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1. 发明申请

US20120107856A1 New Fungal Production System 有权
标题翻译：新型真菌生产系统
公开(公告)号：US20120107856A1
公开(公告)日：2012-05-03
申请号：US13138661
申请日：2010-03-16
申请人： Peter J. Punt , Richard Paul Burlingame , Christine M. Pynnonen , Phillip T. Olson , Jan Wery , Johannes Heinrich Visser , Mark A. Emalfarb , Jacob Visser , Jan Cormelis Verdoes , Jacoba Verdoes
发明人： Peter J. Punt , Richard Paul Burlingame , Christine M. Pynnonen , Phillip T. Olson , Jan Wery , Johannes Heinrich Visser , Mark A. Emalfarb , Jacob Visser , Jan Cormelis Verdoes , Jacoba Verdoes
IPC分类号： C12Q1/34 , C12N1/15 , C12N15/113 , C12N1/14 , C12P21/00
CPC分类号： C12N15/80 , C12N1/14 , C12N9/1205 , C12N9/2402 , C12N9/2428 , C12N9/2437 , C12N9/2442 , C12N9/248 , C12N15/01 , C12P21/02 , C12Q1/40 , C12R1/645 , C12Y302/01003 , C12Y302/01004 , C12Y302/01006 , C12Y302/01008 , C12Y302/01014 , C12Y302/01015 , C12Y302/01091 , C12Y304/21062 , C12Y304/23025 , G01N2333/93
摘要： The present invention provides a new fungal production system comprising a fungal host strain of Chrysosporium lucknowense wherein the endogenous cellulase secretion is less than 20% of the endogenous cellulase secretion of Chrysosporium lucknowense strain UV 18-25. Preferably, also the secretion of endogenous protease, endogenous β-glucanase and endogenous cellobiohydrolase is less than 20% of the secretion of Chrysosporium lucknowense strain UV 18-25. Furthermore, fungal host strains are provided wherein several genes have been disrupted. According to another aspect of the invention a method for homologous and/or heterologous production of a pure protein with a purity of higher than 75%, comprising expressing a gene encoding said protein in the strains according to the invention have been described. Furthermore, a method for production of artificial protein mixes comprising expressing a gene encoding each of said proteins in a strain according to the invention have been disclosed. Finally a method for simplified screening of strains functionally expressing a desired enzyme by application of said strains have been provided.
摘要翻译：本发明提供了一种新的真菌生产系统，其包含真菌宿主菌株Chrysosporium lucknowense，其中内源性纤维素酶分泌少于Chrysosporium lucknowense菌株UV18-25的内源性纤维素酶分泌的20％。内源性蛋白酶，内源性葡聚糖酶和内源纤维二糖水解酶的分泌优选小于Chrysosporium lucknowense菌株UV 18-25分泌的20％。此外，提供真菌宿主菌株，其中几个基因已被破坏。根据本发明的另一方面，已经描述了用于纯化高于75％的纯蛋白质的同源和/或异源产生方法，其包括在本发明的菌株中表达编码所述蛋白质的基因。此外，已经公开了生产人造蛋白质混合物的方法，其包括在根据本发明的菌株中表达编码每种所述蛋白质的基因。已经提供了通过应用所述菌株来功能性表达所需酶的菌株的简化筛选的方法。

