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1. 发明申请

US20140024097A1 YEAST MUTANT OF KLUYVEROMYCES AND METHOD FOR ETHANOL PRODUCTION USING THE SAME 有权
标题翻译：使用该方法生产乙醇的YEAST突变体和乙醇生产方法
公开(公告)号：US20140024097A1
公开(公告)日：2014-01-23
申请号：US14008446
申请日：2012-03-26
申请人： Noriko Shisa , Rinji Akada , Hisashi Hoshida , Takeshi Uemura , Kozue Mutaguchi , Kenro Tokuhiro , Satoshi Katahira
发明人： Noriko Shisa , Rinji Akada , Hisashi Hoshida , Takeshi Uemura , Kozue Mutaguchi , Kenro Tokuhiro , Satoshi Katahira
IPC分类号： C12P7/06 , C12N15/81
CPC分类号： C12P7/06 , C12N9/0006 , C12N15/815 , C12P7/10 , Y02E50/16 , Y02E50/17
摘要： A yeast of the genus Kluyveromyces is modified to improve the ethanol yield from xylose by attenuation of at least one gene selected from the group consisting of the ADH1 gene derived from Kluyveromyces marxianus, a gene functionally equivalent to the ADH1 gene, the ADH4 gene derived from Kluyveromyces marxianus, and a gene functionally equivalent to the ADH4 gene.
摘要翻译：修饰了克鲁维酵母属的酵母以通过减少至少一种选自以下的基因中的至少一种基因来改善来自木糖的乙醇产量，所述基因选自由马克斯克鲁维酵母（Kluyveromyces marxianus）产生的ADH1基因，与ADH1基因功能相同的基因，马克斯克鲁维酵母，和功能上等同于ADH4基因的基因。

2. 发明授权

US09273328B2 Yeast mutant of kluyveromyces and method for ethanol production using the same 有权
标题翻译：克鲁维酵母的酵母突变体和使用其的乙醇生产方法
公开(公告)号：US09273328B2
公开(公告)日：2016-03-01
申请号：US14008446
申请日：2012-03-26
申请人： Noriko Shisa , Rinji Akada , Hisashi Hoshida , Takeshi Uemura , Kozue Mutaguchi , Kenro Tokuhiro , Satoshi Katahira
发明人： Noriko Shisa , Rinji Akada , Hisashi Hoshida , Takeshi Uemura , Kozue Mutaguchi , Kenro Tokuhiro , Satoshi Katahira
IPC分类号： C12P7/06 , C12N15/81 , C12N9/04 , C12P7/10
CPC分类号： C12P7/06 , C12N9/0006 , C12N15/815 , C12P7/10 , Y02E50/16 , Y02E50/17
摘要： A yeast of the genus Kluyveromyces is modified to improve the ethanol yield from xylose by attenuation of at least one gene selected from the group consisting of the ADH1 gene derived from Kluyveromyces marxianus, a gene functionally equivalent to the ADH1 gene, the ADH4 gene derived from Kluyveromyces marxianus, and a gene functionally equivalent to the ADH4 gene.
摘要翻译：修饰了克鲁维酵母属的酵母以通过减少至少一种选自以下的基因中的至少一种基因来改善来自木糖的乙醇产量，所述基因选自由马克斯克鲁维酵母（Kluyveromyces marxianus）产生的ADH1基因，与ADH1基因功能相同的基因，马克斯克鲁维酵母，和功能上等同于ADH4基因的基因。

