公开(公告)号：US08148155B2

公开(公告)日：2012-04-03

申请号：US10420035

申请日：2003-04-21

申请人： Paul Harris ,  Howard Brody

发明人： Paul Harris ,  Howard Brody

IPC分类号： C12N15/00 ,  C12N1/15 ,  C12N15/64 ,  C12N15/80

CPC分类号： C12P21/00 ,  C12N15/80 ,  C12N15/902

摘要： The present invention relates to methods for increasing homologous recombination of a nucleic acid sequence introduced into a host cell, comprising: (a) introducing into a population of filamentous fungal host cells a first nucleic acid sequence encoding a recombination protein and a second nucleic acid sequence comprising one or more regions which are homologous with the genome of the filamentous fungal host cell, wherein (i) the recombination protein promotes the recombination of the one or more regions with the corresponding homologous region in the host's genome to incorporate the second nucleic acid sequence by homologous recombination, and (ii) the number of host cells comprising the incorporated second nucleic acid sequence in the population is increased at least 20% compared to the same population without the first nucleic acid sequence; (b) and isolating from the population a filamentous fungal cell comprising the incorporated second nucleic acid sequence.

摘要翻译： 本发明涉及用于增加引入宿主细胞的核酸序列的同源重组的方法，其包括：（a）向丝状真菌宿主细胞群体中引入编码重组蛋白和第二核酸序列的第一核酸序列 包括与丝状真菌宿主细胞的基因组同源的一个或多个区域，其中（i）重组蛋白促进一个或多个区域与宿主基因组中的相应同源区域的重组，以掺入第二核酸序列 通过同源重组，和（ii）与没有第一核酸序列的相同群体相比，包含所述群体中掺入的第二核酸序列的宿主细胞的数量增加至少20％; （b）并从群体中分离包含掺入的第二核酸序列的丝状真菌细胞。

公开(公告)号：US20130089894A1

公开(公告)日：2013-04-11

申请号：US13710007

申请日：2012-12-10

申请人： Paul Harris ,  Howard Brody

发明人： Paul Harris ,  Howard Brody

IPC分类号： C12N15/90

CPC分类号： C12P21/00 ,  C12N15/80 ,  C12N15/902

摘要： The present invention relates to methods for increasing homologous recombination of a nucleic acid sequence introduced into a host cell, comprising: (a) introducing into a population of filamentous fungal host cells a first nucleic acid sequence encoding a recombination protein and a second nucleic acid sequence comprising one or more regions which are homologous with the genome of the filamentous fungal host cell, wherein (i) the recombination protein promotes the recombination of the one or more regions with the corresponding homologous region in the host's genome to incorporate the second nucleic acid sequence by homologous recombination, and (ii) the number of host cells comprising the incorporated second nucleic acid sequence in the population is increased at least 20% compared to the same population without the first nucleic acid sequence; (b) and isolating from the population a filamentous fungal cell comprising the incorporated second nucleic acid sequence.

公开(公告)号：US08343767B2

公开(公告)日：2013-01-01

申请号：US13434694

申请日：2012-03-29

申请人： Paul Harris ,  Howard Brody

发明人： Paul Harris ,  Howard Brody

IPC分类号： C12N15/00 ,  C12N1/15 ,  C12N15/64 ,  C12N15/84

CPC分类号： C12P21/00 ,  C12N15/80 ,  C12N15/902

摘要： The present invention relates to methods for increasing homologous recombination of a nucleic acid sequence introduced into a host cell, comprising: (a) introducing into a population of filamentous fungal host cells a first nucleic acid sequence encoding a recombination protein and a second nucleic acid sequence comprising one or more regions which are homologous with the genome of the filamentous fungal host cell, wherein (i) the recombination protein promotes the recombination of the one or more regions with the corresponding homologous region in the host's genome to incorporate the second nucleic acid sequence by homologous recombination, and (ii) the number of host cells comprising the incorporated second nucleic acid sequence in the population is increased at least 20% compared to the same population without the first nucleic acid sequence; (b) and isolating from the population a filamentous fungal cell comprising the incorporated second nucleic acid sequence.

