trp Ser Gln Arg Pro Asp Leu Ala Ala Asp Asn Pro Ile Asp Asp
210 215 220 225
cac cat aca atg tat act gtt cac ttc tac act ggt tca cat gct gct 1419
His His Thr Met Tyr Thr Val His Phe Tyr Thr Gly Ser His Ala Ala
230 235 240
tca act gaa agc tat ccg cct gaa act cct aac tct gaa aga gga aac 1467
Ser Thr Glu Ser Tyr Pro Pro Glu Thr Pro Asn Ser Glu Arg Gly Asn
245 250 255
gta atg agt aac act cgt tat gcg tta gaa aac gga gta gca gta ttt 1515
Val Met Ser Asn Thr Arg Tyr Ala Leu Glu Asn Gly Val Ala Val Phe
260 265 270
gca aca gag tgg gga act agc caa gca aat gga gat ggt ggt cct tac 1563
Ala Thr Glu Trp Gly Thr Ser Gln Ala Asn Gly Asp Gly Gly Pro Tyr
275 280 285
ttt gat gaa gca gat gta tgg att gag ttt tta aat gaa aac aac att 1611
Phe Asp Glu Ala Asp Val Trp Ile Glu Phe Leu Asn Glu Asn Asn Ile
290 295 300 305
agc tgg gct aac tgg tct tta acg aat aaa aat gaa gta tct ggt gca 1659
Ser Trp Ala Asn Trp Ser Leu Thr Asn Lys Asn Glu Val Ser Gly Ala
310 315 320
ttt aca cca ttc gag tta ggt aag tct aac gca aca agt ctt gac cca 1707
Phe Thr Pro Phe Glu Leu Gly Lys Ser Asn Ala Thr Ser Leu Asp Pro
325 330 335
ggg cca gac caa gta tgg gta cca gaa gag tta agt ctt tct gga gaa 1755
Gly Pro Asp Gln Val Trp Val Pro Glu Glu Leu Ser Leu Ser Gly Glu
340 345 350
tat gta cgt gct cgt att aaa ggt gtg aac tat gag cca atc gac cgt 1803
Tyr Val Arg Ala Arg Ile Lys Gly Val Asn Tyr Glu Pro Ile Asp Arg
355 360 365
aca aaa tac acg aaa gta ctt tgg gac ttt aat gat gga acg aag caa 1851
Thr Lys Tyr Thr Lys Val Leu Trp Asp Phe Asn Asp Gly Thr Lys Gln
370 375 380 385
gga ttt gga gtg aat gga gat tct cca gtt gaa gat gta gtt att gag 1899
Gly Phe Gly Val Asn Gly Asp Ser Pro Val Glu Asp Val Val Ile Glu
390 395 400
aat gaa gcg ggc gct tta aaa ctt tca gga tta gat gca agt aat gat 1947
Asn Glu Ala Gly Ala Leu Lys Leu Ser Gly Leu Asp Ala Ser Asn Asp
405 410 415
gtt tct gaa ggt aat tac tgg gct aat gct cgt ctt tct gcc gac ggt 1995
Val Ser Glu Gly Asn Tyr Trp Ala Asn Ala Arg Leu Ser Ala Asp Gly
420 425 430
tgg gga aaa agt gtt gat att tta ggt gct gaa aaa ctt act atg gat 2043
Trp Gly Lys Ser Val Asp Ile Leu Gly Ala Glu Lys Leu Thr Met Asp
435 440 445
gtg att gtt gat gag ccg acc acg gta tca att gct gca att cca caa 2091
Val Ile Val Asp Glu Pro Thr Thr Val Ser Ile Ala Ala Ile Pro Gln
450 455 460 465
ggg cca tca gcc aat tgg gtt aat cca aat cgt gca att aag gtt gag 2139
Gly Pro Ser Ala Asn Trp Val Asn Pro Asn Arg Ala Ile Lys Val Glu
470 475 480
cca act aat ttc gta ccg tta gga gat aag ttt aaa gcg gaa tta act 2187
Pro Thr Asn Phe Val Pro Leu Gly Asp Lys Phe Lys Ala Glu Leu Thr
485 490 495
ata act tca gct gac tct cca tcg tta gaa gct att gcg atg cat gct 2235
Ile Thr Ser Ala Asp Ser Pro Ser Leu Glu Ala Ile Ala Met His Ala
500 505 510
gaa aat aac aac atc aac aac atc att ctt ttt gta gga act gaa ggt 2283
Glu Asn Asn Asn Ile Asn Asn Ile Ile Leu Phe Val Gly Thr Glu Gly
515 520 525
gct gat gtt atc tat tta gat aac att aaa gta att gga aca gaa gtt 2331
Ala Asp Val Ile Tyr Leu Asp Asn Ile Lys Val Ile Gly Thr Glu Val
530 535 540 545
gaa att cca gtt gtt cat gat cca aaa gga gaa gct gtt ctt cct tct 2379
Glu Ile Pro Val Val His Asp Pro Lys Gly Glu Ala Val Leu Pro Ser
550 555 560
gtt ttt gaa gac ggt aca cgt caa ggt tgg gac tgg gct gga gag tct 2427
