CN1751124A - CoQ10的微生物生产方法 - Google Patents
CoQ10的微生物生产方法 Download PDFInfo
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- CN1751124A CN1751124A CNA200480004590XA CN200480004590A CN1751124A CN 1751124 A CN1751124 A CN 1751124A CN A200480004590X A CNA200480004590X A CN A200480004590XA CN 200480004590 A CN200480004590 A CN 200480004590A CN 1751124 A CN1751124 A CN 1751124A
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Abstract
本发明涉及辅酶Q－10的合成中涉及到的酶，即癸异戊二烯基二磷酸酯(DPP)合酶和4－羟基苯甲酸聚异戊二烯基转移酶，涉及编码所述酶的经过分离的DNA，还涉及对辅酶Q－10进行微生物生产的方法。
Description
本发明涉及辅酶Q-10的合成中涉及到的酶，涉及编码所述酶的经过分离的DNA，还涉及对辅酶Q-10进行微生物生产的方法。
辅酶Q-10被发现于微生物、植物和动物中。已有确定并日益增加的证据表明，辅酶Q-10是关于人类健康和疾病的重要因子。可以通过化学合成或通过使用微生物发酵来对其进行生产。
癸异戊二烯基二磷酸酯(decaprenyl diphosphate，DPP)合酶和4-羟基苯甲酸聚异戊二烯基转移酶(4-hydroxybenzoate polyprenyltransferase)能催化辅酶Q-10(2，3-二甲氧基-二甲基-6-癸异戊二烯基-1，4-苯醌，也称作泛醌-10)生物合成中的步骤。DPP合酶通过连续的七步将异戊二烯二磷酸酯(IPP)分子加到异戊二烯类中间产物法呢基二磷酸酯(farnesyldiphosphate，FPP)上的步骤来形成DPP。DPP再与4-羟基苯甲酸酯结合；在大多数细菌中(如果不是全部细菌的话)，4-羟基苯甲酸酯来自分支酸(chorismate)。由4-羟基苯甲酸聚异戊二烯基转移酶催化的这种异戊二烯化反应，产生了3-癸异戊二烯基-4-羟基苯甲酸酯(3DP4HP)。3DP4HP的芳环再被进一步的修饰，形成还原型泛醌(ubiquinol)，其是泛醌的还原形式。
异戊二烯类中间产物IPP是通过多步催化途径从乙酰-CoA开始合成的。由phaA编码的乙酰-CoA乙酰基转移酶，催化了两个乙酰-CoA分子的缩合，形成乙酰乙酰-CoA(acetoacetyl-CoA)，这是IPP合成的第一步。乙酰乙酰-CoA还作为phaB基因产物的底物发挥作用，phaB基因产物是乙酰乙酰-CoA还原酶，其催化聚羟基烷酯(PHA)生物合成的第一个关键步骤。在很多细菌中，PHA生物合成中涉及的基因组合成操纵子。在Paracoccus denitrificans中，分别编码乙酰-CoA乙酰基转移酶和乙酰乙酰-CoA还原酶的phaA和phaB基因被串在一个操纵子上，而编码该途径最后一个酶(PHA合酶)的phaC基因，则不是该操纵子的一部分。
本发明涉及经过分离的DNA，其包含(1)编码癸异戊二烯基二磷酸酯(DPP)合酶的核苷酸序列和(2)编码4-羟基苯甲酸聚异戊二烯基转移酶的核苷酸序列，其中，编码DPP合酶的核苷酸序列选自由以下序列构成的组：
(a)SEQ ID NO：16所示的DNA序列或其互补链；
(b)一种DNA序列，其在标准条件下能与(a)中定义的DNA序列或其片段杂交，并编码具有DPP合酶活性的多肽；
(c)一种DNA序列，其与编码SEQ ID NO：17所示的多肽的DNA至少80％，优选至少90％相同，并且其编码具有DPP合酶活性的多肽；和
(d)一种DNA序列，其编码与SEQ ID NO：17所示的氨基酸序列至少80％相同的多肽，并编码具有DPP合酶活性的多肽；并且
其中，编码4-羟基苯甲酸聚异戊二烯基转移酶的核苷酸序列选自由以下序列构成的组：
(a’)SEQ ID NO：23所示的DNA序列或其互补链；
(b’)一种DNA序列，其在标准条件下能与(a’)中定义的DNA序列或其片段杂交，并编码具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽；
(c’)一种DNA序列，其与编码SEQ ID NO：24所示的多肽的DNA至少80％，优选至少90％相同，并且其编码具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽；和
(d’)一种DNA序列，其编码与SEQ ID NO：24所示的氨基酸序列至少80％相同的多肽，并编码具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽。
本文中使用的“标准条件”下的杂交指：在例如Sambrook et al.，Molecular Cloning：A Laboratory Manual，2d Edition，1989，和Shor Protocolsin Molecular Biology，ed.Ausubel，et al.中所述的严谨条件下的杂交，这两份出版物都通过引用的方式被包括进本文中。严谨条件是取决于序列的，在不同环境下有所不同。较长的序列在较高的温度下特异性杂交。通常，严谨条件被选择为较之对于特定序列在设定的离子强度和pH下的热熔解温度(thermal melting point，Tm)要低大约5-10℃的温度。Tm是：(在设定的离子强度、pH和核酸含量的情况下，)达到平衡时，与目标序列互补的序列中的50％与目标序列杂交的温度(目标序列过量存在时，Tm处，达到平衡时50％的探针处于被利用的状态)。严谨条件是以下这样的：盐浓度低于大约1.0M钠离子，典型地是大约0.01至1.0M的钠离子浓度(或其它盐)，pH7.0至8.3，对于短的杂交探针(例如，10至50个核苷酸)温度为至少大约30℃，对于长的杂交探针(例如，多于50个核苷酸)温度为至少大约60℃。严谨条件还可以通过加入去稳定剂例如甲酰胺来获得。就本发明公开的目的而言，用于此类杂交的合适的严谨条件是包括下述条件的，所述条件是：于37℃在40％甲酰胺、1M NaCl、1％十二烷基硫酸钠(SDS)的缓冲液中进行杂交，在至少大约50℃的温度(通常是约55℃至约60℃)下于0.2XSSC中至少进行一次20分钟的洗涤；或者是等同的条件。阳性杂交至少是背景的两倍。普通技术人员将很容易知道，其它替代的杂交和洗涤条件可被用于提供相似严谨度的条件。
本文中所用的术语“片段”指长度为大约10个或更多个核苷酸的核酸序列。
在本文中，如果按照下文所述以最大的对应情况进行比对时，两条序列的核苷酸序列或氨基酸(分别适用的情况下)残基相同，那么两条核酸序列或多肽就被称为是“相同的”。上下文中提到两条或多条核酸或多肽序列时，术语“相同的”或百分比的“相同性”，指：当使用本领域已知的序列比较算法或通过手工比对及视觉检查来进行测量，在比较窗口上按照最大的对应情况来进行比较和比对时，相同的或具有特定百分比的相同氨基酸残基或核苷酸的两条或多条序列或亚序列(subsequence)。当序列相同性的百分比被用于蛋白质或肽，应认识到：不相同的残基位置通常是由于保守氨基酸取代造成的，其中氨基酸残基被具有相似化学性质(例如，电荷或疏水性)的其它氨基酸残基所取代，因此不改变分子的功能属性。当序列因为保守取代而不同时，百分比的序列相同性可被调高，以针对取代的保守本质进行校正。做出此种调节的方法是本领域技术人员公知的。典型地，这包括对保守取代进行评分，将其作为部分而非全部的错配，因此提高百分比的序列相同性。因此，例如，当相同的氨基酸被给为1的分值，非保守取代被给为0的分值时，对保守取代给出0和1之间的分值。根据，例如Meyers & Miller，Computer Applic.Biol.Sci.4：11-17(1988)的算法，例如PC/GENE程序(Intelligenetics，Mountain View，Calif.，USA)所实现的，来计算对保守取代的赋分。在相同序列中间可能会有不相同核苷酸或氨基酸残基的缺口。如果核苷酸或氨基酸序列的不同导致了多结构域的酶的一个结构域的生物活性的降低或改变，而该酶的另一结构域的另一生物活性被保留下来，这样的核苷酸或氨基酸序列仍然是被包括在本发明范围之内的。通常，如果一种多肽与SEQ ID NO：17或SEQ IDNO：24的DPP合酶或4-羟基苯甲酸聚异戊二烯基转移酶的天然存在的氨基酸序列至少80％相同，这种多肽就被认为是在本发明的范围之内。如果一条核酸序列与SEQ ID NO：16或SEQ ID NO：23的编码DPP合酶或4-羟基苯甲酸聚异戊二烯基转移酶的天然存在的核酸序列至少80％，优选至少90％相同，那么，这条核酸序列就被认为是在本发明的范围之内。
