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1. 发明申请

WO1998010080A1 SALT-INDUCIBLE PROMOTER DERIVABLE FROM A LACTIC ACID BACTERIUM, AND ITS USE IN A LACTIC ACID BACTERIUM FOR PRODUCTION OF A DESIRED PROTEIN 审中-公开
标题翻译：从酸乳杆菌衍生的盐诱导促进剂及其用于生产所需蛋白质的酸性细菌
公开(公告)号：WO1998010080A1
公开(公告)日：1998-03-12
申请号：PCT/EP1997004755
申请日：1997-08-20
申请人： UNILEVER N.V. , UNILEVER PLC , SANDERS, Jan, Willem , KOK, Jan , VENEMA, Gerard , LEDEBOER, Adrianus, Marinus
发明人： UNILEVER N.V. , UNILEVER PLC
IPC分类号： C12N15/74
CPC分类号： C12N15/746 , C07K14/335
摘要： The invention provides a salt-inducible promoter present in SEQ ID NO: 10 and derivable from a lactic acid bacterium in isolation from the coding sequence normally controlled by said promoter in a wild-type lactic acid bacterium, with modifications and important parts thereof. Also provided are a recombinant vector and a transformed lactic acid bacterium comprising such promoter, and the production of a desired protein by such transformed bacterium, whereby the gene encoding said desired protein or a precursor thereof is expressed under control of such promoter. The desired protein can be secreted by the bacterium due to the presence of a signal sequence. The action of the salt-inducible promoter is enhanced at a pH of about 4-4.5 and/or by the presence of glutamic acid. Such process can be used in a fermentation process, in which the desired protein is a lytic protein causing lysis of the cells and release of the cell content. Or the desired protein can be an enzyme involved in flavour formation, e.g. in producing dressings, spreads, sausages and sour dough, or a protein functioning in a cheese production process, such as chymosin or a precursor thereof, or an enzyme involved in cheese flavour formation.
摘要翻译：本发明提供了存在于SEQ ID NO：10中的衍生自乳酸菌的盐诱导型启动子，其与野生型乳酸菌通常由所述启动子控制的编码序列分开，其修饰和重要部分。还提供了包含这种启动子的重组载体和转化的乳酸菌，并且通过这种转化细菌产生所需的蛋白质，由此编码所述所需蛋白质或其前体的基因在这种启动子的控制下表达。由于信号序列的存在，所需的蛋白质可被细菌分泌。在约4-4.5的pH和/或谷氨酸的存在下，盐诱导型启动子的作用增强。这种方法可以用于发酵过程，其中所需蛋白质是引起细胞裂解和释放细胞内容物的裂解蛋白。或者所需的蛋白质可以是涉及风味形成的酶，例如在制作敷料，酱油，香肠和酸面团中，或在奶酪生产过程中起作用的蛋白质，例如凝乳酶或其前体，或涉及奶酪香味形成的酶。

2. 发明申请

WO1994006917A1 PRODUCTION OF DESIRED PROTEINS OR POLYPEPTIDES BY CULTURING A TRANSFORMED LACTIC ACID BACTERIUM 审中-公开
标题翻译：通过培养转化的酸性细菌生产所需的蛋白质或多肽
公开(公告)号：WO1994006917A1
公开(公告)日：1994-03-31
申请号：PCT/EP1993002558
申请日：1993-09-20
申请人： UNILEVER PLC , UNILEVER N.V. , SEEGERS, Jozef, Franciscus, M., L. , KIEWIET, Rense , VENEMA, Gerard , BRON, Sierd
发明人： UNILEVER PLC , UNILEVER N.V.
IPC分类号： C12N15/74
CPC分类号： A23K10/18 , A23L29/065 , A23Y2220/00 , C07K14/315 , C12N15/52 , C12N15/746
摘要： The invention provides a food-grade recombinant plasmid comprising (1) a replicon and DNA sequences derived from a lactic acid bacterium (LAB), required for stable maintenance in a LAB, preferably derived from plasmid pWVO2 from (L. lactis) subsp. (cremoris) Wg2, and (2) a structural gene encoding a desired protein or polypeptide or its precursor, not being an antibiotic resistance selection marker, and optionally (3) a food-grade selection marker, whereby said plasmid has a theta-replication mechanism and is stably maintained in the transformed LAB under non-selective conditions. Also provided is a LAB transformed by said recombinant plasmid and the use of such transformed LAB in a process for producing said protein or polypeptide. Said recombinant plasmid can also be used for modifying the metabolic pathway of a LAB, e.g. for producing diacetyl or a precursor thereof by a LAB. Also claimed are food and animal feed products obtained by incorporating therein said protein or polypeptide or precursor thereof produced by said transformed LAB.
摘要翻译：本发明提供了一种食品级重组质粒，其包含（1）来自乳酸杆菌（LAB）的复制子和DNA序列，其需要在LAB中稳定维持，优选来源于来自（乳酸乳球菌）亚种的质粒pWVO2。（cremoris）Wg2，和（2）编码期望的蛋白质或多肽或其前体的结构基因，不是抗生素抗性选择标记，以及任选的（3）食品级选择标记，其中所述质粒具有θ复制机制，并在非选择性条件下稳定地保持在转化的LAB中。还提供了由所述重组质粒转化的LAB和在产生所述蛋白质或多肽的方法中使用这种转化的LAB。所述重组质粒也可用于修饰LAB的代谢途径，例如，用于通过LAB生产二乙酰基或其前体。还要求保护的是通过在其中掺入由所述转化的LAB产生的所述蛋白质或多肽或其前体获得的食物和动物饲料产品。