Val Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp Ala Gly Glu Ser
565 570 575
ggt gtg aaa aca gct tta aca att gaa gaa gca aac ggt tct aac gcg 2475
Gly Val Lys Thr Ala Leu Thr Ile Glu Glu Ala Asn Gly Ser Asn Ala
580 585 590
tta tca tgg gaa ttt gga tac cca gaa gta aaa cct agt gat aac tgg 2523
Leu Ser Trp Glu Phe Gly Tyr Pro Glu Val Lys Pro Ser Asp Asn Trp
595 600 605
gca aca gct cca cgt tta gat ttc tgg aaa tct gac ttg gtt cgc ggt 2571
Ala Thr Ala Pro Arg Leu Asp Phe Trp Lys Ser Asp Leu Val Arg Gly
610 615 620 625
gaa aat gat tat gta act ttt gat ttc tat cta gat cca gtt cgt gca 2619
Glu Asn Asp Tyr Val Thr Phe Asp Phe Tyr Leu Asp Pro Val Arg Ala
630 635 640
aca gaa ggc gca atg aat atc aat tta gta ttc cag cca cct act aac 2667
Thr Glu Gly Ala Met Asn Ile Asn Leu Val Phe Gln Pro Pro Thr Asn
645 650 655
ggg tat tgg gta caa gca cca aaa acg tat acg att aac ttt gat gaa 2715
Gly Tyr Trp Val Gln Ala Pro Lys Thr Tyr Thr Ile Asn Phe Asp Glu
660 665 670
tta gag gaa gcg aat caa gta aat ggt tta tat cac tat gaa gtg aaa 2763
Leu Glu Glu Ala Asn Gln Val Asn Gly Leu Tyr His Tyr Glu Val Lys
675 680 685
att aac gta aga gat att aca aac att caa gat gac acg tta cta cgt 2811
Ile Asn Val Arg Asp Ile Thr Asn Ile Gln Asp Asp Thr Leu Leu Arg
690 695 700 705
aac atg atg atc att ttt gca gat gta gaa agt gac ttt gca ggg aga 2859
Asn Met Met Ile Ile Phe Ala Asp Val Glu Ser Asp Phe Ala Gly Arg
710 715 720
gtc ttt gta gat aat gtt cgt ttt gag ggg gct gct act act gag ccg 2907
Val Phe Val Asp Asn Val Arg Phe Glu Gly Ala Ala Thr Thr Glu Pro
725 730 735
gtt gaa cca gag cca gtt gat cct ggc gaa gag acg ccg cct gtc gat 2955
Val Glu Pro Glu Pro Val Asp Pro Gly Glu Glu Thr Pro Pro Val Asp
740 745 750
gag aag gaa gcg aaa aaa gaa caa aaa gaa gca gag aaa gaa gag aaa 3003
Glu Lys Glu Ala Lys Lys Glu Gln Lys Glu Ala Glu Lys Glu Glu Lys
755 760 765
gaa gca gta aaa gaa gaa aag aaa gaa gct aaa gaa gaa aag aaa gca 3051
Glu Ala Val Lys Glu Glu Lys Lys Glu Ala Lys Glu Glu Lys Lys Ala
770 775 780 785
atc aaa aat gag gct acg aaa aaa taatctaata aactagttat agggttatct 3105
Ile Lys Asn Glu Ala Thr Lys Lys
790
aaaggtctga tgcagatctt ttagataacc tttttttgca taactggaca tagaatggtt 3165
attaaagaaa gcaaggtgtt tatacgatat taaaaaggta gcgattttaa attgaaacct 3225
ttaataatgt cttgtgatag aatgatgaag taatttaaga gggggaaacg aagtgaaaac 3285
ggaaatttct agtagaagaa aaacagacca agaaatactg caagctt 3332
<210>6
<211>793
<212>PRT
<213>芽孢杆菌sp.KSM-64
<400>6
Ala Glu Gly Asn Thr Arg Glu Asp Asn Phe Lys His Leu Leu Gly Asn
1 5 10 15
Asp Asn Val Lys Arg Pro Ser Glu Ala Gly Ala Leu Gln Leu Gln Glu
20 25 30
Val Asp Gly Gln Met Thr Leu Val Asp Gln His Gly Glu Lys Ile Gln
35 40 45
Leu Arg Gly Met Ser Thr His Gly Leu Gln Trp Phe Pro Glu Ile Leu
50 55 60
Asn Asp Asn Ala Tyr Lys Ala Leu Ala Asn Asp Trp Glu Ser Asn Met
65 70 75 80
Ile Arg Leu Ala Met Tyr Val Gly Glu Asn Gly Tyr Ala Ser Asn Pro
85 90 95
Glu Leu Ile Lys Ser Arg Val Ile Lys Gly Ile Asp Leu Ala Ile Glu
100 105 110
Asn Asp Met Tyr Val Ile Val Asp Trp His Val His Ala Pro Gly Asp
115 120 125
Pro Arg Asp Pro Val Tyr Ala Gly Ala Glu Asp Phe Phe Arg Asp Ile
130 135 140
Ala Ala Leu Tyr Pro Asn Asn Pro His Ile Ile Tyr Glu Leu Ala Asn
145 150 155 160