此外，本发明涉及下述多肽：(1)具有氨基酸序列SEQ ID NO：17，或较之SEQ ID NO：17有一个或几个氨基酸缺失、添加或插入的氨基酸序列，并具有DPP合酶活性的多肽，和(2)具有氨基酸序列SEQ IDNO：24，或较之SEQ ID NO：24有一个或几个氨基酸缺失、添加或插入的氨基酸序列，并具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽。
本文中所用的术语“一个或几个氨基酸缺失、添加或插入”表示，各条多肽序列被一个或多个缺失、添加或插入所突变。在本申请的上下文中，术语“几个”指2或更多个。
本发明进一步地提供了包含本发明的新颖的核苷酸序列的构建体，还可以包含调控序列。
本文中所用的“构建体”是具有插入的或添加的额外DNA的DNA片断或序列，例如携带有DNA插入的表达载体或质粒。此类“额外DNA”是指并非在该位置天然存在的DNA，其通过本领域已知的克隆方法被插入或被添加。还包括在内的是通过本领域技术人员已知的标准DNA转化、转导或接合方法被插入到微生物基因组中的DNA序列。因此，在本发明上下文中所用的构建体包括本文公开的通过本领域已知的方法插入到质粒或表达载体或插入到微生物基因组中的DNA序列。合适的质粒或表达载体是本领域技术人员已知的。
术语“调控序列”包括控制或调节编码DNA转录的序列。此类序列可与编码DNA相邻或可定位于编码DNA的上游或下游。调控序列包括但不限于启动子、转录调节子、核糖体结合位点、终止子、mRNA稳定序列以及翻译或转录增强子元件。
在本发明的一种实施方式中，提供了包含上述特定DNA的微生物。因此，本发明提供了包含上述构建体的微生物。微生物优选是细菌或酵母，更优选是辅酶Q-10的天然生产者的细菌或酵母，例如Gluconobactersp.、Sphingomonas trueperi、Schizosaccharomyces pombe、Candida sp.、Pseudomonas sp.或Paracoccus denitrificans。最优选的是Paracoccuszeaxanthinifaciens或Rhodobacter sphaeroides的细菌。
本发明提供了在微生物中生产辅酶Q-10的方法，所述方法包括如下步骤：
(a)增加编码具有DPP合酶的蛋白质的基因和/或编码具有4-羟基苯甲酸聚异戊二烯基转移酶的蛋白质的基因的表达，以增加所述蛋白质的活性，以及
(b)在能生产辅酶Q-10的条件下，在培养基中培养微生物或宿主细胞。编码DPP合酶的基因已知为ddsA，编码4-羟基苯甲酸聚异戊二烯基转移酶的基因已知为ubiA。在一个方面，该方法还包括：通过对所述微生物的crtE基因进行突变，去掉香叶基香叶基二磷酸酯(geranyl geranyldiphosphate，GGPP)合酶的活性。
本发明的酶的活性可被下述方法增加，所述方法包括但不限于增加基因的拷贝数，使用更强的启动子，对mRNA进行稳定，增强翻译，使用较之野生型具有增加的活性和/或增加的稳定性和/或对抑制的抗性和/或对底物或产物抑制的抗性的酶的突变形式，或者本领域技术人员已知的任何其它方法。
用于生产辅酶Q-10的培养基和条件是技术人员已知的，在实施例1的培养基和条件部分中对此有详细描述。
术语“突变”或“对……进行突变”在本文中可与“修饰”互换使用，表示对天然核苷酸序列进行的导致蛋白质氨基酸序列的相应变化的改变。此种改变可导致例如蛋白质功能或活性上的改变。突变可通过若干方法来产生，所述方法包括一个或多个移码、取代、插入和/或缺失，包括无义突变[琥珀型(T/UAG)、赭石型(T/UAA)和乳白型(T/UGA)]。缺失可以是单个核苷酸或多个核苷酸的缺失，也包括整个基因的缺失。用于制造此类突变的方法包括但不限于：使用NTG(N-甲基-N’-硝基-N-亚硝基胍)或EMS(乙基磺酸甲烷)以及亚硝酸进行化学诱变，UV(紫外)照射，以及使用生物试剂例如转座子、插入元件或控制细胞突变速率的特定基因(例如，mutS和mutT)进行的诱变。突变可通过DNA重组方法来产生，其中使用了DNA片段的制剂，其中含有通过PCR制造出来的突变，通过本领域技术人员已知的标准的DNA转化、转导或接合方法，将此类含有突变的DNA片段引入到染色体中(天然的座或次级染色体位点)。
本发明的一种特定的实施方式提供了经过分离的DNA，其选自包含编码癸异戊二烯基二磷酸酯(DPP)合酶的核苷酸序列的DNA，其选自如下序列构成的组：
(a)SEQ ID NO：16所示的DNA序列或其互补链；
(b)一种DNA序列，其在标准条件下能与互补于(a)中定义的DNA序列的DNA序列或其片段杂交，并编码具有癸异戊二烯基二磷酸酯(DPP)合酶活性的多肽；
(c)一种DNA序列，其编码具有由(a)或(b)的DNA序列编码的氨基酸序列的多肽；
(d)一种DNA序列，其与编码包含SEQ ID NO：17的氨基酸序列的多肽的DNA至少80％相同，并且其编码具有癸异戊二烯基二磷酸酯(DPP)合酶活性的多肽；和
(e)一种DNA序列，其编码包含与SEQ ID NO：17的氨基酸序列至少80％相同的氨基酸序列的多肽，并编码具有癸异戊二烯基二磷酸酯(DPP)合酶活性的多肽；以及
包含编码4-羟基苯甲酸聚异戊二烯基转移酶的核苷酸序列的DNA，选自由如下序列构成的组：
(a’)SEQ ID NO：23所示的DNA序列或其互补链；
(b’)一种DNA序列，其在标准条件下能与互补于(a’)中定义的DNA序列的DNA序列或其片段杂交，并编码具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽；
(c’)一种DNA序列，其编码具有由(a’)或(b’)的DNA序列编码的氨基酸序列的多肽；
(d’)一种DNA序列，其与编码包含SEQ ID NO：24的氨基酸序列的多肽的DNA至少80％相同，并且其编码具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽；和
(e’)一种DNA序列，其编码包含与SEQ ID NO：24的氨基酸序列至少80％相同的氨基酸序列的多肽，并编码具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽。
在另一种实施方式中，本发明包括在微生物中生产辅酶Q-10的方法，所述方法包括：提高选自ddsA和ubiA的基因的表达，以增加选自DDP合酶和4-羟基苯甲酸聚异戊二烯基转移酶的酶的活性，以及在能生产辅酶Q-10的条件下，于培养基中培养细胞。
下述实施例被提供来对本发明进行进一步的阐述。这些实施例仅作阐述之用，而非欲以任何方式对本发明的范围进行限制。
实施例1 细菌菌株、微生物方法和分析工序