摘要翻译： 本发明涉及用于增加引入宿主细胞的核酸序列的同源重组的方法，其包括：（a）向丝状真菌宿主细胞群体中引入编码重组蛋白和第二核酸序列的第一核酸序列 包括与丝状真菌宿主细胞的基因组同源的一个或多个区域，其中（i）重组蛋白促进一个或多个区域与宿主基因组中的相应同源区域的重组，以掺入第二核酸序列 通过同源重组，和（ii）与没有第一核酸序列的相同群体相比，包含所述群体中掺入的第二核酸序列的宿主细胞的数量增加至少20％; （b）并从群体中分离包含掺入的第二核酸序列的丝状真菌细胞。

公开(公告)号：US20120231501A1

公开(公告)日：2012-09-13

申请号：US13434694

申请日：2012-03-29

申请人： Paul Harris ,  Howard Brody

发明人： Paul Harris ,  Howard Brody

IPC分类号： C12N15/80 ,  C12P21/02 ,  C12P21/00

CPC分类号： C12P21/00 ,  C12N15/80 ,  C12N15/902

摘要： The present invention relates to methods for increasing homologous recombination of a nucleic acid sequence introduced into a host cell, comprising: (a) introducing into a population of filamentous fungal host cells a first nucleic acid sequence encoding a recombination protein and a second nucleic acid sequence comprising one or more regions which are homologous with the genome of the filamentous fungal host cell, wherein (i) the recombination protein promotes the recombination of the one or more regions with the corresponding homologous region in the host's genome to incorporate the second nucleic acid sequence by homologous recombination, and (ii) the number of host cells comprising the incorporated second nucleic acid sequence in the population is increased at least 20% compared to the same population without the first nucleic acid sequence; (b) and isolating from the population a filamentous fungal cell comprising the incorporated second nucleic acid sequence.

摘要翻译： 本发明涉及用于增加引入宿主细胞的核酸序列的同源重组的方法，其包括：（a）向丝状真菌宿主细胞群体中引入编码重组蛋白和第二核酸序列的第一核酸序列 包括与丝状真菌宿主细胞的基因组同源的一个或多个区域，其中（i）重组蛋白促进一个或多个区域与宿主基因组中的相应同源区域的重组，以掺入第二核酸序列 通过同源重组，和（ii）与没有第一核酸序列的相同群体相比，包含所述群体中掺入的第二核酸序列的宿主细胞的数量增加至少20％; （b）并从群体中分离包含掺入的第二核酸序列的丝状真菌细胞。

公开(公告)号：US08497115B2

公开(公告)日：2013-07-30

申请号：US13350384

申请日：2012-01-13

申请人： Suchindra Maiyuran ,  Ana Fidantsef ,  Howard Brody

发明人： Suchindra Maiyuran ,  Ana Fidantsef ,  Howard Brody

IPC分类号： C12N1/15 ,  C12N15/00 ,  C07H21/04

CPC分类号： C12N15/81 ,  C07K2319/02 ,  C12N15/80 ,  C12P21/02

摘要： The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding the polypeptide, wherein the first nucleotide sequence is foreign to the second nucleotide sequence and the 3′ end of the first nucleotide sequence is immediately upstream of the initiator codon of the second nucleotide sequence. The present invention also relates to the isolated signal peptide sequences and to constructs, vectors, and fungal host cells comprising the signal peptide sequences operably linked to nucleotide sequences encoding polypeptides.

摘要翻译： 本发明涉及产生多肽的方法，包括：（a）在有助于产生多肽的培养基中培养真菌宿主细胞，其中真菌宿主细胞包含核酸构建体，其包含编码信号的第一核苷酸序列 肽，其与编码多肽的第二核苷酸序列可操作地连接，其中第一个核苷酸序列与第二个核苷酸序列是异源的，第一个核苷酸序列的3'末端紧邻第二个核苷酸序列的起始密码子的上游。 本发明还涉及分离的信号肽序列以及包含与编码多肽的核苷酸序列可操作地连接的信号肽序列的构建体，载体和真菌宿主细胞。

公开(公告)号：US06903193B1

公开(公告)日：2005-06-07

申请号：US09710760

申请日：2000-11-10

申请人： John C. Royer ,  Lynne M. Christianson ,  Gregory A. Gambetta ,  Howard Brody ,  Suzanne M. Otani ,  Wendy T. Yoder

发明人： John C. Royer ,  Lynne M. Christianson ,  Gregory A. Gambetta ,  Howard Brody ,  Suzanne M. Otani ,  Wendy T. Yoder

IPC分类号： C12N9/02 ,  C12N9/06 ,  C12N9/24 ,  C12N9/88 ,  C12N15/80 ,  A23J35/78 ,  C07K1/00 ,  C12N1/14 ,  C12N9/00 ,  C12N15/00

CPC分类号： C12N9/0036 ,  C12N9/0044 ,  C12N9/2482 ,  C12N9/88 ,  C12N15/80 ,  C12Y302/01008 ,  C12Y402/03006

摘要： The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a mutant of a parent filamentous fungal cell under conditions conducive for the production of the polypeptide, wherein (i) the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a second nucleic acid sequence comprising a modification of at least one of the genes involved in the production of a trichothecene and (ii) the mutant produces less trichothecene than the parent filamentous fungal cell when cultured under the same conditions; and (b) isolating the polypeptide from the cultivation medium. The present invention also relates to mutants of filamentous fungal cells and methods for obtaining the mutant cells, isolated trichodiene synthases and nucleic acid sequences encoding the trichodiene synthases.