Glu Pro Ser Ser Asn Asn Asn Gly Gly Ala Gly Ile Pro Asn Asn Glu
165 170 175
Glu Gly Trp Asn Ala Val Lys Glu Tyr Ala Asp Pro Ile Val Glu Met
180 185 190
Leu Arg Asp Ser Gly Asn Ala Asp Asp Asn Ile Ile Ile Val Gly Ser
195 200 205
Pro Asn Trp Ser Gln Arg Pro Asp Leu Ala Ala Asp Asn Pro Ile Asp
210 215 220
Asp His His Thr Met Tyr Thr Val His Phe Tyr Thr Gly Ser His Ala
225 230 235 240
Ala Ser Thr Glu Ser Tyr Pro Pro Glu Thr Pro Asn Ser Glu Arg Gly
245 250 255
Asn Val Met Ser Asn Thr Arg Tyr Ala Leu Glu Asn Gly Val Ala Val
260 265 270
Phe Ala Thr Glu Trp Gly Thr Ser Gln Ala Asn Gly Asp Gly Gly Pro
275 280 285
Tyr Phe Asp Glu Ala Asp Val Trp Ile Glu Phe Leu Asn Glu Asn Asn
290 295 300
Ile Ser Trp Ala Asn Trp Ser Leu Thr Asn Lys Asn Glu Val Ser Gly
305 310 315 320
Ala Phe Thr Pro Phe Glu Leu Gly Lys Ser Asn Ala Thr Ser Leu Asp
325 330 335
Pro Gly Pro Asp Gln Val Trp Val Pro Glu Glu Leu Ser Leu Ser Gly
340 345 350
Glu Tyr Val Arg Ala Arg Ile Lys Gly Val Asn Tyr Glu Pro Ile Asp
355 360 365
Arg Thr Lys Tyr Thr Lys Val Leu Trp Asp Phe Asn Asp Gly Thr Lys
370 375 380
Gln Gly Phe Gly Val Asn Gly Asp Ser Pro Val Glu Asp Val Val Ile
385 390 395 400
Glu Asn Glu Ala Gly Ala Leu Lys Leu Ser Gly Leu Asp Ala Ser Asn
405 410 415
Asp Val Ser Glu Gly Asn Tyr Trp Ala Asn Ala Arg Leu Ser Ala Asp
420 425 430
Gly Trp Gly Lys Ser Val Asp Ile Leu Gly Ala Glu Lys Leu Thr Met
435 440 445
Asp Val Ile Val Asp Glu Pro Thr Thr Val Ser Ile Ala Ala Ile Pro
450 455 460
Gln Gly Pro Ser Ala Asn Trp Val Asn Pro Asn Arg Ala Ile Lys Val
465 470 475 480
Glu Pro Thr Asn Phe Val Pro Leu Gly Asp Lys Phe Lys Ala Glu Leu
485 490 495
Thr Ile Thr Ser Ala Asp Ser Pro Ser Leu Glu Ala Ile Ala Met His
500 505 510
Ala Glu Asn Asn Asn Ile Asn Asn Ile Ile Leu Phe Val Gly Thr Glu
515 520 525
Gly Ala Asp Val Ile Tyr Leu Asp Asn Ile Lys Val Ile Gly Thr Glu
530 535 540
Val Glu Ile Pro Val Val His Asp Pro Lys Gly Glu Ala Val Leu Pro
545 550 555 560
Ser Val Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp Ala Gly Glu
565 570 575
Ser Gly Val Lys Thr Ala Leu Thr Ile Glu Glu Ala Asn Gly Ser Asn
580 585 590
Ala Leu Ser Trp Glu Phe Gly Tyr Pro Glu Val Lys Pro Ser Asp Asn
595 600 605
Trp Ala Thr Ala Pro Arg Leu Asp Phe Trp Lys Ser Asp Leu Val Arg
610 615 620
Gly Glu Asn Asp Tyr Val Thr Phe Asp Phe Tyr Leu Asp Pro Val Arg
625 630 635 640
Ala Thr Glu Gly Ala Met Asn Ile Asn Leu Val Phe Gln Pro Pro Thr
645 650 655
Asn Gly Tyr Trp Val Gln Ala Pro Lys Thr Tyr Thr Ile Asn Phe Asp
660 665 670
Glu Leu Glu Glu Ala Asn Gln Val Asn Gly Leu Tyr His Tyr Glu Val
675 680 685
Lys Ile Asn Val Arg Asp Ile Thr Asn Ile Gln Asp Asp Thr Leu Leu
690 695 700
Arg Asn Met Met Ile Ile Phe Ala Asp Val Glu Ser Asp Phe Ala Gly
705 710 715 720
Arg Val Phe Val Asp Asn Val Arg Phe Glu Gly Ala Ala Thr Thr Glu
725 730 735
Pro Val Glu Pro Glu Pro Val Asp Pro Gly Glu Glu Thr Pro Pro Val
740 745 750
Asp Glu Lys Glu Ala Lys Lys Glu Gln Lys Glu Ala Glu Lys Glu Glu
755 760 765
Lys Glu Ala Val Lys Glu Glu Lys Lys Glu Ala Lys Glu Glu Lys Lys
770 775 780
Ala Ile Lys Asn Glu Ala Thr Lys Lys
785 790
<210>7
<211>572
<212>DNA
<213>芽孢杆菌sp.KSM-S237
<400>7
gatttgccga tgcaacaggc ttatatttag aggaaatttc tttttaaatt gaatacggaa 60
taaaatcagg taaacaggtc ctgattttat ttttttgagt tttttagaga actgaagatt 120