在细菌中，包括Paracoccus zeaxanthinifaciens中，对来自异戊二烯类中间产物FPP和IPP的玉米黄质的合成涉及五个酶反应步骤。首先，通过crtE编码的GGPP合酶形成香叶基香叶基二磷酸酯(GGPP)，然后在crtB编码的八氢番茄红素(phytoene)合酶的催化下，两个GGPP分子结合形成八氢番茄红素。下一个酶，crtI编码的番茄红素合酶通过开展四个去饱和步骤，将八氢番茄红素转化为番茄红素。由crtY编码的番茄红素环化酶将番茄红素转化为β-胡萝卜素，其再被由crtZ编码的β-胡萝卜素羟化酶羟化，得到玉米黄质。番茄红素、β-胡萝卜素和玉米黄质是红色或橙色的，而其它中间产物是无色的。
P.zeaxanthinifaciens菌株ATCC 21588可从American Type CultureCollection(ATCC)，10801 University Blvd.，Manassas VA 20110-2201，USA获得，其是生产玉米黄质的野生型海洋(marine)细菌(US3,891,504)。菌株R1534和R114是经过改进的玉米黄质生产突变体，它们是通过经典诱变和筛选工序从ATCC 21588获得的。根据《布达佩斯条约》，菌株R1534和R114之前已被保藏于ATCC，其ATCC菌株编号分别被指定为PTA-3336和PTA-3335。对菌株R1534intE、UV9-4和EMS9-7的构建在实施例2有详细描述。
培养基和条件
E.coli菌株于37℃在LB培养基(Becton Dickinson，Sparks，MD，USA)中生长。为保持重组菌株中的质粒，向培养基中加入氨苄青霉素(100μg/ml)和/或卡那霉素(25-50μg/ml，取决于实验)。对于固体培养基，加入琼脂(1.5％的最终浓度)。液体培养物在旋转式摇床上于200rpm下生长。
P.zeaxanthinifaciens菌株在28℃生长。用于P.zeaxanthinifaciens的培养基的组分在下文中描述(用于每项实验的具体培养基会在后面的实施例部分特别指出)。除非另有指明，生长于瓶中的P.zeaxanthinifaciens的所有液体培养物在旋转式摇床上于200rpm下被振荡。对于固体培养基，加入琼脂(1.5％的最终浓度)。通过高压对培养基灭菌时，在灭菌之后加入葡萄糖(作为浓缩的贮藏溶液)，以获得理想的最终浓度。
F-培养基含有(每升蒸馏水)：胰蛋白胨，10g；酵母提取物，10g；NaCl，30g；D-葡萄糖·H2O，10g；MgSO4·7H2O，5g。在通过过滤或高压灭菌之前，将pH调为7.0。
培养基362F/2含有(每升蒸馏水)：D-葡萄糖·H2O，33g；酵母提取物，10g；胰蛋白胨，10g；NaCl，5g；MgSO4·7H2O，2.5g。在通过过滤或高压灭菌之前，将pH调为7.4。灭菌后，微量元素溶液、NKP溶液和CaFe溶液各加入2.5ml。后三种溶液通过过滤被灭菌。微量元素溶液含有(每升蒸馏水)：(NH4)2Fe(SO4)2·6H2O，80g；ZnSO4·7H2O，6g；MnSO4·H2O，2g；NiSO4·6H2O，0.2g；EDTA，6g。NKP溶液含有(每升蒸馏水)：K2HPO4，250g；(NH4)2PO4，300g。CaFe溶液含有(每升蒸馏水)：CaCl2·2H2O，75g；FeCl3·6H2O，5g；浓HCl，3.75ml。
对P.zeaxanthinifaciens的分批培养
从冷冻的细胞悬浮液(于-80℃贮藏于15-20％的甘油贮液中)来初始化全部培养物。在含有200ml 362F/2培养基的2升带档板摇瓶中，制备双份的用于分批发酵的预培养物。对每只瓶子而言，用一毫升解冻的细胞悬浮物作为接种体。预培养物的初始pH为7.2。在28℃，250rpm的振荡下，将预培养物培养28小时，之后660nm处的光密度(OD660)在14和22个吸收单位之间，这取决于所使用的菌株。主培养物生长于Biostat ED生物反应器(B.Braun Biotech International，Melsungen Germany)中，其中含有具有如下组分的培养基(每升蒸馏水)：D-葡萄糖·H2O，25g；酵母提取物(Tastone 900)，17g；NaCl，4.0g；MgSO4·7H2O，6.25g；(NH4)2Fe(SO4)2·6H2O，0.5g；ZnSO4·7H2O，0.038g；MnSO4·H2O，0.013g；NiSO4·5H2O，0.001g；CaCl2·2H2O，0.47g；FeCl3·6H2O，0.062g；烟酸，0.01g；NH4Cl，0.5g；消泡剂，0.1ml；KP溶液，3.5ml。KP溶液的组分是(每升蒸馏水)：K2HPO4，250g；NaH2PO4·2H2O，200g；(NH4)2HPO4，100g。对于带有质粒的菌株，向培养基中加入卡那霉素(50mg/l终浓度)。所有方法中使用的补料溶液都具有如下组分(每升蒸馏水)：D-葡萄糖·H2O，550g；KP溶液，18.25ml。生物反应器中的初始体积(接种之后)为8L。按照需要用灭菌水对预培养物进行稀释，使得加入50ml到生物反应器中，可获得0.5的初始OD660值。发酵条件被自动控制如下：28℃，pH7.2(加入28％的NH4OH来控制pH)，溶解氧控制为最小为40％的相对值(有叶片搅拌的情况下)，最低搅拌为300rpm，通气速率为1v.v.m.(相对最终体积)。在上述条件不添加补料溶液的情况下，培养进行大约20小时(批次)。这段时间后，搅拌速率降低，基础消耗急剧降低和CO2产量降低是初始葡萄糖被耗尽的指示，补料开始进行。标准的补料曲线是如下定义的(从补料起始点开始)：17小时内，从50g/h斜线上升至80g/h，在80g/h持续7小时，然后在11个小时内斜线下降至55g/h，发酵的剩余阶段都保持在55g/h上。主培养物的最终体积为大约10升。
分析辅酶Q-10的方法
样品制备和提取。将20ml培养物转移到一次性的50ml聚丙烯离心管中，在4000rpm离心20分钟。将上清液移弃。将沉淀重新悬浮于10ml的丙酮中，对样品进行20秒的超声波处理(sonified)。将管子盖上，用IKA Vibrax摇晃器(shaker)对样品进行20分钟的混合。然后在室温对样品进行离心，4000rpm下10分钟，将上清液转移至干净的50ml聚丙烯离心管中。将10ml丙酮和10ml叔丁基甲醚(TMBE)加入到沉淀中，进行第二次提取。重复用于第一次提取的超声波处理、混合和离心步骤。来自该第二次提取的上清液被移出，并与第一次提取的上清液组合。像组合后的提取物中加入五毫升乙醇，以帮助除去水。对样品进行混合之后，用SpeedVac蒸发器(环境温度)除去溶剂。将残余物再次溶解于1ml TMBE加1ml乙醇中。在超声波浴中对样品进行5分钟的超声波处理，以协助溶解。最后，对样品进行离心，将上清液转移到褐色玻璃瓶中，用于高效液相色谱(HPLC)分析。
HPLC。反相HPLC方法被发展出来，用于对泛醌及其相应的对苯二酚的同时测定。该方法可以清楚地将类胡萝卜素八氢番茄红素、β-胡萝卜素、β-隐黄质和玉米黄质与辅酶Q-10分开。用装备有温控自动进样器和二极管阵列检测器的Agilent 1100 HPLC系统色谱来进行色谱分析。方法参数如下：
柱：YMC Carotenoid C30柱；5微米，钢，250mm×4.6mm I.D.(YMC，Part No.CT99S052546WT)
保护柱Pelliguard LC-18 cartridge，20mm(SUPELCO，PartNo.59654)
流动相：乙腈∶甲醇∶TBME(比例58％∶10％∶32％)的混合物，等度洗脱(isocratic elution)
进行时间：25分钟
典型柱压：起始为48bar
流速：1.0ml/分钟
检测：UV，在280nm处
注射体积：20μl
柱温度：15℃
试剂。乙腈和TBME是HPLC级别的，从Fluka获得。乙醇(p.a.)和甲醇(Lichrosolv)是从Merck，Darmstadt购买的。丙酮(Puriss.，p.a.)和辅酶Q-10是从Fluka购买的。
计算。在用外部标准来进行两级校正的情况下，进行定量分析。基于峰的面积进行计算。