2. 发明授权

US09175296B2 Fungal production system 有权
标题翻译：真菌生产系统
公开(公告)号：US09175296B2
公开(公告)日：2015-11-03
申请号：US13138661
申请日：2010-03-16
申请人： Peter J. Punt , Richard Paul Burlingame , Christine M. Pynnonen , Phillip T. Olson , Jan Wery , Johannes Heinrich Visser , Mark A. Emalfarb , Jacob Visser , Jan Cornelis Verdoes , Jacoba Verdoes
发明人： Peter J. Punt , Richard Paul Burlingame , Christine M. Pynnonen , Phillip T. Olson , Jan Wery , Johannes Heinrich Visser , Mark A. Emalfarb , Jacob Visser , Jan Cornelis Verdoes
IPC分类号： C12P21/00 , C12N15/80 , C12N1/14 , C12N9/12 , C12N9/34 , C12N15/01 , C12P21/02 , C12R1/645 , C12N9/42 , C12N9/24
CPC分类号： C12N15/80 , C12N1/14 , C12N9/1205 , C12N9/2402 , C12N9/2428 , C12N9/2437 , C12N9/2442 , C12N9/248 , C12N15/01 , C12P21/02 , C12Q1/40 , C12R1/645 , C12Y302/01003 , C12Y302/01004 , C12Y302/01006 , C12Y302/01008 , C12Y302/01014 , C12Y302/01015 , C12Y302/01091 , C12Y304/21062 , C12Y304/23025 , G01N2333/93
摘要： The present invention provides a new fungal production system comprising a fungal host strain of Chrysosporium lucknowense wherein the endogenous cellulase secretion is less than 20% of the endogenous cellulase secretion of Chrysosporium lucknowense strain UV 18-25. Preferably, also the secretion of endogenous protease, endogenous β-glucanase and endogenous cellobiohydrolase is less than 20% of the secretion of Chrysosporium lucknowense strain UV 18-25. Furthermore, fungal host strains are provided wherein several genes have been disrupted. According to another aspect of the invention a method for homologous and/or heterologous production of a pure protein with a purity of higher than 75%, comprising expressing a gene encoding said protein in the strains according to the invention have been described. Furthermore, a method for production of artificial protein mixes comprising expressing a gene encoding each of said proteins in a strain according to the invention have been disclosed. Finally a method for simplified screening of strains functionally expressing a desired enzyme by application of said strains have been provided.
摘要翻译：本发明提供了一种新的真菌生产系统，其包含真菌宿主菌株Chrysosporium lucknowense，其中内源性纤维素酶分泌少于Chrysosporium lucknowense菌株UV18-25的内源性纤维素酶分泌的20％。内源性蛋白酶，内源性葡聚糖酶和内源纤维二糖水解酶的分泌优选小于Chrysosporium lucknowense菌株UV 18-25分泌的20％。此外，提供真菌宿主菌株，其中几个基因已被破坏。根据本发明的另一方面，已经描述了用于纯化高于75％的纯蛋白质的同源和/或异源产生方法，其包括在本发明的菌株中表达编码所述蛋白质的基因。此外，已经公开了生产人造蛋白质混合物的方法，其包括在根据本发明的菌株中表达编码每种所述蛋白质的基因。已经提供了通过应用所述菌株来功能性表达所需酶的菌株的简化筛选的方法。