gaaataaaag tagaagacaa aggacataag aaaattgcat tagttttaat tatagaaaac 180
gcctttttat aattatttat acctagaacg aaaatactgt ttcgaaagcg gtttactata 240
aaaccttata ttccggctct tttttaaaac agggggtaaa aattcactct agtattctaa 300
tttcaacatg ctataataaa tttgtaagac gcaatatgca tctctttttt tacgatatat 360
gtaagcggtt aaccttgtgc tatatgccga tttaggaagg ggggtagaat atttcaagta 420
gtaataacat acaatactta taagttgttg agaagcagga gagcatctgg gttactcaca 480
agttttttta aaactttaac gaaagcactt tcggtaatgc ttatgaattt agctatttga 540
ttcaattact ttaaaaatat ttaggaggta at 572
<210>8
<211>609
<212>DNA
<213>芽孢杆菌sp.KSM-64
<400>8
agtacttacc attttagagt caaaagatag aagccaagca ggatttgccg atgcaaccgg 60
cttatattta gagggaattt ctttttaaat tgaatacgga ataaaatcag gtaaacaggt 120
cctgatttta tttttttgaa tttttttgag aactaaagat tgaaatagaa gtagaagaca 180
acggacataa gaaaattgta ttagttttaa ttatagaaaa cgcttttcta taattattta 240
tacctagaac gaaaatactg tttcgaaagc ggtttactat aaaaccttat attccggctc 300
tttttttaaa cagggggtga aaattcactc tagtattcta atttcaacat gctataataa 360
atttgtaaga cgcaatatac atcttttttt tatgatattt gtaagcggtt aaccttgtgc 420
tatatgccga tttaggaagg gggtagaata tttcaagtag taataacata caatacttat 480
aagttgttga gaagcaggag agaatctggg ttactcacaa gttttttaaa acattatcga 540
aagcactttc ggttatgctt atgaatttag ctatttgatt caattacttt aataatttta 600
ggaggtaat 609
<210>9
<211>37
<212>DNA
<213>人工序列
<220>
<223>设计自用于纤维素酶的芽孢杆菌sp.KSM-S237基因的核苷酸序列的作为PCR
引物的低聚核苷酸;在5’-端***有BamHI限制酶切位点的序列
<400>9
ttgcggatcc aacaggctta tatttagagg aaatttc
<210>10
<211>40
<212>DNA
<213>人工序列
<220>
<223>设计自纤维素酶的芽孢杆菌sp.KSM-S237基因的核苷酸序列的作为PCR引物的
低聚核苷酸;包含8个SigmaE识别的核苷酸取代基的序列
<400>10
gtatgttatt actacttgaa atattctacc ccccttccta
<210>11
<211>39
<212>DNA
<213>人工序列
<220>
<223>设计自纤维素酶的芽孢杆菌sp.KSM-S237基因的核苷酸序列的作为PCR引物的
低聚核苷酸;包含8个SigmaE识别的核苷酸取代基的序列
<400>11
atatttcaag tagtaataac atacaatact tataagttg
<210>12
<211>20
<212>DNA
<213>人工序列
<220>
<223>设计自纤维素酶的芽孢杆菌sp.KSM-S237基因的核苷酸序列的作为PCR引物的
低聚核苷酸
<400>12
tcgctaccct tttattatcg
<210>13
<211>1795
<212>DNA
<213>芽孢杆菌sp.KSM-K38
<220>
<221>CDS
<222>(212)..(1714)
<223>
<220>
<221>sig_peptide
<222>(212)..(274)
<223>
<220>
<221>mat_peptide
<222>(275)..(1714)
<223>
<400>13
caggccagcc aaagtagcca ccaactaagt aacatcgatt caggataaaa gtatgcgaaa 60
cgatgcgcaa aactgcgcaa ctactagcac tcttcaggga ctaaaccacc ttttttccaa 120
aaatgacatc atataaacaa atttgtctac caatcactat ttaaagctgt ttatgatata 180
tgtaagcgtt atcattaaaa ggaggtattt g atg aga aga tgg gta gta gca 232
Met Arg Arg Trp Val Val Ala
-21 -20 -15
atg ttg gca gtg tta ttt tta ttt cct tcg gta gta gtt gca gat gga 280
Met Leu Ala Val Leu Phe Leu Phe Pro Ser Val Val Val Ala Asp Gly
-10 -5 1
ttg aac ggt acg atg atg cag tat tat gag tgg cat ttg gaa aac gac 328
Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu Asn Asp
5 10 15
ggg cag cat tgg aat cgg ttg cac gat gat gcc gca gct ttg agt gat 376
Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Ala Ala Leu Ser Asp
20 25 30
gct ggt att aca gct att tgg att ccg cca gcc tac aaa ggt aat agt 424
Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly Asn Ser
35 40 45 50
cag gcg gat gtt ggg tac ggt gca tac gat ctt tat gat tta gga gag 472
Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu
55 60 65