3. 发明授权

US09796973B2 Terminator sequence-containing reverse primer for overexpression and linear DNA 有权
公开(公告)号：US09796973B2
公开(公告)日：2017-10-24
申请号：US14113674
申请日：2012-04-27
申请人： Rinji Akada , Hisashi Hoshida , Mikiko Nakamura
发明人： Rinji Akada , Hisashi Hoshida , Mikiko Nakamura
IPC分类号： C12N15/113 , C12N15/64 , C12N15/10 , C12N15/85 , C12N15/11
CPC分类号： C12N15/113 , C12N15/1075 , C12N15/111 , C12N15/64 , C12N15/85 , C12N2310/531 , C12N2330/51
摘要： Plasmid vectors have been widely used as a carrier of a DNA sequence capable of expressing a target RNA in cells. However, construction of these plasmid vectors requires technical skill and time. Thus, a quicker and easier method is required therefor. To solve this problem, a method using a linear DNA that has been amplified by the PCR method is examined. However, this method is disadvantageous in that RNA expression in cells is extremely low. Under these circumstances, the present inventors attempted to develop an RNA expression method using a linear DNA which can be produced mainly by using the PCR method alone and which enables a high level of RNA expression. As the results of intensive studies on terminator sequences to be used in a linear DNA, the present inventors found a smallest unit of a terminator sequence enabling linear DNA expression equivalent to that when using a plasmid vector. A linear DNA including the aforesaid terminator sequence can be produced quickly and easily, and enables RNA expression at a higher level. The present invention has been completed based on these findings.

4. 发明授权

US09453233B2 Method for producing Kluyveromyces marxianus transformant 有权
标题翻译：生产马克斯克鲁维酵母转化株的方法
公开(公告)号：US09453233B2
公开(公告)日：2016-09-27
申请号：US13634891
申请日：2011-02-01
申请人： Rinji Akada , Hisashi Hoshida , Babiker Mohamed Ahmed Abdel-Banat , Jun Asakawa
发明人： Rinji Akada , Hisashi Hoshida , Babiker Mohamed Ahmed Abdel-Banat , Jun Asakawa
IPC分类号： C12N15/74 , C12N15/81 , C12N15/64 , C07K14/37 , C12N1/19
CPC分类号： C12N15/815 , C07K14/37 , C12N15/64
摘要： An object to be solved by the present invention is to provide, for example, a method for producing a Kluyveromyces marxianus transformant by a method for conveniently and efficiently connecting the ends of DNA fragments without using specific restriction enzymes or their recognition sequences, and a method for producing a useful substance using the transformant. The present inventors have found, as means to solve the object, a method comprising introducing two or more linear double-stranded DNA fragments free from a Kluyveromyces marxianus autonomously replicating sequence into Kluyveromyces marxianus and selecting a transformant comprising a desired DNA ligation product with marker gene expression by the desired DNA ligation product as an index, or comprising introducing a linear double-stranded DNA fragment comprising a Kluyveromyces marxianus autonomously replicating sequence, alone or in combination with one or more linear double-stranded DNA fragment(s) different therefrom into Kluyveromyces marxianus and selecting a transformant comprising a desired circular DNA ligation product with marker gene expression by the desired circular DNA ligation product as an index.
摘要翻译：本发明要解决的问题是提供例如通过方法有效连接DNA片段末端而不使用特异性限制酶或其识别序列的方法来生产马克斯克鲁维酵母转化体，以及方法用于使用转化体生产有用物质。作为解决目的的手段，本发明人已经发现，将不含马克斯克鲁维酵母自主复制序列的两条或更多条线性双链DNA片段引入马克斯克鲁维酵母（Kluyveromyces marxianus）并选择含有标记基因的所需DNA连接产物的转化体通过所需的DNA连接产物作为指标表达，或包括将包含马克斯克鲁维酵母自主复制序列的线性双链DNA片段单独或与其不同的一个或多个线性双链DNA片段组合引入克鲁维酵母并且通过所需的环状DNA连接产物作为指标选择包含具有标记基因表达的所需环状DNA连接产物的转化体。

