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1. 发明授权

US08093036B2 Methods for producing hyaluronan in a recombinant host cell 有权
公开(公告)号：US08093036B2
公开(公告)日：2012-01-10
申请号：US12891548
申请日：2010-09-27
申请人： Alan Sloma , Leslie Naggiar, legal representative , Regine Behr , William Widner , Maria Tang , David Sternberg , Linda Sternberg, legal representative , Stephen Brown
发明人： Alan Sloma , Regine Behr , William Widner , Maria Tang , David Sternberg , Stephen Brown
IPC分类号： C12N1/20 , C12N15/74 , C12N15/00 , C12P21/06 , C12P19/04 , C12P19/36 , C12P1/00 , C12N9/00 , C12N9/24 , C12N9/88 , C07H21/04
CPC分类号： C12P19/26 , C12N9/1051 , C12N15/52
摘要： The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase.

2. 发明授权

US08137951B2 Methods for producing hyaluronan in a recombinant host cell 有权
标题翻译：在重组宿主细胞中产生透明质酸的方法
公开(公告)号：US08137951B2
公开(公告)日：2012-03-20
申请号：US13084230
申请日：2011-04-11
申请人： Alan Sloma , Leslie Naggiar, legal representative , Regine Behr , William Widner , Maria Tang , David Sternberg , Linda Sternberg, legal representative , Stephen Brown
发明人： Alan Sloma , Regine Behr , William Widner , Maria Tang , David Sternberg , Stephen Brown
IPC分类号： C12N1/20 , C12N15/74 , C12N15/00 , C12P21/00 , C12P9/26 , C12P1/00 , C12N9/00 , C12N9/24 , C12N9/88 , C07H21/04
CPC分类号： C12P19/26 , C12N9/1051 , C12N15/52
摘要： The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase.
摘要翻译：本发明涉及透明质酸的制备方法，其包括：（a）在适于产生透明质酸的条件下培养芽孢杆菌属宿主细胞，其中所述芽孢杆菌宿主细胞包含可操作地连接的透明质酸合酶编码序列的核酸构建体涉及透明质酸合酶编码序列外源的启动子序列; 和（b）从培养基中回收透明质酸。本发明还涉及编码包含透明质酸合酶基因和UDP-葡萄糖-6-脱氢酶基因的透明质酸合酶操纵子的分离的核酸序列，以及任选地一种或多种选自UDP-葡萄糖焦磷酸化酶基因， UDP-N-乙酰葡糖胺焦磷酸化酶基因和葡萄糖-6-磷酸异构酶基因。本发明还涉及编码UDP-葡萄糖-6-脱氢酶，UDP-葡萄糖焦磷酸化酶和UDP-N-乙酰葡糖胺焦磷酸化酶的分离的核酸序列。

3. 发明申请

US20110189737A1 Methods for producing hyaluronan in a recombinant host cell 有权
公开(公告)号：US20110189737A1
公开(公告)日：2011-08-04
申请号：US13084230
申请日：2011-04-11
申请人： Alan Sloma , Regine Behr , William Widner , Maria Tang , David Sternberg , Stephen Brown , Leslie Naggiar , Linda Sternberg
发明人： Alan Sloma , Regine Behr , William Widner , Maria Tang , David Sternberg , Stephen Brown , Leslie Naggiar , Linda Sternberg
IPC分类号： C12P19/04
CPC分类号： C12P19/26 , C12N9/1051 , C12N15/52
摘要： The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase

4. 发明申请

US20110014662A1 Methods for producing hyaluronan in a recombinant host cell 有权
标题翻译：在重组宿主细胞中产生透明质酸的方法
公开(公告)号：US20110014662A1
公开(公告)日：2011-01-20
申请号：US12891548
申请日：2010-09-27
申请人： Alan Sloma , Leslie Naggiar , Regine Behr , William Widner , Maria Tang , David Sternberg , Linda Sternberg , Stephen Brown
发明人： Alan Sloma , Leslie Naggiar , Regine Behr , William Widner , Maria Tang , David Sternberg , Linda Sternberg , Stephen Brown
IPC分类号： C12P19/04
CPC分类号： C12P19/26 , C12N9/1051 , C12N15/52
摘要： The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase
摘要翻译：本发明涉及透明质酸的制备方法，其包括：（a）在适于产生透明质酸的条件下培养芽孢杆菌属宿主细胞，其中所述芽孢杆菌宿主细胞包含可操作地连接的透明质酸合酶编码序列的核酸构建体涉及透明质酸合酶编码序列外源的启动子序列; 和（b）从培养基中回收透明质酸。本发明还涉及编码包含透明质酸合酶基因和UDP-葡萄糖-6-脱氢酶基因的透明质酸合酶操纵子的分离的核酸序列，以及任选的一种或多种选自UDP-葡萄糖焦磷酸化酶基因， UDP-N-乙酰葡糖胺焦磷酸化酶基因和葡萄糖-6-磷酸异构酶基因。本发明还涉及编码UDP-葡萄糖-6-脱氢酶，UDP-葡萄糖焦磷酸化酶和UDP-N-乙酰葡糖胺焦磷酸化酶的分离的核酸序列