选择性。通过注射相关参考化合物的标准溶液来检验本发明的选择性。目标化合物(辅酶Q-10和还原型泛醌-10)被完全分开，没有显示任何干扰。被选出的化合物的保留时间为：全反式玉米黄质，8.0分钟；15Z-八氢番茄红素，10.4分钟；辅酶Q-9，11.0分钟；β-隐黄质，12.3分钟；还原型泛醌-10，13.8分钟；辅酶Q-10，14.8分钟；β-胡萝卜素，19.0分钟。
线性。制备辅酶Q-10在TBME/乙醇(1∶1)中的一系列稀释溶液(最终浓度为300、100、30和3μg/ml)，用上述HPLC方法进行分析。线性范围被发现为从3μg/ml至300μg/ml。相关系数为0.9999。
检测极限。通过HPLC对辅酶Q-10进行检测的低限被测定为1μg/ml。
实施例2 构建P.zeaxanthinifaciens的crtE突变体
在P.zeaxanthinifaciens菌株R1534中对crtE基因进行插入失活。Pasamontes et al.[Gene 185：37-41(1997)]报道了对来自P.zeaxanthinifaciens菌株R1534的crtE基因进行的克隆和测序。使用质粒pRSF1010-Ampr-crt2(US6,291,204)作为模板，通过聚合酶链式反应(PCR)来制造在其5’和3’末端具有PstI限制性酶识别位点的crtE特异性DNA片段。所用的正向和反向引物具有分别如SEQ ID NO：1和SEQ ID NO：2所示的序列。PCR反应条件是：94℃，1分钟(一个循环)；94℃，1分钟接着是72℃，1.5分钟(30个循环)；72℃，7分钟(一个循环)。用PstI去消化通过PCR反应制造出来的460个碱基对(bp)的DNA片段，并用QIAquick柱(Qiagen，Hilden，德国)从琼脂糖胶对其进行纯化。经过分离的片段被连接到之前已用PstI进行过消化的“自杀型”质粒载体pSUP202[Simon et al.，Bio/Technology 1：784-791(1983)]上。使用标准工序，用经连接的混合物去转化E.coli S-17细胞。对转化子的筛选验证了对期待的含有内部crtE片段的质粒(被命名为质粒pSUPcrtE)的构建。使用US6,291,204中公开的接合方法，将质粒pSUPcrtE转移到P.zeaxanthinifaciens菌株R1534中。在含有100μg/ml利福平和3μg/ml四环素的琼脂培养基上筛选出转移接合子。大约获得103个菌落。较之菌株R1534(由于玉米黄质的产生呈现黄色)，上述菌落中的大量都显示出了颜色的变化(变白)，这与由于对crtE基因进行插入失活造成的玉米黄质生产能力丢失一致。
十个有代表性的假定crtE整合子(integrant)被选出来，用于下述通过菌落PCR进行的分析。将菌落转移到1.5ml的微量离心管中。在微波炉中对样品进行加热(1分钟，900瓦)，然后转移至冰上。然后将PCR混合物加入，其中含有PCR缓冲液、15％的甘油、聚合酶、核苷酸和引物。PCR条件是：94℃，1分钟，接着57℃，30秒，接着72℃，1分钟(总共25个循环)；然后72℃7分钟(一个循环)。所用的寡核苷酸引物经过了设计，所述设计使得：仅当pSUPcrtE质粒的期待整合发生于P.zeaxanthinifaciens菌株R1534染色体上的crtE座时才可获得PCR产物。正向引物，pCrtEampF(SEQ ID NO：3)是crtE特异性的，而反向引物pCrtEampR(SEQ ID NO：4)是bla(氨苄青霉素抗性基因)特异性的。使用该菌落PCR方法，我们发现，待测的10个菌落全部都含有在crtE座上的自杀型载体pSUPcrtE的整合(全部都给出了期待大小的PCR产物)。此外，用对应于载体pSUP202bla基因的特定片段而特别设计的PCR引物，检测到，这10个crtE整合子中的8个都含有多个拷贝的pSUPcrtE，按照头尾相连的方式排列。
通过Southern杂交对crtE整合子中的三个(和作为对照的P.zeaxanthinifaciens菌株R1534)进行进一步的分析。这些菌株生长于液体F-培养基上，并用标准方法从细胞中提取染色体DNA。用限制性酶AlwNI、XhoI、BglII、PvuII以及BglII加PvuII来对染色体DNA进行消化，并将其用于琼脂糖凝胶电泳。片段到膜的转移和用探针进行的杂交都按照标准工序来进行。用32P对来自P.zeaxanthinifaciens菌株R1534的crtE基因进行标记，并将其用作为探针。在所有利用来自对照菌株R1534的经消化的染色体DNA进行的杂交中，与crtE探针杂交的片段的数量和大小都如预期一样基于从R1534染色体中分离出来的crtE基因的核苷酸序列。用来自三个crtE整合子的经消化的DNA进行的杂交的结果就与用R1534对照所获得的结果明显不同，并且(与菌落PCR分析的情况一样)，上述结果与pSUPcrtE的多个拷贝在染色体crtE座的整合一致。一个被命名为R1534intE的整合子(表1中列出)，被保留下来用于进一步的实验(实施例4中所述的)。
制造P.zeaxanthinifaciens菌株R114的crtE突变体。使用标准工序对P.zeaxanthinifaciens菌株R114进行诱变(用紫外[UV]照射或乙基磺酸甲烷[EMS]进行处理)。简言之，对(生长于F-培养基上的)菌株R114的过夜培养物进行亚培养至OD660为0.1，并在振荡下培养3小时。对试验用量的(aliquots)培养物进行离心，通过重新悬浮于20mM磷酸钾缓冲液(pH7.2)来对细胞沉淀进行洗涤，悬浮液被再次离心以收集细胞。然后将经过洗涤的细胞沉淀重新悬浮于20mM的磷酸钾缓冲液(pH7.2)中，至OD660为0.1。对UV诱变而言，将10ml细胞悬浮液样品转移至100ml的玻璃烧杯中。用磁力搅拌器对悬浮液进行持续的轻柔混合(烧杯中置有回形针)。混合中的细胞悬浮液被UV光照射一段预定的时间，照射流量为1450μW/cm2。对EMS诱变而言，将0.1ml的EMS加入到10ml经洗涤的细胞悬浮液的样品中。在旋转式摇床上对混合物进行多达90分钟的振荡。然后通过离心收集细胞，洗两次以去除诱变剂。
若干项预备实验被用来完善与百分比存活率、用于涂板的稀释率等相关的诱变工序。一旦这些条件已建立起来，经过UV或EMS诱变的P.zeaxanthinifaciens菌株R114的细胞就被涂布到362F/2琼脂平板上，以分离白色(无玉米黄质生产的)菌落。很多的此类突变体被获得，将其在362F/2琼脂平板上重新划线以分离单个菌落，并确证低返祖率/无返祖率。按照下面部分所述，对总共43个稳定的突变体进行进一步的检测。
P.zeaxanthinifaciens的crtE、crtB或crtI基因突变体预期会给出白色菌落，因为番茄红素是提供可视颜色的玉米黄质途径的第一个中间产物。为从菌株R114的43个白色的无玉米黄质生产的突变体中鉴定出crtE突变体，使用了两步筛选方法。首先，针对八氢番茄红素的积累对突变体进行筛选，这是crtI基因产物，番茄红素环化酶，活性丢失的指标。因为八氢番茄红素在crtE被破坏的突变体中是不可能被生产出来的，所以任何能积累八氢番茄红素的突变体都可被认定为不是crtE突变体，因此也不作进一步的考虑。第二，用携带有来自P.zeaxanthinifaciens菌株R114的克隆crtE基因的质粒pBBR-K-PcrtE-crtER114，对第一步中鉴定出来的无八氢番茄红素积累的白色突变体进行转化，以验证遗传互补。引入克隆crtE基因之后玉米黄质生产的恢复(即黄色菌落)强烈表明，白色菌落表型的基础是crtE的突变，它使香叶基香叶基二磷酸酯(GGPP)合酶失活。
