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11. 发明申请

US20110014709A1 Method of Modifying Target Region in Host DNA and Selectable Marker Cassette 有权
标题翻译：修改宿主DNA和可选择标记盒中目标区域的方法
公开(公告)号：US20110014709A1
公开(公告)日：2011-01-20
申请号：US12933766
申请日：2009-03-23
申请人： Katsutoshi Ara , Takuya Morimoto , Naotake Ogasawara
发明人： Katsutoshi Ara , Takuya Morimoto , Naotake Ogasawara
IPC分类号： C12N15/87 , C12N15/63
CPC分类号： C12N15/1082 , C12N15/102
摘要： A method of modifying a target region in a host DNA using a donor DNA: wherein the donor DNA having regions homologous to a 5′-side region outside of the target region in the host DNA, a 3′-side region outside of the target region in the host DNA and a first homologous recombination region inside of the target region in the host DNA, respectively, in this order, and further having a first selectable marker gene, an expression-inducing promoter and a second selectable marker gene expressed under the control of the expression-inducing promoter between the region homologous to the 3′-side region and the region homologous to the first homologous recombination region; which method has the steps of: a first step of performing homologous recombination between the donor DNA and the host DNA at the regions of the 5′-side region and the first homologous recombination region, to conduct selection of a host integrated with the donor DNA based on expression of the first selectable marker gene; and a second step of performing homologous recombination, within the host DNA integrated with the donor DNA by the first step, between two regions of the 3′-side region derived from the host DNA and the 3′-side region derived from the donor DNA, to conduct selection of a host whose target region is modified based on expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter: and a selectable marker cassette for use in the method.
摘要翻译：使用供体DNA修饰宿主DNA中的靶区域的方法：其中具有与宿主DNA中的靶区域外侧的5'-侧区域同源的区域的供体DNA，靶标外的3'侧区域区域，宿主DNA中的靶区域内的第一同源重组区域，并且还具有第一选择标记基因，表达诱导启动子和第二选择标记基因控制与3'侧区域同源的区域与与第一同源重组区域同源的区域之间的表达诱导启动子; 该方法具有以下步骤：在5'-侧区域和第一同源重组区域的区域上进行供体DNA和宿主DNA之间的同源重组的第一步骤，以进行与供体DNA整合的宿主的选择基于第一选择标记基因的表达; 以及在源自宿主DNA的3'-侧区域的两个区域和源自供体DNA的3'侧区域之间，通过第1步在与供体DNA整合的宿主DNA内进行同源重组的第2步骤，用于在表达诱导启动子的表达诱导条件下，基于第二选择标记基因的表达来选择其目标区域被修饰的宿主;以及用于该方法的可选择标记盒。

12. 发明授权

US08852943B2 Method of modifying target region in host DNA and selectable marker cassette 有权
标题翻译：修改宿主DNA和选择性标记盒中靶区的方法
公开(公告)号：US08852943B2
公开(公告)日：2014-10-07
申请号：US12933766
申请日：2009-03-23
申请人： Katsutoshi Ara , Takuya Morimoto , Naotake Ogasawara
发明人： Katsutoshi Ara , Takuya Morimoto , Naotake Ogasawara
IPC分类号： C12N15/70 , C12N15/74 , C12N15/10 , C12N1/21 , C07H21/04
CPC分类号： C12N15/1082 , C12N15/102
摘要： A method of modifying a target region in a host DNA using a donor DNA: wherein the donor DNA having regions homologous to a 5′-side region outside of the target region in the host DNA, a 3′-side region outside of the target region in the host DNA and a first homologous recombination region inside of the target region in the host DNA, respectively, in this order, and further having a first selectable marker gene, an expression-inducing promoter and a second selectable marker gene expressed under the control of the expression-inducing promoter between the region homologous to the 3′-side region and the region homologous to the first homologous recombination region; which method has the steps of: a first step of performing homologous recombination between the donor DNA and the host DNA at the regions of the 5′-side region and the first homologous recombination region, to conduct selection of a host integrated with the donor DNA based on expression of the first selectable marker gene; and a second step of performing homologous recombination, within the host DNA integrated with the donor DNA by the first step, between two regions of the 3′-side region derived from the host DNA and the 3′-side region derived from the donor DNA, to conduct selection of a host whose target region is modified based on expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter; and a selectable marker cassette for use in the method.
摘要翻译：使用供体DNA修饰宿主DNA中的靶区域的方法：其中具有与宿主DNA中的靶区域外侧的5'-侧区域同源的区域的供体DNA，靶标外的3'侧区域区域，宿主DNA中的靶区域内的第一同源重组区域，并且还具有第一选择标记基因，表达诱导启动子和第二选择标记基因控制与3'侧区域同源的区域与与第一同源重组区域同源的区域之间的表达诱导启动子; 该方法具有以下步骤：在5'-侧区域和第一同源重组区域的区域上进行供体DNA和宿主DNA之间的同源重组的第一步骤，以进行与供体DNA整合的宿主的选择基于第一选择标记基因的表达; 以及在源自宿主DNA的3'-侧区域的两个区域和源自供体DNA的3'侧区域之间，通过第1步在与供体DNA整合的宿主DNA内进行同源重组的第2步骤基于表达诱导启动子的表达诱导条件进行基于第二选择标记基因的表达修饰其靶区的宿主的选择; 以及用于该方法的可选标记盒。