3. 发明申请

WO1995031563A1 COMPLEX INDUCIBLE PROMOTER SYSTEM DERIVABLE FROM A PHAGE OF A LACTIC ACID BACTERIUM (LAB), AND ITS USE IN A LAB FOR PRODUCTION OF A DESIRED PROTEIN 审中-公开
标题翻译：从酸乳杆菌（LAB）的噬菌体衍生的复合可诱导促进剂体系及其用于生产所需蛋白质的实验室
公开(公告)号：WO1995031563A1
公开(公告)日：1995-11-23
申请号：PCT/NL1995000172
申请日：1995-05-12
申请人： QUEST INTERNATIONAL B.V. , NAUTA, Arjen , VENEMA, Gerard , KOK, Jan , LEDEBOER, Aat, M.
发明人： QUEST INTERNATIONAL B.V.
IPC分类号： C12N15/74
CPC分类号： C12N15/746
摘要： The invention provides a complex inducible promoter system derivable from a phage of a lactic acid bacterium, especially one having the DNA sequence of SEQ. ID. NO: 3 given in Figure 2, or a DNA sequence essentially corresponding to those sequences, and a modification of (an essential part of) such promoter system in which the mitomycin C induction system is replaced by a food-grade system, e.g. a temperature-initiated induction system or a salt-initiated induction system. Also is provided a recombinant vector and a transformed lactic acid bacterium comprising (an essential part of) such promoter system. Further a process for producing a desired protein by such transformed bacterium is provided, comprising expressing a gene encoding said desired protein or a precursor thereof under control of such promoter system or an essential part thereof. Preferably the transformed lactic acid bacterium is made food-grade due to using food-grade DNA sequences and/or removing non-food-grade DNA sequences. When required, the desired protein can be secreted by the lactic acid bacterium if a DNA sequence fused to the gene encoding the desired protein is present which effects secretion of the desired protein or its precursor. The process can be used in a fermentation process, in which the desired protein causes lysis of the bacterial cells so that the contents of the cells can be released, or in which the desired protein is an enzyme involved in flavour formation, or in which the desired protein has a function in a cheese production process, such as chymosin or a precursor thereof, or an enzyme involved in flavour formation.
摘要翻译：本发明提供了可从乳酸菌噬菌体衍生的复合诱导型启动子系统，特别是具有SEQ ID NO： ID。或者与这些序列基本相对应的DNA序列，以及其中丝裂霉素C诱导系统被食品级系统替代的这种启动子系统的（主要部分）的修饰，例如，温度启动的诱导系统或盐引发的诱导系统。还提供了重组载体和包含这种启动子系统的（必要部分）的转化的乳酸菌。此外，提供了通过这种转化细菌产生所需蛋白质的方法，包括在该启动子系统或其主要部分的控制下表达编码所述期望蛋白质或其前体的基因。优选地，由于使用食品级DNA序列和/或去除非食品级DNA序列，转化的乳酸菌被制成食品级。当需要时，如果存在与编码所需蛋白质的基因融合的DNA序列，则所需的蛋白质可以由乳酸菌分泌，其影响所需蛋白质或其前体的分泌。该方法可用于发酵过程中，其中期望的蛋白质引起细菌细胞的裂解，使得细胞的内容物可以释放，或其中所需蛋白质是涉及风味形成的酶，或其中所需蛋白质在奶酪生产过程中具有功能，例如凝乳酶或其前体，或涉及风味形成的酶。