在摇瓶培养物中对突变体进行关于八氢番茄红素积累的试验。白色突变体(加亲本对照菌株R114)生长于500ml带档板瓶中的110ml 362F/2培养基中。培养物的初始OD660是0.16。在24、48和72小时取出30ml的样品，置于50ml的聚丙烯管中，离心以收集细胞。用水对细胞沉淀洗涤，并再次离心。然后用与实施例1中所述用于分析辅酶Q-10相同的方法，通过HPLC来对最终的细胞沉淀进行关于八氢番茄红素积累的分析。因为目前还没有八氢番茄红素的标准物，对应于八氢番茄红素的色谱峰只能通过其UV谱和由质谱分析测定的化合物质量来鉴定。对八氢番茄红素浓度的判断基于八氢番茄红素和辅酶Q-10的比吸收系数的比例来进行。这种方法能够清楚地辨别出在摇瓶培养物中积累八氢番茄红素的那些突变体。六个不积累八氢番茄红素的突变体(UV6-1、UV7-6、UV9-4、EMS3-6、EMS3-15和EMS9-7)被用来进行下一阶段的试验。
按照下述内容，通过克隆的crtE基因，对6个白色的无八氢番茄红素积累的突变体UV6-1、UV7-6、UV9-4、EMS3-6、EMS3-15和EMS9-7进行遗传互补的试验。对每个突变体，用体积为1.5ml的固定相(stationaryphase)培养物来接种100ml的F-培养基。在28℃、200rpm下培养该培养物，直到OD660达到大约0.5。然后通过在4℃下以7000xg离心15分钟来收获细胞，在100ml冰冷的1mM HEPES缓冲液(pH7.0)中洗两次，将最终的沉淀重新悬浮于0.1ml冰冷的1mM HEPES缓冲液(pH7.0)中，或者立即使用电感受态(eletrocompetent)细胞进行电穿孔，或者加入甘油至终浓度为15％，将细胞分装成50μl的小份贮藏于-70℃。为进行转化，将1-5μl的质粒pBBR-K-PcrtE-crtER114(在无盐溶液中)加入到0.1ml的电感受态细胞中，进行电穿孔(条件：18KV/cm，129ohms，在冰冻的1mm小管中；脉冲长度典型地在4至5毫秒之间)。然后加入1ml的F-培养基，在28℃伴随振荡下对细胞悬浮液进行1小时的培养。然后将悬浮液的稀释物涂布到含有50μg/ml卡那霉素的F-琼脂平板上，在28℃对平板进行培养。
突变体UV6-1、UV9-4、EMS3-6和EMS9-7的所有抗卡那霉素的菌落(即转化子)颜色都是深黄色的，而UV7-6和EMS3-15突变体的转化子保留了白色。该遗传互补试验表明，突变体UV6-1、UV9-4、EMS3-6和EMS9-7含有使GGPP合酶失活的crtE突变。突变体UV9-4和EMS9-7被用于进一步的实验，以评价辅酶Q-10的生产。
实施例3 对来自P.zeaxanthinifaciens菌株R114和ATCC 21588的ddsA
基因进行克隆和测序
方法
分离基因组DNA。在4℃下，以10000xg对600ml P.zeaxanthinifaciens菌株R114的培养物(生长于F-培养基中)进行10分钟的离心，用200ml溶菌缓冲液(0.1M NaCl、50mM EDTA、10mM Tris-HCl，pH7.5)洗一次沉淀，再用100ml溶菌缓冲液洗一次。最终的沉淀被重新悬浮于20ml含有50mg溶菌酶和1mg RNase A的溶菌缓冲液中(无DNase)。在37℃培养15分钟之后，加入1.5ml 20％的N-月桂酰肌氨酸钠和2.25mg蛋白酶K。在50℃培养30-60分钟之后，通过轻柔但彻底的混合，用一倍体积的经缓冲液饱和的苯酚(pH7.5-7.8)来对溶菌产物进行抽提。以30000xg对乳化液离心20分钟，用苯酚对水相进行再次抽提。如前所述对这些相进行分离，再用一倍体积的苯酚∶氯仿(1∶1)对水相抽提两次。在该步骤，在浮桶式转子离心机(swinging bucket rotor)中于3200xg离心20分钟足以获得令人满意的相分离结果。用一倍体积的氯仿进行最后抽提之后，加入0.1倍体积的3M乙酸钠(pH5.2)，用两倍体积冰冷的乙醇覆盖溶液。将沉淀的DNA缠绕在玻璃棒上，用70％的乙醇浸泡5分钟，用氯仿洗一下，然后空气干燥5-10分钟。将DNA重新悬浮于5ml TE缓冲液(10mM Tris-HCl，pH7.5，1mM EDTA)中过夜。因为由于痕量玉米黄质的存在溶液是黄色的，所以重复进行上述有机抽提和缠绕，以获得纯净的制剂。
λ-文库。从Stratagene(La Jolla，CA，USA)购买lambda FIXII中的带有经Sau3AI部分消化的P.zeaxanthinifaciens菌株R114DNA的顾客定制文库。
PCR。按照厂商说明书，使用富含GC的PCR系统(Roche MolecularBiochemicals，Mannheim，德国)，在GeneAmpPCR系统9700(PEApplied Biosystems，Foster City，CA，USA)中进行PCR。典型地，使用的MgCl2浓度为1.5mM，溶解溶液(resolution solution)被加入至终浓度为1M。
DNA标记和检测。PCR DIG Probe Synthesis试剂盒和DIGLuminescent Detection试剂盒被分别用于DNA标记和检测(都从RocheMolecular Biochemicals，Manneheim，德国获得)。
分离λ-DNA。按照厂商说明书，使用QiagenLambda试剂盒(Qiagen，Hilden，德国)。
DNA测序。按照厂商说明书，用BigDyeDNA测序试剂盒(PEApplied Biosystems，Foster City，CA，USA)来进行测序反应。在DyeExTM旋转柱上来纯化测序反应产物，用ABI PrismTM 310 GeneticAnalyzer(PE Applied Biosystems，Foster City，CA，USA)来进行检测和片段分离。
对来自P.zeaxanthinifaciens菌株R114的ddsA基因进行克隆和测序。P.zeaxanthinifaciens菌株R114的ddsA基因(编码DPP合酶)最初是在对phaAB基因簇进行克隆和测序时被偶然鉴定出来的。使用基于P.denitrificans的phaA和phaC基因序列的引物，获得含有来自P.zeaxanthinifaciens菌株R1534的phaA和来自P.zeaxanthinifaciens菌株R114的phaC的部分的PCR片段。然后用这些PCR片段针对phaA和phaC基因对P.zeaxanthinifaciens菌株R114的λ文库进行筛选。从该文库中鉴定并克隆出与探针杂交的DNA片段。测序验证了phaA基因和phaC基因确实存在于被克隆出的片段上。此外，phaB基因被发现处于phaA基因的下游。因此，与P.denitrificans的情况一样，P.zeaxanthinifaciens中，phaA和phaB基因是串在一起的，而phaC基因处于基因组的其它地方。对phaAB下游区域的测序揭示了被鉴定为ddsA基因的开放阅读框，这是基于与来自P.denitrificans的ddsA基因的相似性来鉴定的。P.zeaxanthinifaciens菌株R114的ddsA基因的核苷酸序列(SEQ ID NO：5)及其编码的相应的DPP合酶的氨基酸序列(SEQ ID NO：6)于2002年2月21日被提交至EMBL，自2002年5月2日起可被公众所获得，编号为AJ431695。
对来自P.zeaxanthinifaciens菌株ATCC 21588的ddsA基因进行测序。使用来自P.zeaxanthinifaciens菌株ATCC 21588的基因组DNA作为模板，用正向引物7R-01(SEQ ID NO：7)和反向引物dds-3(SEQ IDNO：8)来扩增ddsA基因，对PCR产物进行测序。对来自野生型菌株ATCC 21588和来自其经典获得的突变体R114的ddsA序列的比较揭示，R114中的ddsA基因在编码区域的核苷酸698含有G到A的突变。这将密码子233中的中间核苷酸从G
GC改变为G
AC，这导致了氨基酸233从甘氨酸(ATCC 21588中)变为天冬氨酸(R114中)。
实施例4 P.zeaxanthinifaciens的ddsA基因的过量表达对P.