5. 发明授权

US07811806B2 Methods for producing hyaluronan in a recombinant host cell 有权
标题翻译：在重组宿主细胞中产生透明质酸的方法
公开(公告)号：US07811806B2
公开(公告)日：2010-10-12
申请号：US10326185
申请日：2002-12-20
申请人： Alan Sloma , Regine Behr , William Widner , Maria Tang , David Sternberg , Stephen Brown
发明人： Alan Sloma , Regine Behr , William Widner , Maria Tang , David Sternberg , Stephen Brown
IPC分类号： C12N1/20 , C12N15/74 , C12N15/00 , C12P21/06 , C12P19/04 , C12P19/26
CPC分类号： C12P19/26 , C12N9/1051 , C12N15/52
摘要： The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase.
摘要翻译：本发明涉及透明质酸的制备方法，其包括：（a）在适于产生透明质酸的条件下培养芽孢杆菌属宿主细胞，其中所述芽孢杆菌宿主细胞包含可操作地连接的透明质酸合酶编码序列的核酸构建体涉及透明质酸合酶编码序列外源的启动子序列; 和（b）从培养基中回收透明质酸。本发明还涉及编码包含透明质酸合酶基因和UDP-葡萄糖-6-脱氢酶基因的透明质酸合酶操纵子的分离的核酸序列，以及任选地一种或多种选自UDP-葡萄糖焦磷酸化酶基因， UDP-N-乙酰葡糖胺焦磷酸化酶基因和葡萄糖-6-磷酸异构酶基因。本发明还涉及编码UDP-葡萄糖-6-脱氢酶，UDP-葡萄糖焦磷酸化酶和UDP-N-乙酰葡糖胺焦磷酸化酶的分离的核酸序列。

6. 发明申请

US20120149067A1 Methods for producing hyaluronan in a recombinant host cell 有权
标题翻译：在重组宿主细胞中产生透明质酸的方法
公开(公告)号：US20120149067A1
公开(公告)日：2012-06-14
申请号：US13401663
申请日：2012-02-21
申请人： Alan Sloma , Leslie Naggiar , Regine Behr , William Widner , Maria Tang , David Sternberg , Linda Sternberg , Stephen Brown
发明人： Alan Sloma , Leslie Naggiar , Regine Behr , William Widner , Maria Tang , David Sternberg , Linda Sternberg , Stephen Brown
IPC分类号： C12P19/28
CPC分类号： C12P19/26 , C12N9/1051 , C12N15/52
摘要： The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase
摘要翻译：本发明涉及透明质酸的制备方法，其包括：（a）在适于产生透明质酸的条件下培养芽孢杆菌属宿主细胞，其中所述芽孢杆菌宿主细胞包含可操作地连接的透明质酸合酶编码序列的核酸构建体涉及透明质酸合酶编码序列外源的启动子序列; 和（b）从培养基中回收透明质酸。本发明还涉及编码包含透明质酸合酶基因和UDP-葡萄糖-6-脱氢酶基因的透明质酸合酶操纵子的分离的核酸序列，以及任选地一种或多种选自UDP-葡萄糖焦磷酸化酶基因， UDP-N-乙酰葡糖胺焦磷酸化酶基因和葡萄糖-6-磷酸异构酶基因。本发明还涉及编码UDP-葡萄糖-6-脱氢酶，UDP-葡萄糖焦磷酸化酶和UDP-N-乙酰葡糖胺焦磷酸化酶的分离的核酸序列