摘要翻译： 本发明涉及产生多肽的方法，包括：（a）在有利于产生多肽的条件下培养亲本丝状真菌细胞的突变体，其中（i）突变细胞包含编码多肽的第一核酸序列 多肽和第二核酸序列，其包含参与单端孢霉烯生产的至少一个基因的修饰，和（ii）当在相同条件下培养时，所述突变体产生比亲本丝状真菌细胞少的单端孢霉素; 和（b）从培养基中分离多肽。 本发明还涉及丝状真菌细胞的突变体以及获得突变细胞，分离的二恶烯合酶和编码二氢合酶的核酸序列的方法。

公开(公告)号：US06180366B2

公开(公告)日：2001-01-30

申请号：US09316080

申请日：1999-05-20

申请人： John C. Royer ,  Lynne M. Christianson ,  Gregory A. Gambetta ,  Howard Brody ,  Suzanne M. Otani ,  Wendy T. Yoder

发明人： John C. Royer ,  Lynne M. Christianson ,  Gregory A. Gambetta ,  Howard Brody ,  Suzanne M. Otani ,  Wendy T. Yoder

IPC分类号： C12P2106

CPC分类号： C12N9/0036 ,  C12N9/0044 ,  C12N9/2482 ,  C12N9/88 ,  C12N15/80 ,  C12Y302/01008 ,  C12Y402/03006

摘要： The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a mutant of a parent filamentous fungal cell under conditions conducive for the production of the polypeptide, wherein (i) the mutant cell comprises a first nucleic acid sequence encoding the polypeptide and a second nucleic acid sequence comprising a modification of at least one of the genes involved in the production of a trichothecene and (ii) the mutant produces less of the trichothecene than the parent filamentous fungal cell when cultured under the same conditions; and (b) isolating the polypeptide from the cultivation medium. The present invention also relates to mutants of filamentous fungal cells and methods for obtaining the mutant cells. The present invention also relates to isolated trichodiene synthases and isolated nucleic acid sequences encoding the trichodiene synthases. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the trichodiene synthases. The present invention further relates to mutants cells comprising a marker-free modification of a gene, and methods for obtaining and using such mutant cells.

摘要翻译： 本发明涉及产生多肽的方法，包括：（a）在有利于产生多肽的条件下培养亲本丝状真菌细胞的突变体，其中（i）突变细胞包含编码多肽的第一核酸序列 多肽和第二核酸序列，其包含参与单端孢霉烯生产的至少一个基因的修饰;和（ii）当在相同条件下培养时，所述突变体产生比亲本丝状真菌细胞少的单端孢霉烯; 和（b）从培养基中分离多肽。 本发明还涉及丝状真菌细胞的突变体和获得突变细胞的方法。 本发明还涉及分离的二恶烯合成酶和分离的编码二萜烯合酶的核酸序列。 本发明还涉及包含核酸序列的核酸构建体，载体和宿主细胞以及产生二氢合酶合酶的方法。 本发明还涉及包含基因的无标记物修饰的突变体细胞，以及获得和使用这种突变细胞的方法。

公开(公告)号：US20120122191A1

公开(公告)日：2012-05-17

申请号：US13350384

申请日：2012-01-13

申请人： Suchindra Maiyuran ,  Ana Fidantsef ,  Howard Brody

发明人： Suchindra Maiyuran ,  Ana Fidantsef ,  Howard Brody

IPC分类号： C12N1/19 ,  C12N15/63 ,  C07H21/04 ,  C12N1/15

CPC分类号： C12N15/81 ,  C07K2319/02 ,  C12N15/80 ,  C12P21/02

摘要： The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding the polypeptide, wherein the first nucleotide sequence is foreign to the second nucleotide sequence and the 3′ end of the first nucleotide sequence is immediately upstream of the initiator codon of the second nucleotide sequence. The present invention also relates to the isolated signal peptide sequences and to constructs, vectors, and fungal host cells comprising the signal peptide sequences operably linked to nucleotide sequences encoding polypeptides.