5. 发明授权

US08846343B2 High-expression promoter derived from Kluyveromyces marxianus 有权
标题翻译：来源于马克斯克鲁维酵母的高表达启动子
公开(公告)号：US08846343B2
公开(公告)日：2014-09-30
申请号：US13577607
申请日：2011-02-07
申请人： Rinji Akada , Hisashi Hoshida , Masamitsu Ide
发明人： Rinji Akada , Hisashi Hoshida , Masamitsu Ide
IPC分类号： C07H21/04 , C12N15/11 , C12N15/81 , C12N15/113 , C12N9/12 , C12P21/02
CPC分类号： C12N15/113 , C12N9/1205 , C12N15/11 , C12N15/81 , C12N15/815 , C12P21/02 , C12Y207/01006
摘要： Provided is a novel high-expression promoter, namely a GAL1 promoter, derived from Kluyveromyces marxianus. Also provided are the following, characterized by the use of the provided high-expression promoter; a recombinant polynucleotide containing said high-expression promoter; a vector containing said recombinant polynucleotide; a transformant obtained by introducing said recombinant polynucleotide or vector into yeast; a method using said transformant for high expression of a target gene; and a method using said transformant to manufacture the gene product of a target gene.
摘要翻译：提供了一种新型的高表达启动子，即来源于马克斯克鲁维酵母的GAL1启动子。还提供以下特征，其特征在于使用所提供的高表达启动子; 含有所述高表达启动子的重组多核苷酸; 含有所述重组多核苷酸的载体; 通过将所述重组多核苷酸或载体引入酵母而获得的转化体; 使用所述转化体用于靶基因高表达的方法; 以及使用所述转化体制造靶基因的基因产物的方法。

6. 发明申请

US20130210107A1 HIGH-EXPRESSION PROMOTER DERIVED FROM KLUYVEROMYCES MARXIANUS 有权
标题翻译：从KLUYVEROMYCES MARXIANUS衍生的高表达促进剂
公开(公告)号：US20130210107A1
公开(公告)日：2013-08-15
申请号：US13577607
申请日：2011-02-07
申请人： Rinji Akada , Hisashi Hoshida , Masamitsu Ide
发明人： Rinji Akada , Hisashi Hoshida , Masamitsu Ide
IPC分类号： C12N15/113
CPC分类号： C12N15/113 , C12N9/1205 , C12N15/11 , C12N15/81 , C12N15/815 , C12P21/02 , C12Y207/01006
摘要： Provided is a novel high-expression promoter, namely a GAL1 promoter, derived from Kluyveromyces marxianus. Also provided are the following, characterized by the use of the provided high-expression promoter; a recombinant polynucleotide containing said high-expression promoter; a vector containing said recombinant polynucleotide; a transformant obtained by introducing said recombinant polynucleotide or vector into yeast; a method using said transformant for high expression of a target gene; and a method using said transformant to manufacture the gene product of a target gene.
摘要翻译：提供了一种新型的高表达启动子，即来源于马克斯克鲁维酵母的GAL1启动子。还提供以下特征，其特征在于使用所提供的高表达启动子; 含有所述高表达启动子的重组多核苷酸; 含有所述重组多核苷酸的载体; 通过将所述重组多核苷酸或载体引入酵母而获得的转化体; 使用所述转化体用于靶基因高表达的方法; 以及使用所述转化体制造靶基因的基因产物的方法。