zeaxanthinifaciens中辅酶Q-10的生产的影响
克隆来自P.zeaxanthinifaciens的ddsA基因用于在P.zeaxanthinifaciens 中过量表达。用ddsA/NdeI/for(SEQ ID NO：9)引物和ddsA/BamHI/rev(SEQ ID NO：10)引物，通过PCR从P.zeaxanthinifaciens菌株R114中扩增P.zeaxanthinifaciens菌株R114的ddsA基因的编码区域。引物ddsA/NdeI/for含有NdeI(CATATG)限制性位点，其被定位成使得第二条一半的链(second half)与ddsA基因的ATG起始密码子一致。引物ddsA/BamHI/rev含有紧接在终止密码子之后的BamHI位点。用NdeI和BamHI来切割PCR片段，并与经NdeI-BamHI切过的载体pXI12主链连接(Hümbelin et al.，J.Ind.Microbiol.Biotechnol.22：1-7，1999)，得到质粒pTH36。接着，用BstXI和Bsu36I来切割载体pBBR1MCS-2(GenBank编号#U23751)，将较大的片段与退火的(annealed)寡核苷酸MCS-2上游(SEQ ID NO：11)和MCS-2下游(SEQ ID NO：12)相连，得到载体pBBR-K-Nde。将经过EcoRI-NdeI切割的具有SEQ ID NO：13序列的片段(该片段含有来自Rhodobacter sphaeroides的核糖体RNA操纵子的rrnB启动子)插入到经EcoRI-NdeI切割的pBBR-K-Nde主链中，产生质粒pBBR-K-PrrnB。最后，用NdeI和BamHI从pTH36下切下来自菌株R114的ddsA基因，将其与经过NdeI-BamHI切割的载体pBBR-K-PrrnB的主链相连，得到质粒pBBR-K-PrrnB-ddsAR114。
为制造用于在P.zeaxanthinifaciens中过量表达来自P.zeaxanthinifaciens ATCC 21588的野生型ddsA基因的载体，用QuikChangeTM定点突变试剂盒(Stratagene)和引物dds-wt-1(SEQ IDNO：14)和dds-wt-2(SEQ ID NO：15)来对质粒pBBR-K-PrrnB-ddsAR114进行突变。对得到的被命名为pBBR-K-PrrnB-ddsAwt的质粒中的ddsA基因进行了完全测序，证实了想要的改变。来自P.zeaxanthinifaciens菌株ATCC 21588的ddsA基因的完整核苷酸序列在SEQ ID NO：16中示出，对应的氨基酸序列在SEQ ID NO：17中示出。用于评价在重组P.zeaxanthinifaciens菌株中辅酶Q-10生产的预备实验表明，用于质粒pBBR-K-PrrnB-ddsAR114和pBBR-K-PrrnB-ddsAwt的rrnB启动子导致了结果的不一致。因此，用P.zeaxanthinifaciens中处于crtE基因上游的DNA序列对rrnB启动子进行了替换。该序列含有crtE启动子。因此得到了质粒pBBR-K-PcrtE-ddsAwt和pBBR-K-PcrtE-ddsAR114，它们携带有具有ddsA基因上游的SEQ ID NO：18(代替SEQ ID NO：13)序列的EcoRI-NdeI片段。
在过量表达ddsA基因的P.zeaxanthinifaciens菌株中生产辅酶Q-10。通过电穿孔，将质粒pBBR-K-PcrtE-ddsAwt和pBBR-K-PcrtE-ddsAR114引入到P.zeaxanthinifaciens菌株R114、UV9-4、EMS9-7和R1534intE中。得到的重组菌株及其各自的亲本(对照)菌株(以双份)生长于摇瓶培养物中(培养基362F/2)，测量辅酶Q-10的产量。最初的结果显示，P.zeaxanthinifaciens ATCC 21588的野生型ddsA基因编码DPP合酶的活性高于来自P.zeaxanthinifaciens菌株R114的突变体ddsA基因。因此，仅有过量表达野生型ddsA基因的菌株被用于进一步研究。表1中的数据清楚地显示：(通过克隆多拷贝质粒)增加野生型ddsA基因的表达，显著地增加了P.zeaxanthinifaciens所有菌株中辅酶Q-10的产量。在不同被测菌株中，辅酶Q-10的单位生产率提高，在2倍至8倍的范围内。
表1：
| 辅酶Q-10 | ||||||
| mg/l(标准方差) | 单位形成率(SpecificFormation)(mg/g细胞干重) | |||||
| 菌株/质粒 | 24h | 48h | 72h | 24h | 48h | 72h |
| R114/pBBR-K(空载体) | 3.95(0.71) | 3.9(0.85) | 4.65(0.78) | 0.21 | 0.19 | 0.26 |
| R114/pBBR-K-PcrtE-ddsAWT | 19.45(3.04) | 31.95(0.64) | 40.3(1.41) | 0.98 | 1.27 | 2.05 |
| UV9-4 | 4.1(0.42) | 3.25(0.21) | 3.2(0.14) | 0.21 | 0.17 | 0.20 |
| UV9-4/pBBR-K-PcrtE-ddsAWT | 25.05(0.64) | 25.6(0.42) | 24.2(4.38) | 0.89 | 1.22 | 1.21 |
| EMS9-7 | nd | nd | nd | |||
| EMS9-7/pBBR-K-PcrtE-ddsAWT | 18.1(2.26) | 24.2(0.93) | 28.71(1.40) | 0.85 | 1.27 | 1.58 |
| R1534intE | 13.81(1.17) | 18.43(1.63) | 24.26(3.19) | 0.33 | 0.58 | 1.12 |
| R1534intE/pBBR-K-PcrtE-ddsAWT | 19.64(3.97) | 34.6(0.23) | 38.28(0.93) | 0.94 | 1.61 | 2.27 |
nd：未检测到
实施例5 对来自P.zeaxanthinifaciens菌株R114和ATCC 21588的nbiA
基因进行克隆和测序
通过将P.zeaxanthinifaciens菌株R114的基因组序列与已知的细菌4-羟基苯甲酸聚异戊二烯基转移酶序列进行比较，来鉴定P.zeaxanthinifaciens菌株R114的ubiA基因(编码4-羟基苯甲酸聚异戊二烯基转移酶)。P.zeaxanthinifaciens菌株R114的ubiA基因的核苷酸序列(SEQ ID NO：19)被用于设计正向引物ubiA-Nde(SEQ ID NO：21)和反向引物ubiA-Bam(SEQ ID NO：22)。引物ubiA-Nde含有NdeI(CATATG)限制性位点，其被定位成使得第二条一半的链(secondhalf)与ubiA基因的ATG起始密码子一致。引物ubiA-BamHI含有紧接在终止密码子之后的BamHI位点。使用来自P.zeaxanthinifaciens菌株R114或P.zeaxanthinifaciens ATCC 21588的基因组DNA作为模板，用这两条引物来扩增ubiA基因(分离基因组DNA、PCR和DNA测序的方法在实施例3中描述)。用TOPO TA Cloning试剂盒(Invitrogen，Carlsbad，CA，USA)将两条PCR片段克隆进载体pCR2.1-TOPO，产生质粒pCR2.1-TOPO-ubiAwt(含有来自野生型菌株ATCC 21588的ubiA基因)和pCR2.1-TOPO-ubiAR114(含有来自突变体菌株R114的ubiA基因)。对每种质粒的克隆插入进行测序。对来自菌株ATCC 21588的ubiA基因的核苷酸序列(SEQ ID NO：23)和来自菌株R114的ubiA基因的核苷酸序列(SEQ IDNO：19)进行的比较揭示了改变了密码子220的一个核苷酸不同。对从两种菌株基因组DNA扩增出来的未克隆PCR片段的相关区域进行测序，证实了这种不同，排除了PCR人为错误的可能性。在来自ATCC 21588菌株的野生型ubiA基因中，密码子220是ACC(编码苏氨酸)，而在来自菌株R114的突变体ubiA基因中，密码子220是ATC(编码异亮氨酸)。SEQ ID NO：20和SEQ ID NO：24中分别给出了来自P.zeaxanthinifaciens菌株R114和ATCC 21588的4-羟基苯甲酸聚异戊二烯基转移酶的相应氨基酸序列。
实施例6 P.zeaxanthinifaciens的ubiA基因的过量表达对P.