4. 发明申请

WO1995031561A1 PROCESS FOR THE LYSIS OF A CULTURE OF LACTIC ACID BACTERIA BY MEANS OF A LYSIN, AND USES OF THE RESULTING LYSED CULTURE 审中-公开
标题翻译：利用LYSIN手段研究乳酸菌培养物的方法，以及结果良好的文化利用
公开(公告)号：WO1995031561A1
公开(公告)日：1995-11-23
申请号：PCT/NL1995000170
申请日：1995-05-12
申请人： QUEST INTERNATIONAL B.V. , BUIST, Girbe , VENEMA, Gerard , KOK, Jan , LEDEBOER, Aat, M.
发明人： QUEST INTERNATIONAL B.V.
IPC分类号： C12N15/74
CPC分类号： C12N9/14 , A23C19/0323 , C12N15/746 , C12P21/06
摘要： The invention provides a process for the lysis of a culture of lactic acid bacteria, or a product containing such culture e.g. cheese, by means of a lysin through the in situ production of a homologous autolysin, or a heterologous autolysin obtainable from Gram-positive bacteria esp. from lactic acid bacteria. The gene encoding said autolysin is controlled by a promoter, preferably regulated by food-grade ingredients or parameters, to achieve an enhanced lysis after induction resulting in an enhanced production of total autolysin compared with the natural production level of the homologous autolysin during fermentation or shortly thereafter. Other uses of the invention include preparing a mixture of peptides which are modified by peptidases freed after the lysis, using the autolysin as a bactericidal agent against spoiling bacteria or pathogenic bacteria for improving the shelf life of a product containing the lysed culture.
摘要翻译：本发明提供了一种乳酸菌培养物的裂解方法，或含有这种培养物的产物。干酪，通过原位产生同源自溶素的溶素，或可从革兰氏阳性菌esp获得的异源自溶素。来自乳酸菌。编码所述自溶素的基因由促进剂控制，优选由食品级成分或参数调节，以在诱导后实现增强的裂解，从而与发酵期间的同源自溶胶的天然生产水平相比较，或者短期内其后。本发明的其它用途包括制备由裂解后释放的肽酶修饰的肽的混合物，使用自溶素作为防止破坏细菌或病原菌的杀菌剂，以改善含有裂解培养物的产品的保质期。