7. 发明授权

US08574886B2 Methods for producing hyaluronan in a recombinant host cell 有权
标题翻译：在重组宿主细胞中产生透明质酸的方法
公开(公告)号：US08574886B2
公开(公告)日：2013-11-05
申请号：US13401663
申请日：2012-02-21
申请人： Regine Behr , William Widner , Maria Tang , Stephen Brown , Leslie Naggiar , Linda Sternberg
发明人： Alan Sloma , Regine Behr , William Widner , Maria Tang , David Sternberg , Stephen Brown
IPC分类号： C12N1/20 , C12N15/74 , C12N15/00 , C12P19/04 , C12P19/26 , C12P19/28 , C12P1/00 , C12P21/06 , C07H21/04
CPC分类号： C12P19/26 , C12N9/1051 , C12N15/52
摘要： The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase.
摘要翻译：本发明涉及透明质酸的制备方法，其包括：（a）在适于产生透明质酸的条件下培养芽孢杆菌属宿主细胞，其中所述芽孢杆菌宿主细胞包含可操作地连接的透明质酸合酶编码序列的核酸构建体涉及透明质酸合酶编码序列外源的启动子序列; 和（b）从培养基中回收透明质酸。本发明还涉及编码包含透明质酸合酶基因和UDP-葡萄糖-6-脱氢酶基因的透明质酸合酶操纵子的分离的核酸序列，以及任选地一种或多种选自UDP-葡萄糖焦磷酸化酶基因， UDP-N-乙酰葡糖胺焦磷酸化酶基因和葡萄糖-6-磷酸异构酶基因。本发明还涉及编码UDP-葡萄糖-6-脱氢酶，UDP-葡萄糖焦磷酸化酶和UDP-N-乙酰葡糖胺焦磷酸化酶的分离的核酸序列。

8. 发明申请

US20050221446A1 Methods for producing hyaluronic acid in a Bacillus cell 审中-公开
标题翻译：在芽孢杆菌细胞中生产透明质酸的方法
公开(公告)号：US20050221446A1
公开(公告)日：2005-10-06
申请号：US11096190
申请日：2005-03-31
申请人： William Widner , Alan Sloma , Michael Thomas , Maria Tang
发明人： William Widner , Alan Sloma , Michael Thomas , Maria Tang
IPC分类号： C12N1/21 , C12N9/28 , C12N15/52 , C12P19/26 , C12P19/28
CPC分类号： C12N9/2417 , C12N15/52 , C12P19/26
摘要： The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell in a medium conducive for the production of the hyaluronic acid, wherein the Bacillus cell comprises a nucleic acid construct comprising a triple promoter comprising a variant amyL promoter having a mutation corresponding to position 590 of SEQ ID NO: 1, a consensus promoter having the sequence TTGACA for the “−35” region and TATAAT for the “−10” region, and a cryIIIA promoter, in which each promoter sequence of the triple promoter is operably linked to one or more coding sequences involved in the biosynthesis of the hyaluronic acid; and (b) isolating the hyaluronic acid from the cultivation medium. The present invention also relates to Bacillus cells comprising a nucleic acid construct which comprises (i) a triple promoter comprising a variant amyL promoter having a mutation corresponding to position 590 of SEQ ID NO: 1, a consensus promoter having the sequence TTGACA for the “−35” region and TATAAT for the “−10” region, and a cryIIIA promoter, in which each promoter sequence of the triple promoter is operably linked to one or more coding sequences involved in the biosynthesis of the hyaluronic acid.
摘要翻译：本发明涉及生产透明质酸的方法，其包括：（a）在有利于产生透明质酸的培养基中培养芽孢杆菌宿主细胞，其中芽孢杆菌细胞包含包含三重启动子的核酸构建体，其包含变体具有对应于SEQ ID NO：1的位置590的突变的amyL启动子，具有“-35”区域的TTGACA序列和“-10”区域的TATAAT的共有启动子和cryIIIA启动子，其中每个启动子序列的三重启动子可操作地连接到参与透明质酸生物合成的一个或多个编码序列; 和（b）从培养基中分离透明质酸。本发明还涉及包含核酸构建体的芽孢杆菌细胞，其包含（i）包含具有对应于SEQ ID NO：1的位置590的突变的变体amyL启动子的三重启动子，具有SEQ ID NO：1的序列TTGACA的共有启动子， -35“区域和”-10“区域的TATAAT，以及cryIIIA启动子，其中三重启动子的每个启动子序列可操作地连接到参与透明质酸生物合成的一个或多个编码序列。