3. 发明授权

US07205123B2 Recombinant materials for carotenoid production 有权
标题翻译：用于类胡萝卜素生产的重组材料
公开(公告)号：US07205123B2
公开(公告)日：2007-04-17
申请号：US10001192
申请日：2001-10-30
申请人： Albert Johannes Joseph Van Ooijen , Jan Cornelis Verdoes , Jan Wery
发明人： Albert Johannes Joseph Van Ooijen , Jan Cornelis Verdoes , Jan Wery
IPC分类号： C12P23/00
CPC分类号： C12P23/00 , C07K14/39 , C12N9/00 , C12N9/0004 , C12N9/0008 , C12N9/1085 , C12N9/90 , C12N15/52 , C12N15/815 , C12Y102/01012
摘要： The present invention provides recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed, in operable linkage therewith, wherein the transcription promoter comprises a region found upstream of the open reading frame of a highly expressed Phaffia gene, preferably a glycolytic pathway gene, more preferably the gene coding for Glyceraldehyde-3-Phosphate Dehydrogenase. Further preferred recombinant DNAs according to the invention contain promoters of ribosomal protein encoding genes, more preferably wherein the transcription promoter comprises a region found upstream of the open reading frame encoding a protein as represented by one of the amino acid sequences depicted in any one of SEQIDNOs: 24 to 50. According to a further aspect of the invention an isolated DNA sequence coding for an enzyme involved in the carotenoid biosynthetic pathway of Phaffia rhodozyma is provided, preferably wherein said enzyme has an activity selected from isopentenyl pyrophosphate isomerase activity, geranylgeranyl pyrophosphate synthase activity, phytoene synthase activity, phytoene desaturase activity and lycopene cyclase activity, still more preferably those coding for an enzyme having an amino acid sequence selected from the one represented by SEQIDNO: 13, SEQIDNO: 15, SEQIDNO: 17, SEQIDNO: 19, SEQIDNO: 21 or SEQIDNO: 23. Further embodiments concern vectors, transformed host organisms, methods for making proteins and/or carotenoids, such as astaxanthin, and methods for isolating highly expressed promoters from Phaffia.
摘要翻译：本发明提供了包含转录启动子和待表达的下游序列的重组DNA，其可操作地连接于其中，其中转录启动子包含在高度表达的菲菲亚基因，优选糖酵解途径基因的开放阅读框的上游发现的区域，更优选为编码甘油醛-3-磷酸脱氢酶的基因。更优选的根据本发明的重组DNA包含核糖体蛋白质编码基因的启动子，更优选地，其中转录启动子包含编码如SEQ ID NO中任一个所示的氨基酸序列之一的开放阅读框上游的区域根据本发明的另一方面，提供了编码参与红发夫酵母的类胡萝卜素生物合成途径的酶的分离的DNA序列，优选其中所述酶具有选自异戊烯基焦磷酸异构酶活性，香叶基香叶基焦磷酸合酶活性，八氢番茄红素合成酶活性，八氢番茄红素去饱和酶活性和番茄红素环化酶活性，还更优选编码具有选自SEQ ID NO：13，SEQ ID NO：15，SEQ ID NO：17，SEQ ID NO：19，SEQ ID NO ：21或SEQ ID NO：23.其他实施方案涉及载体，转化宿主生物，用于制备蛋白质和/或类胡萝卜素的方法，例如虾青素，以及从Phaffia分离高表达启动子的方法。

4. 发明申请

US20070259409A1 Microbial Production of Aromatic Acids 审中-公开
标题翻译：微生物生产芳香酸
公开(公告)号：US20070259409A1
公开(公告)日：2007-11-08
申请号：US11578824
申请日：2004-12-23
申请人： Jan Wery
发明人： Jan Wery
IPC分类号： C12P7/40 , C12N1/00 , C12P7/42 , C12N15/01
CPC分类号： C12P7/42 , C12P7/40
摘要： The invention relates to the enzymatic production of aromatic acids using renewable carbon sources, such as sugars. Provided is a method for the microbial production of aromatic acids from a fermentable carbon substrate using a host cell capable of producing said aromatic acid, for instance cinnamic acid, para-hydroxycinnamic acid and para-hydroxybenzoic acid, and comprising an efflux pump for said aromatic acid. A preferred host cell comprises a member of the proton-dependent resistance/nodulation/cell division (RND) family of efflux pumps, preferably the solvent resistance pump srpABC of P. putida strain S12.
摘要翻译：本发明涉及使用可再生碳源（例如糖）的芳族酸的酶促生产。提供了使用能够产生所述芳族酸的宿主细胞从可发酵碳底物微生物生产芳族酸的方法，例如肉桂酸，对羟基肉桂酸和对羟基苯甲酸，并且包含用于所述芳族化合物的外排泵酸。优选的宿主细胞包括质子依赖性电阻/结瘤/细胞分裂（RND）家族外排泵的成员，优选恶臭假单胞菌菌株S12的耐溶剂泵srpABC。