7. 发明申请

US20130059389A1 Method for Producing Kluyveromyces Marxianus Transformant 有权
标题翻译：生产马克斯克鲁维酵母转化体的方法
公开(公告)号：US20130059389A1
公开(公告)日：2013-03-07
申请号：US13634891
申请日：2011-02-01
申请人： Rinji Akada , Hisashi Hoshida , Babiker Mohamed Ahmed Abdel-Banat , Jun Asakawa
发明人： Rinji Akada , Hisashi Hoshida , Babiker Mohamed Ahmed Abdel-Banat , Jun Asakawa
IPC分类号： C12N15/81 , C12N15/31
CPC分类号： C12N15/815 , C07K14/37 , C12N15/64
摘要： An object to be solved by the present invention is to provide, for example, a method for producing a Kluyveromyces marxianus transformant by a method for conveniently and efficiently connecting the ends of DNA fragments without using specific restriction enzymes or their recognition sequences, and a method for producing a useful substance using the transformant. The present inventors have found, as means to solve the object, a method comprising introducing two or more linear double-stranded DNA fragments free from a Kluyveromyces marxianus autonomously replicating sequence into Kluyveromyces marxianus and selecting a transformant comprising a desired DNA ligation product with marker gene expression by the desired DNA ligation product as an index, or comprising introducing a linear double-stranded DNA fragment comprising a Kluyveromyces marxianus autonomously replicating sequence, alone or in combination with one or more linear double-stranded DNA fragment(s) different therefrom into Kluyveromyces marxianus and selecting a transformant comprising a desired circular DNA ligation product with marker gene expression by the desired circular DNA ligation product as an index.
摘要翻译：本发明要解决的问题是提供例如通过方法有效连接DNA片段末端而不使用特异性限制酶或其识别序列的方法来生产马克斯克鲁维酵母转化体，以及方法用于使用转化体生产有用物质。作为解决目的的手段，本发明人已经发现，将不含马克斯克鲁维酵母自主复制序列的两条或更多条线性双链DNA片段引入马克斯克鲁维酵母（Kluyveromyces marxianus）并选择含有标记基因的所需DNA连接产物的转化体通过所需的DNA连接产物作为指标表达，或包括将包含马克斯克鲁维酵母自主复制序列的线性双链DNA片段单独或与其不同的一个或多个线性双链DNA片段组合引入克鲁维酵母并且通过所需的环状DNA连接产物作为指标选择包含具有标记基因表达的所需环状DNA连接产物的转化体。

8. 发明授权

US08198089B2 Flocculent yeast and method for production thereof 有权
标题翻译：絮状酵母及其生产方法
公开(公告)号：US08198089B2
公开(公告)日：2012-06-12
申请号：US12922602
申请日：2009-03-18
申请人： Rinji Akada , Sanom Nonklang , Hisashi Hoshida , Babiker Mohamed Ahmed Abdel-Banat
发明人： Rinji Akada , Sanom Nonklang , Hisashi Hoshida , Babiker Mohamed Ahmed Abdel-Banat
IPC分类号： C12N15/70 , C12N1/19 , C12N1/00
CPC分类号： C07K14/395 , C12N15/81 , C12N15/815 , C12P7/06 , Y02E50/17
摘要： It is to provide a novel Kluyveromyces marxianus transformant having thermotolerance and flocculation property, suitable for the industrial production of bioethanol, by introducing a foreign flocculation gene into Kluyveromyces marxianus, and an efficient method for producing the transformant. The present inventors focused on the flocculation gene FLO of Saccharomyces cerevisiae as a foreign gene to confer flocculation property to Kluyveromyces marxianus and produced a linear DNA fragment comprising a known expression promoter sequence and a FLO gene sequence derived from Saccharomyces cerevisiae. As a result of introducing this linear DNA fragment into Kluyveromyces marxianus, the present inventors have confirmed that Kluyveromyces marxianus transformant can be obtained efficiently, and that the flocculation property of the above transformant is unexpectedly and significantly enhanced. The present invention has been thus completed.
摘要翻译：通过将外源絮凝基因引入马克斯克鲁维酵母（Kluyveromyces marxianus）中，提供了具有耐热性和絮凝性的新型克鲁维酵母转化体，适用于生物乙醇的工业化生产，也是生产转化体的有效方法。本发明人着重于酿酒酵母的絮凝基因FLO作为外源基因以赋予马克斯克鲁维酵母的絮凝性，并产生包含已知表达启动子序列和源自酿酒酵母的FLO基因序列的线性DNA片段。作为将线性DNA片段引入马克斯克鲁维酵母的结果，本发明人已经证实，可以有效地获得马克斯克鲁维酵母转化株，并且意外地显着提高了上述转化体的絮凝性。从而完成了本发明。