ttc aat caa aag ggt act gtt cga acg aaa tac gga act aag gca cag 520
Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ala Gln
70 75 80
ctt gaa cga gct att ggg tcc ctt aaa tct aat gat atc aat gta tac 568
Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn Val Tyr
85 90 95
gga gat gtc gtg atg aat cat aaa atg gga gct gat ttt acg gag gca 616
Gly Asp Val Val Met Asn His Lys Met Gly Ala Asp Phe Thr Glu Ala
100 105 110
gtg caa gct gtt caa gta aat cca acg aat cgt tgg cag gat att tca 664
Val Gln Ala Val Gln Val Asn Pro Thr Asn Arg Trp Gln Asp Ile Ser
115 120 125 130
ggt gcc tac acg att gat gcg tgg acg ggt ttc gac ttt tca ggg cgt 712
Gly Ala Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Ser Gly Arg
135 140 145
aac aac gcc tat tca gat ttt aag tgg aga tgg ttc cat ttt aat ggt 760
Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe Asn Gly
150 155 160
gtt gac tgg gat cag cgc tat caa gaa aat cat att ttc cgc ttt gca 808
Val Asp Trp Asp Gln Arg Tyr Gln Glu Asn His Ile Phe Arg Phe Ala
165 170 175
aat acg aac tgg aac tgg cga gtg gat gaa gag aac ggt aat tat gat 856
Asn Thr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn Tyr Asp
180 185 190
tac ctg tta gga tcg aat atc gac ttt agt cat cca gaa gta caa gat 904
Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val Gln Asp
195 200 205 210
gag ttg aag gat tgg ggt agc tgg ttt acc gat gag tta gat ttg gat 952
Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp Leu Asp
215 220 225
ggt tat cgt tta gat gct att aaa cat att cca ttc tgg tat aca tct 1000
Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr Thr Ser
230 235 240
gat tgg gtt cgg cat cag cgc aac gaa gca gat caa gat tta ttt gtc 1048
Asp Trp Val Arg His Gln Arg Asn Glu Ala Asp Gln Asp Leu Phe Val
245 250 255
gta ggg gaa tat tgg aag gat gac gta ggt gct ctc gaa ttt tat tta 1096
Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe Tyr Leu
260 265 270
gat gaa atg aat tgg gag atg tct cta ttc gat gtt cca ctt aat tat 1144
Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu Asn Tyr
275 280 285 290
aat ttt tac cgg gct tca caa caa ggt gga agc tat gat atg cgt aat 1192
Asn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met Arg Asn
295 300 305
att tta cga gga tct tta gta gaa gcg cat ccg atg cat gca gtt acg 1240
Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Met His Ala Val Thr
310 315 320
ttt gtt gat aat cat gat act cag cca ggg gag tca tta gag tca tgg 1288
Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu Ser Trp
325 330 335
gtt gct gat tgg ttt aag cca ctt gct tat gcg aca att ttg acg cgt 1336
Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu Thr Arg
340 345 350
gaa ggt ggt tat cca aat gta ttt tac ggt gat tac tat ggg att cct 1384
Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly Ile Pro
355 360 365 370
aac gat aac att tca gct aaa aaa gat atg att gat gag ctg ctt gat 1432
Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu Leu Asp
375 380 385
gca cgt caa aat tac gca tat ggc acg cag cat gac tat ttt gat cat 1480
Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp His
390 395 400
tgg gat gtt gta gga tgg act agg gaa gga tct tcc tcc aga cct aat 1528
Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg Pro Asn
405 410 415