5. 发明申请

WO1995031562A1 PROCESS FOR INHIBITING THE GROWTH OF A CULTURE OF LACTIC ACID BACTERIA, AND OPTIONALLY LYSING THE BACTERIAL CELLS, AND USES OF THE RESULTING LYSED CULTURE 审中-公开
标题翻译：抑制酸性细菌培养物生长的方法，以及细菌感染细菌，以及结果良好的文化的使用
公开(公告)号：WO1995031562A1
公开(公告)日：1995-11-23
申请号：PCT/NL1995000171
申请日：1995-05-12
申请人： QUEST INTERNATIONAL B.V. , NAUTA, Arjen , VENEMA, Gerard , KOK, Jan , LEDEBOER, Aat, M.
发明人： QUEST INTERNATIONAL B.V.
IPC分类号： C12N15/74
CPC分类号： A23C19/0323 , A23C2220/202 , C07K14/315 , C12N9/14 , C12N15/746
摘要： The invention provides a process for inhibiting the growth of a culture of lactic acid bacteria, or a product containing such culture e.g. a cheese product, in which in the cells of the lactic acid bacteria a holin obtainable from bacteriophages of Gram-positive bacteria, esp. from bacteriophages of lactic acid bacteria is produced in situ, the gene encoding said holin being under control of a first regulatable promoter, said holin being capable of exerting a bacteriostatic effect on the cells in which it is produced by means of a system, whereby the cell membrane is perforated, while preferably the natural production of autolysin is not impaired. It is preferable that additionally a lysin obtainable from lactic acid bacteria or their bacteriophages is produced in situ in the cells of the lactic acid bacteria, the gene encoding said lysin being under control of a second regulatable promoter, whereby the produced lysin effects lysis of the cells of the lactic acid bacteria. The second regulatable promoter can be the same as the first regulatable promoter and the genes encoding the holin and the lysin, respectively can be placed under the same regulatable promoter in one operon. Preferably the promoters are regulatable by the food-grade ingredients or parameters. Other uses of the invention include preparing a mixture of peptides which are modified by peptidases freed after the lysis, using the lysed culture as a bactericidal agent against spoiling bacteria or pathogenic bacteria for improving the shelf life of a product containing the lysed culture.
摘要翻译：本发明提供一种抑制乳酸菌培养物生长的方法，或含有这种培养物的产品，例如，乳酪产品，其中在乳酸菌的细胞中可以从革兰氏阳性菌的噬菌体，特别是，从原位产生乳酸菌的噬菌体，编码所述的蛋白的基因在第一可调节启动子的控制之下，所述蛋白能够通过系统对其产生的细胞施加抑菌作用，细胞膜被穿孔，而优选自愈肌酐的自然产生不受损害。另外，由乳酸菌或其噬菌体可获得的赖氨酸在乳酸菌的细胞中原位产生，编码所述溶素的基因在第二可调节启动子的控制下，由此所产生的赖氨酸会影响乳酸菌细胞。第二个可调节的启动子可以与第一个可调节的启动子相同，编码霍宁和赖氨酸的基因分别可以放在一个操纵子的相同的可调节启动子之下。优选地，启动子可通过食品级成分或参数调节。本发明的其它用途包括制备由裂解后释放的肽酶修饰的肽混合物，使用裂解培养物作为抗破坏细菌或病原菌的杀菌剂，以改善含有裂解培养物的产品的保质期。

6. 发明申请

WO1985003945A1 RECOMBINANT PLASMIDS; BACTERIA CONTAINING SUCH RECOMBINANT PLASMIDS; PROCESS FOR PREPARING FERMENTED FOOD PRODUCTS, ANIMAL FEEDSTUFFS, INGREDIENTS THEREOF OR PROTEINS USING SUCH BACTERIA 审中-公开
标题翻译：重组PLASMIDS; 含有这种重组体的细菌; 制备发酵食品，动物饲料，其成分或使用这种细菌的蛋白质的方法
公开(公告)号：WO1985003945A1
公开(公告)日：1985-09-12
申请号：PCT/EP1985000077
申请日：1985-03-04
申请人： UNILEVER N.V. , KOK, Jan , MAAT, Jan , VAN DER VOSSEN, Josephus, Mauritius, Bernardus, ... , VENEMA, Gerard
发明人： UNILEVER N.V.
IPC分类号： C12N15/00
CPC分类号： C12N9/6481 , C07K2319/02 , C12N9/52 , C12N15/70 , C12N15/746 , C12N15/75
摘要： The cryptic Streptococcus cremoris Wg2 plasmid pWV01 (1.5 Md) was genetically marked with the chloramphenicol resistance (CmR) gene from pC194, the erythromycin resistance (EmR) gene from pE194 cop-6, the chloramphenicol acetyl transferase (CAT) gene, or combinations thereof to provide recombinant vector plasmids which can replicate in B. subtilis, E. coli and lactic streptococci. The CmR and EmR genes were expressed under regie of their own promoters, whereas the CAT gene was expressed under regie of the b. subtilis SPO2 promoter. Instead of antibiotic resistance markers, the use of auxotrophic markers, e.g. the genes for lactose metabolism, is envisaged. Moreover, other genes were inserted into these plasmids and expressed in Streptococcus lactis, namely the bovine prochymosin gene described in EP-PA 0 077 109 (A2) under regie of B. subtilis SPO2 promoter and the protease gene(s) from Streptococcus cremoris Wg2 plasmids pWV05. These and similar plasmids can be used to transform lactic acid bacteria to provide them with improved or new properties for the use in the fermentative preparation of food products, animal feedstuffs and ingredients thereof, as well as of proteins. A method for isolating other protease genes is provided which can be used instead of the pW05-derived protease gene(s).

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