13. 发明申请

US20090221055A1 Novel Bacillus Subtilis Mutant Strain 有权
标题翻译：新型枯草芽孢杆菌突变株
公开(公告)号：US20090221055A1
公开(公告)日：2009-09-03
申请号：US12083539
申请日：2006-09-25
申请人： Ryosuke Kadoya , Keiji Endo , Masatoshi Tohata , Katsutoshi Ara , Naotake Ogasawara
发明人： Ryosuke Kadoya , Keiji Endo , Masatoshi Tohata , Katsutoshi Ara , Naotake Ogasawara
IPC分类号： C12N1/21
CPC分类号： C12N15/75 , C12N9/2417 , C12N9/54 , C12P21/02
摘要： Novel Bacillus subtilis mutant strains having good productivity of various enzymes are provided through extensive analysis of strains that are derived from Bacillus subtilis via gene disruption. The Bacillus subtilis mutant strains according to the present invention have genomic structures prepared by deletion of regions listed in the columns for deficient regions. Each of these Bacillus subtilis mutant strains exerts significantly improved secretory productivity of a protein when a gene encoding such a secretory target protein is introduced so that it can be expressed, compared with a case in which the same gene is introduced into a wild-type strain.
摘要翻译：通过对通过基因破坏衍生自枯草芽孢杆菌的菌株的广泛分析，提供具有各种酶的良好生产力的新型枯草芽孢杆菌突变菌株。根据本发明的枯草芽孢杆菌突变菌株具有通过缺失缺陷区列中列出的区域而制备的基因组结构。当与将相同基因导入野生型菌株的情况相比，当引入编码这种分泌型靶蛋白的基因以使其能够被表达时，这些枯草芽孢杆菌突变菌株中的每一种都能显着提高蛋白质的分泌生产力。

14. 发明申请

US20080009039A1 Recombinant Microorganism 审中-公开
标题翻译：重组微生物
公开(公告)号：US20080009039A1
公开(公告)日：2008-01-10
申请号：US10578613
申请日：2004-11-05
申请人： Masatoshi Tohata , Kazuhisa Sawada , Katsuya Ozaki , Kazuo Kobayashi , Naotake Ogasawara
发明人： Masatoshi Tohata , Kazuhisa Sawada , Katsuya Ozaki , Kazuo Kobayashi , Naotake Ogasawara
IPC分类号： C12P21/04 , C12N1/21
CPC分类号： C12N15/75 , C12P21/00
摘要： A recombinant microorganism obtained by transferring, into a host microorganism capable of producing protein or polypeptide with increased productivity, a gene encoding a protein or polypeptide, and a method for producing a protein or polypeptide by use of the recombinant microorganism. The recombinant microorganism is prepared by transferring, to a mutant strain of microorganism from which any of Bacillus subtilis genes comA, yopO, treR, yvbA, cspB, yvaN, yttP, yurK, yozA, licR, sigL, mntR, glcT, yvdE, ykvE, sir, rocR, ccpA, yaaT, yyaA, yycH, yacP, hprK, rsiX, yhdK, and yibO, or one or more genes functionally equivalent to any of these genes have been deleted or knocked out, a gene encoding a heterologous protein or polypeptide.
摘要翻译：通过将能够产生生产能力的蛋白质或多肽的宿主微生物，编码蛋白质或多肽的基因，以及通过使用重组微生物产生蛋白质或多肽的方法而获得的重组微生物。通过转移到枯草芽孢杆菌基因comA，yopO，treR，yvbA，cspB，yvaN，yttP，yurK，yozA，licR，sigL，mntR，glcT，yvdE，ykvE中的任一种的微生物突变菌株来制备重组微生物，先生，rocR，ccpA，yaaT，yyaA，yycH，yacP，hprK，rsiX，yhdK和yibO，或者与这些基因中的任何一个功能等同的一个或多个基因已被缺失或敲除，编码异源蛋白的基因或多肽。

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