tca ggc ctt gcg act att atg tcg aat gga cct ggt ggt tcc aag tgg 1576
Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser Lys Trp
420 425 430
atg tat gta gga cgt cag aat gca gga caa aca tgg aca gat tta act 1624
Met Tyr Val Gly Arg Gln Asn Ala Gly Gln Thr Trp Thr Asp Leu Thr
435 440 445 450
ggt aat aac gga gcg tcc gtt aca att aat ggc gat gga tgg ggc gaa 1672
Gly Asn Asn Gly Ala Ser Val Thr Ile Asn Gly Asp Gly Trp Gly Glu
455 460 465
ttc ttt acg aat gga gga tct gta tcc gtg tac gtg aac caa taacaaaaa 1723
Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln
470 475 480
gccttgagaa gggattcctc cctaactcaa ggctttcttt atgtcgctta gctttacgct 1783
tctacgactt tg 1795
<210>14
<211>480
<212>PRT
<213>芽孢杆菌sp.KSM-K38
<400>14
Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr Glu Trp His Leu Glu
1 5 10 15
Asn Asp Gly Gln His Trp Asn Arg Leu His Asp Asp Ala Ala Ala Leu
20 25 30
Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45
Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu
50 55 60
Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
65 70 75 80
Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn
85 90 95
Val Tyr Gly Asp Val Val Met Asn His Lys Met Gly Ala Asp Phe Thr
100 105 110
Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr Asn Arg Trp Gln Asp
115 120 125
Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr Gly Phe Asp Phe Ser
130 135 140
Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp Arg Trp Phe His Phe
145 150 155 160
Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu Asn His Ile Phe Arg
165 170 175
Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn
180 185 190
Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val
195 200 205
Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp
210 215 220
Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp Tyr
225 230 235 240
Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu Ala Asp Gln Asp Leu
245 250 255
Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val Gly Ala Leu Glu Phe
260 265 270
Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu
275 280 285
Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met
290 295 300
Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Met His Ala
305 310 315 320
Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu
325 330 335
Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu
340 345 350
Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr Tyr Gly
355 360 365
Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu
370 375 380
Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe
385 390 395 400
Asp His Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg
405 410 415
Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser
420 425 430
Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly Gln Thr Trp Thr Asp
435 440 445
Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile Asn Gly Asp Gly Trp
450 455 460
Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn Gln
465 470 475 480
<210>15
<211>25
<212>DNA
<213>人工序列
<220>
<223>设计自纤维素酶的芽孢杆菌sp.KSM-S237基因的核苷酸序列的作为PCR引物的
低聚核苷酸;在5’-端***有BamHI限制酶切位点的序列
<400>15
cccggatcca acaggcttat attta 25
<210>16
<211>29
<212>DNA
<213>人工序列
<220>
<223>作为PCR引物的低聚核苷酸;其3’-部分设计自纤维素酶的芽孢杆菌sp.