zeaxanthinifaciens中辅酶Q-10的生产的影响
克隆来自P.zeaxanthinifaciens的ubiA基因用于在P.zeaxanthinifaciens 中过量表达。来自P.zeaxanthinifaciens菌株ATCC 21588和R114的ubiA基因分别被来自质粒pCR2.1-TOPO-ubiAwt和pCR2.1-TOPO-ubiAR114的限制性内切酶NdeI和BamHI切割(见实施例5)，并被连接到经NdeI和BamHI切割的来自表达质粒pBBR-K-PcrtE的载体主链上。这使得nbiA基因处于crtE启动子的控制下，并制造出了与实施例4中所述的表达质粒pBBR-K-PcrtE-ddsAwt和pBBR-K-PcrtE-ddsAR114类似的质粒。用新质粒pBBR-K-PcrtE-ubiAwt和pBBR-K-PcrtE-ubiAR114，通过电穿孔来转化P.zeaxanthinifaciens菌株R1534。选择菌株R1534是因为其含有ddsA基因的野生型染色体版本。
辅酶Q-10在过量表达ubiA基因的P.zeaxanthinifaciens菌株R1534中 的生产。在生物反应器中(双份，条件如实施例1所述)对P.zeaxanthinifaciens菌株R1534(对照菌株)、R1534/pBBR-K-PcrtE-ubiAwt和R1534/pBBR-K-PcrtE-ubiAR114进行培养，以比较辅酶Q-10的产量。表2中的结果显示，来自P.zeaxanthinifaciensR114或ATCC 21588的ubiA基因的过量表达增加了P.zeaxanthinifaciens中辅酶Q-10的产量。
表2：
| 辅酶Q-10 | ||||
| mg/l(标准方差) | 单位形成率(Specific Formation)(mg/g细胞干重) | |||
| 43h | 70h | 43h | 70h | |
| R1534 | 58.8(0.54) | 65.8(0.19) | 1.61(0.09) | 1.79(0.01) |
| R1534/pBBR-K-PcrtE-ubiAWT | 63.8(4.74) | 72.8(0.62) | 1.77(0.11) | 2.07(0.08) |
| R1534/pBBR-K-PcrtE-ubiAR114 | 65.5(0.47) | 85.3(4.55) | 1.73(0.01) | 2.14(0.16) |
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gagtcgccgc gcaggtcggg cgaaaggggc gggtcgcggc tccgcggcaa cgaaaaacgc 120
caagatttct tggctgcgac atgaaatgtt acggagccca aaaaatccgc ttgcgcccgg 180
ggccgtctgc tcctagaacc gcttcaccga gacgaagacc ggcagcgccg gacggagacg 240
agggagggat gacagaaacg tcggccgcga caattgaaga tgaggcggac gggatgctgg 300
ttgtctgtcg sctctagacg atcgcctacc ggagtacata tg 342
<210>14
<211>31
<212>DNA
<213>人工
<220>
<221>misc_feature
<222>(1)..(31)
<223>PCR引物dds-wt-1
<400>14
ccgccgaggc gatcggcaag aataccggcg a 31
<210>15
<211>31
<212>DNA
<213>人工
<220>
<221>misc_feature
<222>(1)..(31)
<223>PCR引物dds-wt-2
<400>15
tcgccggtat tcttgccgat cgcctcggcg g 31
<210>16
<211>1002
<212>DNA
<213>Paracoccus zeaxanthinifaciens
<220>
<221>CDS
<222>(1)..(1002)
<223>
<400>16
atg aac gtg cag gaa gac gtc cgc aaa cca ctg gac cgg ctg gcc gag 48
Met Asn Val Gln Glu Asp Val Arg Lys Pro Leu Asp Arg Leu Ala Glu
1 5 10 15
gcg ctg gca ccc gag atg gag gcc gtg aac gcg ctg atc cgc gaa cgc 96
Ala Leu Ala Pro Glu Met Glu Ala Val Asn Ala Leu Ile Arg Glu Arg
20 25 30
atg gcc agc agg cat gcg ccg cgc atc ccc gag gtg acc gcc cac ctg 144
Met Ala Ser Arg His Ala Pro Arg Ile Pro Glu Val Thr Ala His Leu
35 40 45
atc gag gcc ggc ggc aag cgc ctg cgc ccg atg ctg acc ctg gcc gcg 192
Ile Glu Ala Gly Gly Lys Arg Leu Arg Pro Met Leu Thr Leu Ala Ala
50 55 60
gcg aag ctg ctt ggc tat ggc ggc ccc tat cac gtg cat ctg gcc gcg 240
Ala Lys Leu Leu Gly Tyr Gly Gly Pro Tyr His Val His Leu Ala Ala
65 70 75 80
acg gtc gaa ttc atc cac acc gcg acc ctg ctg cat gac gac gtg gtc 288
Thr Val Glu phe Ile His Thr Ala Thr Leu Leu His Asp Asp Val Val
85 90 95
gac gaa agc cgc cag cgc cgc ggg cgt ccg acg gcg aac ctg ctg tgg 336
Asp Glu Ser Arg Gln Arg Arg Gly Arg Pro Thr Ala Asn Leu Leu Trp
100 105 110
gac aac aag tcc agc gtg ctg gtc ggc gat tac ctg ttc gcg cgc agc 384
Asp Asn Lys Ser Ser Val Leu Val Gly Asp Tyr Leu Phe Ala Arg Ser
115 120 125
ttc cag ctg atg gtc gaa ccc ggc agc atg cgc acg ctc gag atc ctg 432
Phe Gln Leu Met Val Glu Pro Gly Ser Met Arg Thr Leu Glu Ile Leu
130 135 140
tcg aac gcc gcc gcc acc atc gcc gag ggc gag gtg ctg cag ctg acc 480
Ser Asn Ala Ala Ala Thr Ile Ala Glu Gly Glu Val Leu Gln Leu Thr
145 150 155 160
gcg gcg cag gat ctg gcc acg aac gag gac atc tat ctg cag gtc gtg 528
Ala Ala Gln Asp Leu Ala Thr Asn Glu Asp Ile Tyr Leu Gln Val Val
165 170 175
cgc ggc aag acg gca gcg ctg ttc tcg gcc gcg acc gag gtg ggc ggc 576
Arg Gly Lys Thr Ala Ala Leu Phe Ser Ala Ala Thr Glu Val Gly Gly
180 185 190
gtc atc gcg ggc gtc ccc gat gcg cag gtc cgc gcg ctg ttc gat tac 624
Val Ile Ala Gly Val Pro Asp Ala Gln Val Arg Ala Leu Phe Asp Tyr
195 200 205
ggc gac gcg ctt ggc atc gcc ttc cag atc gtg gac gac ctg ctg gat 672
Gly Asp Ala Leu Gly Ile Ala Phe Gln Ile Val Asp Asp Leu Leu Asp
210 215 220
tac ggc ggc acc gcc gag gcg atc ggc aag aat acc ggc gac gat ttc 720
Tyr Gly Gly Thr Ala Glu Ala Ile Gly Lys Asn Thr Gly Asp Asp Phe
225 230 235 240
cgc gaa cgc aag ctg acg ctg ccg gtg atc aag gcc gtg gcc cgc gcc 768
Arg Glu Arg Lyg Leu Thr Leu Pro Val Ile Lys Ala Val Ala Arg Ala
245 250 255
acc ccc gag gaa cgc gcc ttc tgg tcg cgc acc atc gag aag ggc gac 816
Thr Pro Glu Glu Arg Ala Phe Trp Ser Arg Thr Ile Glu Lys Gly Asp
260 265 270
cag aag gac ggc gac ctt gaa cac gcg ctg gaa ctg ctg gcc cgc cac 864
Gln Lys Asp Gly Asp Leu Glu His Ala Leu Glu Leu Leu Ala Arg His
275 280 285
ggc gcg atg gcc gat gcc cgc cgc gac gcg ctg gac tgg gcg gcc agg 912
Gly Ala Met Ala Asp Ala Arg Arg Asp Ala Leu Asp Trp Ala Ala Arg
290 295 300
gcc cgc gcc tcc ctg cag atc ctg ccc gag cat ccg atc cgc gac atg 960
Ala Arg Ala Ser Leu Gln Ile Leu Pro Glu His Pro Ile Arg Asp Met
305 310 315 320
ctg tcg gac ctg gcc gat ttc gtg gtc gaa cgc atc gcc tga 1002
Leu Ser Asp Leu Ala Asp Phe Val Val Glu Arg Ile Ala
325 330
<210>17
<211>333
<212>PRT
<213>Paracoccus zeaxanthinifaciens
<400>17
Met Asn Val Gln Glu Asp Val Arg Lys Pro Leu Asp Arg Leu Ala Glu
1 5 10 15
Ala Leu Ala Pro Glu Met Glu Ala Val Asn Ala Leu Ile Arg Glu Arg
20 25 30
Met Ala Ser Arg His Ala Pro Arg Ile Pro Glu Val Thr Ala His Leu
35 40 45
Ile Glu Ala Gly Gly Lys Arg Leu Arg Pro Met Leu Thr Leu Ala Ala
50 55 60