9. 发明授权

US06255076B1 Methods for producing a polypeptide in a Bacillus cell 有权
标题翻译：用于在芽孢杆菌细胞中产生多肽的方法
公开(公告)号：US06255076B1
公开(公告)日：2001-07-03
申请号：US09258377
申请日：1999-02-26
申请人： William Widner , Alan Sloma , Michael D. Thomas
发明人： William Widner , Alan Sloma , Michael D. Thomas
IPC分类号： C12P2106
CPC分类号： C12N15/75
摘要： The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a Bacillus host cell in a medium conducive for the production of the polypeptide, wherein the Bacillus cell comprises a nucleic acid construct comprising (i) a tandem promoter in which each promoter sequence of the tandem promoter is operably linked to a single copy of a nucleic acid sequence encoding the polypeptide and alternatively also (ii) an mRNA processing/stabilizing sequence located downstream of the tandem promoter and upstream of the nucleic acid sequence encoding the polypeptide; and (b) isolating the polypeptide from the cultivation medium. The present invention also relates to methods for producing a polypeptide, comprising: (a) cultivating a Bacillus host cell in a medium conducive for the production of the polypeptide, wherein the Bacillus cell comprises a nucleic acid construct comprising (i) a “consensus” promoter having the sequence TTGACA for the “−35” region and TATAAT for the “−10” region operably linked to a single copy of a nucleic acid sequence encoding the polypeptide and (ii) an mRNA processing/stabilizing sequence located downstream of the “consensus” promoter and upstream of the nucleic acid sequence encoding the polypeptide; and (b) isolating the polypeptide from the cultivation medium.
摘要翻译：本发明涉及产生多肽的方法，其包括：（a）在有助于产生多肽的培养基中培养芽孢杆菌宿主细胞，其中所述芽孢杆菌细胞包含核酸构建体，其包含（i）串联启动子，其中串联启动子的每个启动子序列可操作地连接到编码多肽的核酸序列的单拷贝，或者还有（ii）位于串联启动子下游的mRNA加工/稳定序列和编码多肽的核酸序列的上游 ; 和（b）从培养基中分离多肽。本发明还涉及产生多肽的方法，其包括：（a）在有助于产生多肽的培养基中培养芽孢杆菌宿主细胞，其中所述芽孢杆菌细胞包含核酸构建体，其包含（i）“共有” 具有“-35”区域的TTGACA序列的启动子和“-10”区域的TATAAT可操作地连接到编码多肽的核酸序列的单拷贝，和（ii）位于“ 共有“启动子和编码多肽的核酸序列的上游; 和（b）从培养基中分离多肽。

10. 发明授权

US5955310A Methods for producing a polypeptide in a bacillus cell 失效
标题翻译：在芽孢杆菌细胞中产生多肽的方法
公开(公告)号：US5955310A
公开(公告)日：1999-09-21
申请号：US31442
申请日：1998-02-26
申请人： William Widner , Alan Sloma , Michael D. Thomas
发明人： William Widner , Alan Sloma , Michael D. Thomas
IPC分类号： C12N15/09 , C07K14/325 , C12N1/21 , C12N9/54 , C12N15/32 , C12N15/75 , C12R1/07 , C12P21/06 , C12N1/20 , C12N9/00 , C12N9/88
CPC分类号： C12N15/75
摘要： The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a Bacillus host cell in a medium conducive for the production of the polypeptide, wherein the Bacillus cell comprises a nucleic acid construct comprising (i) a tandem promoter in which each promoter sequence of the tandem promoter is operably linked to a single copy of a nucleic acid sequence encoding the polypeptide and (ii) an mRNA processing/stabilizing sequence located downstream of the tandem promoter and upstream of the nucleic acid sequence encoding the polypeptide; and (b) isolating the polypeptide from the cultivation medium. The present invention also relates to Bacillus cells for producing a polypeptide comprising a nucleic acid construct which comprises (i) a tandem promoter in which each promoter sequence of the tandem promoter is operably linked to a single copy of a nucleic acid sequence encoding the polypeptide and (ii) an mRNA processing/stabilizing sequence located downstream of the tandem promoter and upstream of the nucleic acid sequence encoding the polypeptide.
摘要翻译：本发明涉及产生多肽的方法，其包括：（a）在有助于产生多肽的培养基中培养芽孢杆菌宿主细胞，其中所述芽孢杆菌细胞包含核酸构建体，其包含（i）串联启动子，其中串联启动子的每个启动子序列可操作地连接到编码多肽的核酸序列的单拷贝，和（ii）位于串联启动子下游的mRNA加工/稳定序列和编码多肽的核酸序列的上游; 和（b）从培养基中分离多肽。本发明还涉及用于产生包含核酸构建体的多肽的芽孢杆菌细胞，其包含（i）串联启动子，其中串联启动子的每个启动子序列可操作地连接到编码多肽的核酸序列的单拷贝，以及（ii）位于串联启动子下游并编码该多肽的核酸序列上游的mRNA加工/稳定序列。

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