KSM-S237基因的核苷酸序列,其5’-部分设计自淀粉酶的芽孢杆菌sp.
KSM-K38基因的核苷酸序列
<400>16
ttcaatccat ctgctgcaag agctgccgg 29
<210>17
<211>30
<212>DNA
<213>人工序列
<220>
<223>作为PCR引物的低聚核苷酸;其3’-部分设计自淀粉酶的芽孢杆菌sp.KSM-
K38基因的核苷酸序列,其5’-部分设计自纤维素酶的芽孢杆菌sp.KSM-S237
基因的核苷酸序列
<400>17
gctcttgcag cagatggatt gaacggtacg 30
<210>18
<211>30
<212>DNA
<213>人工序列
<220>
<223>设计自淀粉酶的芽孢杆菌sp.KSM-K38基因的核苷酸序列的作为PCR引物的低
聚核苷酸;在5’-端***有Xbal限制酶切位点的序列
<400>18
ttggtctaga ccccaagctt caaagtcgta 30
Claims (15)
1.一种启动子DNA,其特征在于,
是对含有由SigA识别的启动子及其附近的碱基的碱基序列进行修饰以使得被SigA和SigE识别而形成的启动子DNA。
2.如权利要求1所述的启动子DNA,其特征在于,
是通过构建被SigE识别的共有序列而进行修饰的启动子DNA。
3.如权利要求2所述的启动子DNA,其特征在于,
被SigE识别的共有序列为,由用ATAHTT所表示的-35区域和用在其13或14碱基之后连接的CATAYAHT所表示的-10区域形成的碱基序列,其中,H为A或C或T,Y为C或T。
4.如权利要求1~3的任意一项所述的启动子DNA,其特征在于,
含有被SigA识别的启动子及其附近的碱基的碱基序列为含有如下碱基序列的碱基序列:由序列号1所表示的碱基序列的碱基号92~552的碱基序列,由序列号2所表示的碱基序列的碱基号133~589的碱基序列,或者对该碱基序列具有80%以上的同一性、且具有SigA的共有序列和/或具有相同的启动子功能的碱基序列。
5.如权利要求1~3的任意一项所述的启动子DNA,其特征在于,
含有被SigA识别的启动子及其附近的碱基的碱基序列为,由序列号1所表示的碱基序列,由序列号2所表示的碱基序列,或对该碱基序列具有90%以上的同一性、且具有SigA的共有序列和/或具有相同的启动子功能的碱基序列。
6.如权利要4或5所述的启动子DNA,其特征在于,
含有由SigA识别的启动子及其附近的碱基的碱基序列为610bp以下的大小。
7.一种启动子DNA,其特征在于,
是连接2个以上的权利要求1~6的任意一项所述的启动子DNA的启动子DNA。
8.一种表达载体,其特征在于,
是含有权利要求1~7的任意一项所述的启动子DNA的表达载体。
9.一种重组微生物,其特征在于,
是含有权利要求8所述的表达载体的重组微生物。
10.一种重组微生物,其特征在于,
是在基因组上导入了权利要求1~7的任意一项所述的启动子DNA的重组微生物。
11.一种蛋白质或多肽的制造方法,其特征在于,
培养权利要求9或10所述的重组微生物。
12.如权利要求11所述的制造方法,其特征在于,
蛋白质为纤维素酶或淀粉酶。
13.如权利要求12所述的制造方法,其特征在于,
纤维素酶为由用序列号4所表示的氨基酸序列所组成的碱性纤维素酶,或者对该氨基酸序列具有70%以上的同源性、并且具有碱性纤维素酶活性的蛋白质。
14.如权利要求12所述的制造方法,其特征在于,
淀粉酶为由用序列号14所表示的氨基酸序列所组成的碱性淀粉酶,或者对该氨基酸序列具有70%以上的同源性、并且具有碱性淀粉酶活性的蛋白质。
15.一种启动子DNA的构建方法,其特征在于,
对含有由SigA识别的启动子及其附近的碱基的碱基序列进行修饰以使得可由SigA和SigE识别。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2004062853 | 2004-03-05 | ||
JP062853/2004 | 2004-03-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1930291A true CN1930291A (zh) | 2007-03-14 |
Family
ID=34918130
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CNA2005800071171A Pending CN1930291A (zh) | 2004-03-05 | 2005-03-04 | 经过修饰的启动子 |
Country Status (6)
Country | Link |
---|---|
US (1) | US8623631B2 (zh) |
EP (1) | EP1721976B1 (zh) |
CN (1) | CN1930291A (zh) |
DE (1) | DE602005017469D1 (zh) |
DK (1) | DK1721976T3 (zh) |
WO (1) | WO2005085441A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102575253A (zh) * | 2009-10-21 | 2012-07-11 | 花王株式会社 | 经过修饰的启动子 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4485341B2 (ja) * | 2004-12-20 | 2010-06-23 | 花王株式会社 | 組換え微生物 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2000210081A (ja) * | 1999-01-21 | 2000-08-02 | Kao Corp | 耐熱性アルカリセルラ―ゼ遺伝子 |