9. 发明申请

US20110020937A1 FLOCCULENT YEAST AND METHOD FOR PRODUCTION THEREOF 有权
标题翻译：浮游植物及其生产方法
公开(公告)号：US20110020937A1
公开(公告)日：2011-01-27
申请号：US12922602
申请日：2009-03-18
申请人： Rinji Akada , Sanom Nonklang , Hisashi Hoshida , Babiker Mohamed Ahmed Abdel-Banat
发明人： Rinji Akada , Sanom Nonklang , Hisashi Hoshida , Babiker Mohamed Ahmed Abdel-Banat
IPC分类号： C12N15/74 , C12N1/19
CPC分类号： C07K14/395 , C12N15/81 , C12N15/815 , C12P7/06 , Y02E50/17
摘要： It is to provide a novel Kluyveromyces marxianus transformant having thermotolerance and flocculation property, suitable for the industrial production of bioethanol, by introducing a foreign flocculation gene into Kluyveromyces marxianus, and an efficient method for producing the transformant. The present inventors focused on the flocculation gene FLO of Saccharomyces cerevisiae as a foreign gene to confer flocculation property to Kluyveromyces marxianus and produced a linear DNA fragment comprising a known expression promoter sequence and a FLO gene sequence derived from Saccharomyces cerevisiae. As a result of introducing this linear DNA fragment into Kluyveromyces marxianus, the present inventors have confirmed that Kluyveromyces marxianus transformant can be obtained efficiently, and that the flocculation property of the above transformant is unexpectedly and significantly enhanced. The present invention has been thus completed.
摘要翻译：通过将外源絮凝基因引入马克斯克鲁维酵母（Kluyveromyces marxianus）中，提供了具有耐热性和絮凝性的新型克鲁维酵母转化体，适用于生物乙醇的工业化生产，是生产转化体的有效方法。本发明人着重于酿酒酵母的絮凝基因FLO作为外源基因以赋予马克斯克鲁维酵母的絮凝性，并产生包含已知表达启动子序列和源自酿酒酵母的FLO基因序列的线性DNA片段。作为将线性DNA片段引入马克斯克鲁维酵母的结果，本发明人已经证实，可以有效地获得马克斯克鲁维酵母转化株，并且意外地显着提高了上述转化体的絮凝性。从而完成了本发明。

10. 发明授权

US08633029B2 Agent for improving gene transfer efficiency to mammalian cells 有权
标题翻译：用于提高哺乳动物细胞基因转移效率的药剂
公开(公告)号：US08633029B2
公开(公告)日：2014-01-21
申请号：US13583847
申请日：2011-03-13
申请人： Rinji Akada , Mikiko Nakamura
发明人： Rinji Akada , Mikiko Nakamura
IPC分类号： C12N15/88 , C09K3/00
CPC分类号： C12N15/88
摘要： Provided is an efficiency improving agent for gene transfer to mammalian cells, a method for improving efficiency of gene transfer to mammalian cells, and a method for transforming mammalian cells. The method is characterized in that tRNA is used in combination with a lipofection reagent. Preferably, the agent may be used so that the tRNA concentration in a lipofection solution falls within the range of 3 to 50 μg/mL, and the concentration in a culture is approximately 1/10. More preferably, tRNA and PEG may be used in combination with a lipofection reagent. According to the present invention, gene transfer to mammalian cells with high efficiency can be achieved.
摘要翻译：提供了用于向哺乳动物细胞的基因转移的效率提高剂，提高哺乳动物细胞的基因转移效率的方法和转化哺乳动物细胞的方法。该方法的特征在于tRNA与脂转染试剂组合使用。优选地，可以使用试剂，使得脂转染溶液中的tRNA浓度在3至50ug / mL的范围内，培养物中的浓度约为1/10。更优选地，tRNA和PEG可以与脂转染试剂组合使用。根据本发明，可以实现以高效率转运到哺乳动物细胞的基因。

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