摘要翻译： 本发明涉及产生多肽的方法，包括：（a）在有助于产生多肽的培养基中培养真菌宿主细胞，其中真菌宿主细胞包含核酸构建体，其包含编码信号的第一核苷酸序列 肽，其与编码多肽的第二核苷酸序列可操作地连接，其中第一个核苷酸序列与第二个核苷酸序列是异源的，第一个核苷酸序列的3'末端紧邻第二个核苷酸序列的起始密码子的上游。 本发明还涉及分离的信号肽序列以及包含与编码多肽的核苷酸序列可操作地连接的信号肽序列的构建体，载体和真菌宿主细胞。

公开(公告)号：US07771971B2

公开(公告)日：2010-08-10

申请号：US11837279

申请日：2007-08-10

申请人： Mariah Connelly ,  Howard Brody

发明人： Mariah Connelly ,  Howard Brody

IPC分类号： C12P21/04

CPC分类号： C07K14/37 ,  C07K14/38 ,  C07K14/40 ,  C12N9/00 ,  C12N15/52 ,  C12P1/02 ,  C12P21/02

摘要： The present invention relates to methods of producing a heterologous biological substance, comprising: (a) cultivating a mutant of a parent Aspergillus niger strain in a medium suitable for the production of the heterologous biological substance, wherein (i) the mutant strain comprises a first nucleotide sequence encoding the heterologous biological substance and one or more second nucleotide sequences comprising a modification of glaA and at least one of the genes selected from the group consisting of asa, amyA, amyB, prtT, and oah, and (ii) the mutant strain is deficient in the production of glucoamylase and at least one enzyme selected from the group consisting of acid stable alpha-amylase, neutral alpha-amylase A, and neutral alpha-amylase B, protease, and oxalic acid hydrolase compared to the parent Aspergillus niger strain when cultivated under identical conditions; and (b) recovering the heterologous biological substance from the cultivation medium. The present invention also relates to enzyme-deficient mutants of Aspergillus niger strains and methods for producing such mutants.

摘要翻译： 本发明涉及产生异源生物物质的方法，包括：（a）在适于生产异源生物物质的培养基中培养母本黑曲霉菌株的突变体，其中（i）突变株包含第一 编码异源生物物质的核苷酸序列和一个或多个第二核苷酸序列，其包含glaA的修饰和至少一种选自asa，amyA，amyB，prtT和oah的基因，以及（ii）突变株 与母本黑曲霉菌株相比，生产葡糖淀粉酶和至少一种选自酸稳定的α-淀粉酶，中性α-淀粉酶A和中性α-淀粉酶B，蛋白酶和草酸水解酶的酶不足 在相同的条件下耕种; 和（b）从培养基中回收异源生物物质。 本发明还涉及黑曲霉菌株的酶缺陷型突变体及其生产方法。

公开(公告)号：US07303877B2

公开(公告)日：2007-12-04

申请号：US10815495

申请日：2004-03-31

申请人： Mariah Connelly ,  Howard Brody

发明人： Mariah Connelly ,  Howard Brody

IPC分类号： C12Q1/68

CPC分类号： C07K14/37 ,  C07K14/38 ,  C07K14/40 ,  C12N9/00 ,  C12N15/52 ,  C12P1/02 ,  C12P21/02

摘要： The present invention relates to methods of producing a heterologous biological substance, comprising: (a) cultivating a mutant of a parent Aspergillus niger strain in a medium suitable for the production of the heterologous biological substance, wherein (i) the mutant strain comprises a first nucleotide sequence encoding the heterologous biological substance and one or more second nucleotide sequences comprising a modification of glaA and at least one of the genes selected from the group consisting of asa, amyA, amyB, prtT, and oah, and (ii) the mutant strain is deficient in the production of glucoamylase and at least one enzyme selected from the group consisting of acid stable alpha-amylase, neutral alpha-amylase A, and neutral alpha-amylase B, protease, and oxalic acid hydrolase compared to the parent Aspergillus niger strain when cultivated under identical conditions; and (b) recovering the heterologous biological substance from the cultivation medium. The present invention also relates to enzyme-deficient mutants of Aspergillus niger strains and methods for producing such mutants.

摘要翻译： 本发明涉及产生异源生物物质的方法，包括：（a）在适于生产异源生物物质的培养基中培养母本黑曲霉菌株的突变体，其中（i）突变株包含第一 编码异源生物物质的核苷酸序列和一个或多个第二核苷酸序列，其包含glaA的修饰和至少一种选自asa，amyA，amyB，prtT和oah的基因，以及（ii）突变株 与母本黑曲霉菌株相比，生产葡糖淀粉酶和至少一种选自酸稳定的α-淀粉酶，中性α-淀粉酶A和中性α-淀粉酶B，蛋白酶和草酸水解酶的酶不足 在相同的条件下耕种; 和（b）从培养基中回收异源生物物质。 本发明还涉及黑曲霉菌株的酶缺陷型突变体及其生产方法。