公开(公告)号：US20080311631A1

公开(公告)日：2008-12-18

申请号：US11628459

申请日：2005-06-02

申请人： Marcel Gerhardus Wubbolts ,  Roelof Ary Lans Bovenberg ,  Georg Sprenger ,  Johannes Josef Bongaerts ,  Stefan Kozak ,  Michael Muller ,  Volker Lorbach

发明人： Marcel Gerhardus Wubbolts ,  Roelof Ary Lans Bovenberg ,  Georg Sprenger ,  Johannes Josef Bongaerts ,  Stefan Kozak ,  Michael Muller ,  Volker Lorbach

IPC分类号： C12P13/22 ,  C12P13/00 ,  C12N15/63 ,  C12N1/20 ,  C07C61/22

CPC分类号： C12P13/04

摘要： The invention relates to a process for the biosynthetic production of 4-amino-4-deoxychorismate (ADC) performed fermentatively in vivo with a 4-amino-4-deoxychorismate synthase, preferably a PabAB bipartite protein (which may be a fusion protein), at an increased level of activity, thereby obtaining a broth comprising ADC and 4-amino-4-deoxyprephenate (ADP), that are recovered. The invention also relates to a further process of converting the ADP into p-aminophenylalanine. The invention, moreover relates to biosynthetic production of [3R,4R]-4-amino-3-hydroxycyclohexa-1,5-diene-1-carboxylic acid (3,4-CHA), by concerted action of such 4-amino-4-deoxychorismate synthase and of an enzyme capable of converting isochorismate into [5S,6S]-5,6 dihydroxycyclohexa-1,3-diene-1-carboxylic acid (2,3-CHD), preferably a phenazine biosynthesis protein PhzD, including recovery of 3,4-CHA. The invention also relates to expression vectors and host cells for use in any of such processes. The invention further relates to the use of 3,4-CHA as a catalytically active product, in particular as a chiral catalyst. And the invention finally relates to synthesis of oseltamivir phosphate from 3,4-CHA.

摘要翻译： 本发明涉及用4-氨基-4-脱氧激酶合酶，优选PabAB二分体蛋白（可以是融合蛋白）在体内发酵生物合成生产4-氨基-4-脱氧胸苷酸（ADC）的方法。 在增加的活性水平上，从而获得包含ADC和4-氨基-4-脱氧降钙素（ADP）的肉汤，其被回收。 本发明还涉及将ADP转化为对氨基苯丙氨酸的另一种方法。 本发明还涉及通过这种4-氨基-3-羟基环己基-1,5-二烯-1-羧酸（3,4-CHA）的共同作用生产合成[3R，4R] -4-氨基-3-羟基环己-1,5-二烯-1-羧酸（3,4-CHA） 能够将等分子酸转化为[5S，6S] -5,6二羟基环己-1,3-二烯-1-羧酸（2,3-CHD），优选吩嗪生物合成蛋白PhzD的酶的酶，包括4-脱氧胸苷酸合酶 回收3,4-CHA。 本发明还涉及用于任何这些方法的表达载体和宿主细胞。 本发明还涉及3,4-CHA作为催化活性产物，特别是作为手性催化剂的用途。 本发明最终涉及从3,4-CHA合成磷酸奥司他韦。

公开(公告)号：US20090209001A1

公开(公告)日：2009-08-20

申请号：US11628232

申请日：2005-06-02

申请人： Martin Schüermann ,  Marcel Gerhardus Wubbolts ,  Daniel Mink ,  Michael Wolberg ,  Johannes Helena Michael Mommers ,  Stefan Martin Jennewein

发明人： Martin Schüermann ,  Marcel Gerhardus Wubbolts ,  Daniel Mink ,  Michael Wolberg ,  Johannes Helena Michael Mommers ,  Stefan Martin Jennewein

IPC分类号： C12P21/00 ,  C12N9/88 ,  C40B30/00 ,  C07H21/00 ,  C12N15/63 ,  C12N1/00 ,  C12P19/02

CPC分类号： C12N9/88 ,  C12P19/02

摘要： The invention relates to isolated mutants of enzymes from the group of 2-deoxy-D-ribose 5-phosphate aldolase wild-type enzymes having a productivity factor (as determined by a specific test) which is at least 10% higher than the productivity factor for the corresponding wild-type enzyme from which it is a mutant. The mutants have at least one amino acid substitution at one or more of the positions corresponding to K13, T19, Y49, N80, D84, A93, E127, A128, K146, K160, I166, A174, M185, K196, F200, and S239 in Escherichia coli K12 (EC 4.1.2.4) wild-type enzyme sequence, and/or a deletion of at least one amino acid at the positions corresponding to S258 and Y259 therein, optionally combined with, specific, C-terminal extension and/or N terminal extension.The invention also relates to screening processes to find 2-deoxy-D-ribose 5-phosphate aldolase enzymes (either as such or as mutants) having a productivity factor (as determined by said specific test, which forms an essential part of the screening) which is at least 10% higher than the reference value.Moreover, the invention relates to mutant enzymes obtained by the screening process, and to nucleic acids encoding such mutants, and to vectors and host cells comprising, respectively, such nucleic acids or mutants.Finally the invention relates to the use of such (preferably mutant) enzymes, nucleic acids, vectors and host cells in the production of, for instance, 6-chloro-2,4,6-trideoxy-D-erythrohexapyranoside.

摘要翻译： 本发明涉及分离的2-脱氧-D-核糖5-磷酸醛缩酶野生型酶的突变体，其具有比生产率高至少高10％的生产率因子（通过特异性测试确定） 对于其为突变体的相应野生型酶。 突变体在对应于K13，T19，Y49，N80，D84，A93，E127，A128，K146，K160，I166，A174，M185，K196，F200和S239的一个或多个位置具有至少一个氨基酸取代 在大肠杆菌K12（EC 4.1.2.4）野生型酶序列中，和/或在其对应于S258和Y259的位置上的至少一个氨基酸的缺失，任选地与特异性C末端延伸和/或 N终端扩展。 本发明还涉及筛选方法以找到具有生产率因子的2-脱氧-D-核糖-5-磷酸醛缩酶（或者作为突变体）（通过所述具体试验确定，其形成筛选的重要部分） 其比参考值高至少10％。 此外，本发明涉及通过筛选方法获得的突变酶，以及编码这种突变体的核酸，以及分别含有这些核酸或突变体的载体和宿主细胞。 最后，本发明涉及这样的（优选突变的）酶，核酸，载体和宿主细胞在例如6-氯-2,4,6-三脱氧-D-赤藓酮糖苷的生产中的用途。