5. 发明申请

US20090311760A1 HOST CELLS AND USES THEREOF IN THE MICROBIAL PRODUCTION OF HYDROXYLATED AROMATICS 审中-公开
标题翻译：主体细胞及其在羟化微生物微生物生产中的应用
公开(公告)号：US20090311760A1
公开(公告)日：2009-12-17
申请号：US12301105
申请日：2007-05-18
申请人： Jan Wery , Regina Gerda Maaike Westerhof , Karin Nijkamp
发明人： Jan Wery , Regina Gerda Maaike Westerhof , Karin Nijkamp
IPC分类号： C12P7/40 , C12P7/22 , C12N1/21
CPC分类号： C12P7/42 , C12N9/88 , C12P7/22
摘要： The invention relates to the field of the microbial production of substituted aromatics. In particular, it relates to the production of hydroxylated aromatics from renewable carbon stocks, like sugars or glycerol, via the metabolic intermediate L-tyrosine. Provided is a microbial host cell capable of producing at least one hydroxylated aromatic from a renewable carbon source, wherein at least one enzyme of said host cell that is involved in the degradation of said at least one hydroxylated aromatic is disabled and wherein the de novo synthesis of L-phenylalanine (L-Phe) in said host cell is impeded. Also provided is a method for the microbial production of at least one hydroxylated aromatic from a renewable carbon source, comprising culturing a host cell in the presence of exogenous L-Phe and a renewable carbon source and allowing said host cell to produce said at least one hydroxylated aromatic.
摘要翻译：本发明涉及取代芳烃的微生物生产领域。特别地，它涉及通过代谢中间体L-酪氨酸从可再生碳原料（例如糖或甘油）生产羟基化芳族化合物。提供了能够从可再生碳源产生至少一种羟基化芳族化合物的微生物宿主细胞，其中所述宿主细胞中涉及所述至少一种羟基化芳族化合物降解的至少一种酶被禁用，并且其中从头合成的所述宿主细胞中的L-苯丙氨酸（L-Phe）受阻。还提供了用于从可再生碳源微生物生产至少一种羟基化芳族化合物的方法，包括在外源L-Phe和可再生碳源的存在下培养宿主细胞，并允许所述宿主细胞产生所述至少一种羟基化芳香族。

6. 发明授权

US06329141B1 Methods for transforming Phaffia strains, transformed Phaffia strains so obtained and recombinant DNA in said methods 有权
标题翻译：用于在所述方法中转化Phaffia菌株，如此获得的转化的Phaffia菌株和重组DNA的方法
公开(公告)号：US06329141B1
公开(公告)日：2001-12-11
申请号：US09091725
申请日：1998-11-19
申请人： Albert Johannes Joseph Van Ooijen , Jan Cornelis Verdoes , Jan Wery
发明人： Albert Johannes Joseph Van Ooijen , Jan Cornelis Verdoes , Jan Wery
IPC分类号： C12Q168
CPC分类号： C12P23/00 , C07K14/39 , C12N9/00 , C12N9/0004 , C12N9/0008 , C12N9/1085 , C12N9/90 , C12N15/52 , C12N15/815 , C12Y102/01012
摘要： The present invention provides recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed, in operable linkage therewith, wherein the transcription promoter comprises a region found upstream of the open reading frame of a highly expressed Phaffia gene, preferably a glycolytic pathway gene, more preferably the gene coding for Glyceraldehyde-3-Phosphate Dehydrogenase. Further preferred recombinant DNAs according to the invention contain promoters of ribosomal protein encoding genes, more preferably wherein the transcription promoter comprises a region found upstream of the open reading frame encoding a protein as represented by one of the disclosed amino acid sequences. According to a further aspect of the invention an isolated DNA sequence coding for an enzyme involved in the carotenoid biosynthetic pathway of Phaffia rhodozyma is provided.
摘要翻译：本发明提供了包含转录启动子和待表达的下游序列的重组DNA，其可操作地连接于其中，其中转录启动子包含在高度表达的菲菲亚基因，优选糖酵解途径基因的开放阅读框的上游发现的区域，更优选为编码甘油醛-3-磷酸脱氢酶的基因。更优选的根据本发明的重组DNA包含核糖体蛋白质编码基因的启动子，更优选地，其中转录启动子包含编码由所公开的氨基酸序列之一表示的蛋白质的开放阅读框上游的区域。根据本发明的另一方面，提供了编码参与红发夫酵母的类胡萝卜素生物合成途径的酶的分离的DNA序列。

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