JP4336082B2 (ja) | 2001-05-29 | 2009-09-30 | 花王株式会社 | 宿主微生物 |
-
2005
- 2005-03-04 WO PCT/JP2005/003757 patent/WO2005085441A1/ja not_active Application Discontinuation
- 2005-03-04 DE DE602005017469T patent/DE602005017469D1/de active Active
- 2005-03-04 CN CNA2005800071171A patent/CN1930291A/zh active Pending
- 2005-03-04 US US10/589,960 patent/US8623631B2/en not_active Expired - Fee Related
- 2005-03-04 EP EP05720029A patent/EP1721976B1/en not_active Expired - Fee Related
- 2005-03-04 DK DK05720029T patent/DK1721976T3/da active
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102575253A (zh) * | 2009-10-21 | 2012-07-11 | 花王株式会社 | 经过修饰的启动子 |
US9029519B2 (en) | 2009-10-21 | 2015-05-12 | Kao Corporation | Modified promoter |
Also Published As
Publication number | Publication date |
---|---|
US8623631B2 (en) | 2014-01-07 |
EP1721976B1 (en) | 2009-11-04 |
WO2005085441A1 (ja) | 2005-09-15 |
DK1721976T3 (da) | 2010-01-04 |
US20080014608A1 (en) | 2008-01-17 |
EP1721976A4 (en) | 2007-08-15 |
DE602005017469D1 (de) | 2009-12-17 |
EP1721976A1 (en) | 2006-11-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1311075C (zh) | 表达超热稳定蛋白的*** | |
CN1115413C (zh) | 合成前导肽序列 | |
CN1513057A (zh) | 宿主微生物 | |
CN1108383C (zh) | 提高了耐热性的脱氨基甲酰酶及其制造方法 | |
CN1163611C (zh) | 在棒状细菌中可自主复制的质粒 | |
CN1151252C (zh) | 生产微生物转谷氨酰胺酶的方法 | |
CN1908174A (zh) | 生产l-赖氨酸的方法 | |
CN1890367A (zh) | 耐表面活性剂的纤维素酶及其修饰方法 | |
CN1492043A (zh) | 新型腈水合酶 | |
CN1875106A (zh) | 重组微生物 | |
CN1192037C (zh) | 用于由大肠杆菌向培养基中分泌来制备Leu-水蛭素的信号序列 | |
CN1160465C (zh) | 其中areA、pepC和/或pepE基因已被灭活的真菌 | |
CN1502689A (zh) | 生产l-谷氨酰胺的方法和生产l-谷氨酰胺的细菌 | |
CN1930289A (zh) | 变异芽孢杆菌属细菌 | |
CN1153831C (zh) | 编码神经酰胺糖内切酶激活物的基因 | |
CN1966678A (zh) | 突变的木糖异构酶及其基因和用途 | |
CN1150323C (zh) | 具有碱性支链淀粉酶和碱性α-淀粉酶两种活性的酶的基因 | |
CN1183248C (zh) | 环状缩肽合成酶及其基因、以及环状缩肽的大规模生产*** | |
CN1610740A (zh) | 具有改变产物产量特性的真细菌rna聚合酶突变体 | |
CN1160459C (zh) | 乳发酵短杆菌细胞色素bd型醌醇氧化酶基因 | |
CN1875095A (zh) | 重组微生物 | |
CN1930291A (zh) | 经过修饰的启动子 | |
CN1894404A (zh) | 有机酸存在下的启动子及其用途 | |
CN1756835A (zh) | 用作选择标记的转位酶 | |
CN1950505A (zh) | 生产多肽的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C12 | Rejection of a patent application after its publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20070314 |