Ala Lys Leu Leu Gly Tyr Gly Gly Pro Tyr His Val His Leu Ala Ala
65 70 75 80
Thr Val Glu Phe Ile His Thr Ala Thr Leu Leu His Asp Asp Val Val
85 90 95
Asp Glu Ser Arg Gln Arg Arg Gly Arg Pro Thr Ala Asn Leu Leu Trp
100 105 110
Asp Asn Lys Ser Ser Val Leu Val Gly Asp Tyr Leu Phe Ala Arg Ser
115 120 125
Phe Gln Leu Met Val Glu Pro Gly Ser Met Arg Thr Leu Glu Ile Leu
130 135 140
Ser Asn Ala Ala Ala Thr Ile Ala Glu Gly Glu Val Leu Gln Leu Thr
145 150 155 160
Ala Ala Gln Asp Leu Ala Thr Asn Glu Asp Ile Tyr Leu Gln Val Val
165 170 175
Arg Gly Lys Thr Ala Ala Leu Phe Ser Ala Ala Thr Glu Val Gly Gly
180 185 190
Val Ile Ala Gly Val Pro Asp Ala Gln Val Arg Ala Leu Phe Asp Tyr
195 200 205
Gly Asp Ala Leu Gly Ile Ala Phe Gln Ile Val Asp Asp Leu Leu Asp
210 215 220
Tyr Gly Gly Thr Ala Glu Ala Ile Gly Lys Asn Thr Gly Asp Asp Phe
225 230 235 240
Arg Glu Arg Lyg Leu Thr Leu Pro Val Ile Lys Ala Val Ala Arg Ala
245 250 255
Thr Pro Glu Glu Arg Ala Phe Trp Ser Arg Thr Ile Glu Lys Gly Asp
260 265 270
Gln Lys Asp Gly Asp Leu Glu His Ala Leu Glu Leu Leu Ala Arg His
275 280 285
Gly Ala Met Ala Asp Ala Arg Arg Asp Ala Leu Asp Trp Ala Ala Arg
290 295 300
Ala Arg Ala Ser Leu Gln Ile Leu Pro Glu His Pro Ile Arg Asp Met
305 310 315 320
Leu Ser Asp Leu Ala Asp Phe Val Val Glu Arg Ile Ala
325 330
<210>18
<211>277
<212>DNA
<213>人工
<220>
<221>misc_feature
<222>(1)..(277)
<223>含有PcrtE启动子的EcoRI-NdeI片段
<400>18
gaattcgctg ctgaacgcga tggcggcgcg gggcgcgacg cgcggggccg catccgtctg 60
catcggcggg ggcgaggcga cggccatcgc gctggaacgg ctgagctaat tcatttgcgc 120
gaatccgcgt ttttcgtgca cgatggggga accggaaacg gccacgcctg ttgtggttgc 180
gtcgacctgt cttcgggcca tgcccgtgac gcgatgtggc aggcgcatgg ggcgttgccg 240
atccggtcgc atgactgacg caacgaaggc acatatg 277
<210>19
<211>975
<212>DNA
<213>Paracoccus zeaxanthinifaciens
<220>
<221>CDS
<222>(1)..(975)
<223>
<400>19
atg aac aat cgt atc ttc gcc atg atg ggc aac gct atg caa agc agc 48
Met Asn Asn Arg Ile Phe Ala Met Met Gly Asn Ala Met Gln Ser Ser
1 5 10 15
acg gaa aga cca gac gcg gtc gtc gac gcg ccg aag gga aac tgg gtc 96
Thr Glu Arg Pro Asp Ala Val Val Asp Ala Pro Lys Gly Asn Trp Val
20 25 30
gat gag atc gcc ccg cca tgg tcg cgc ccc tgg ctg cgg ctc agc cgc 144
Asp Glu Ile Ala Pro Pro Trp Ser Arg Pro Trp Leu Arg Leu Ser Arg
35 40 45
gcg gac cgg ccc atc ggg aca tgg ctg ctg ctg ctg ccc tgc tgg tgg 192
Ala Asp Arg Pro Ile Gly Thr Trp Leu Leu Leu Leu Pro Cys Trp Trp
50 55 60
ggg atc ggg ctg gcg atg atg gca gac ggg ccg cgc tgg ttc gat gcg 240
Gly Ile Gly Leu Ala Met Met Ala Asp Gly Pro Arg Trp Phe Asp Ala
65 70 75 80
tgg atc gcg ctg gcc tgc acc atc ggc gcc ttc gtc atg cgg ggc gcg 288
Trp Ile Ala Leu Ala Cys Thr Ile Gly Ala Phe Val Met Arg Gly Ala
85 90 95
ggc tgc acc tgg aac gac atc acc gac cgc c gg atc gac gcg cag gtc 336
Gly Cys Thr Trp Asn Asp Ile Thr Asp Arg Arg Ile Asp Ala Gln Val
100 105 110
gca cgc acc cgc tcg cgc ccg ctg cca agc gga cag gtc acg ctg cgg 384
Ala Arg Thr Arg Ser Arg Pro Leu Pro Ser Gly Gln Val Thr Leu Arg
115 120 125
ggc gcc tat ggc tgg ctg atc gcg cag ggg ctg atc ggg ctg gcg atc 432
Gly Ala Tyr Gly Trp Leu Ile Ala Gln Gly Leu Ile Gly Leu Ala Ile
130 135 140
ctg ctg acc ctg ggg cag gcc gcg atc tgg atg ggc gtc gcc tcg ctg 480
Leu Leu Thr Leu Gly Gln Ala Ala Ile Trp Met Gly Val Ala Ser Leu
145 150 155 160
gcg ctg gtc gcg atc tat ccc ttc gcg aaa cgc ttc acc tgg tgg ccg 528
Ala Leu Val Ala Ile Tyr Pro Phe Ala Lys Arg Phe Thr Trp Trp Pro
165 170 175
cag atc ttc ctg ggg ctg gcc ttc aac tgg ggc gtc atg ctg gcc tat 576
Gln Ile Phe Leu Gly Leu Ala Phe Asn Trp Gly Val Met Leu Ala Tyr
180 185 190
gcc gcg cat gcg ggc cgt gtc gat gcg gcc cct gtc gtg gca tgg ctg 624
Ala Ala His Ala Gly Arg Val Asp Ala Ala Pro Val Val Ala Trp Leu
195 200 205
ggg gcc atc gcc tgg acg atc ttc tac gac acc atc tat gcc cac cag 672
Gly Ala Ile Ala Trp Thr Ile Phe Tyr Asp Thr Ile Tyr Ala His Gln
210 215 220
gac gcc gag gac gac gcc ctg atc ggg gtg aaa tcc acc gcg cgg ctg 720
Asp Ala Glu Asp Asp Ala Leu Ile Gly Val Lys Ser Thr Ala Arg Leu
225 230 235 240
ttc ggc gac aaa agc ccg cgc atc ctt gcg gga ttc gcc ctg ggc gcg 768
Phe Gly Asp Lys Ser Pro Arg Ile Leu Ala Gly Phe Ala Leu Gly Ala
245 250 255
gtc ctg gtg ctg atg ctg gcc acc gcg ctg ccc ggt cgc aat ctg ttg 816
Val Leu Val Leu Met Leu Ala Thr Ala Leu Pro Gly Arg Asn Leu Leu
260 265 270
att gcc tgg gcg ggc gtc gcg ggt ttc ggc ctg cac cta ggc tgg cag 864
Ile Ala Trp Ala Gly Val Ala Gly Phe Gly Leu His Leu Gly Trp Gln
275 280 285
ctt cgc aaa ttc cag ccg gat cag ggc gat acc tgc ctg cgc ctg ttc 912
Leu Arg Lys Phe Gln Pro Asp Gln Gly Asp Thr Cys Leu Arg Leu Phe
290 295 300
cgg tcc aac cgc gat gcg ggg ctg atc cttgcg ctg ttt ctt gcc gtg 960
Arg Ser Asn Arg Asp Ala Gly Leu Ile Leu Ala Leu Phe Leu Ala Val
305 310 315 320
gcg ggc ctc gca tga 975
Ala Gly Leu Ala
<210>20
<211>324
<212>PRT
<213>Paracoccus zeaxanthinifaciens
<400>20
Met Asn Asn Arg Ile Phe Ala Met Met Gly Asn Ala Met Gln Ser Ser
1 5 10 15
Thr Glu Arg Pro Asp Ala Val Val Asp Ala Pro Lys Gly Asn Trp Val
20 25 30
Asp Glu Ile Ala Pro Pro Trp Ser Arg Pro Trp Leu Arg Leu Ser Arg
35 40 45
Ala Asp Arg Pro Ile Gly Thr Trp Leu Leu Leu Leu Pro Cys Trp Trp
50 55 60
Gly Ile Gly Leu Ala Met Met Ala Asp Gly Pro Arg Trp Phe Asp Ala
65 70 75 80
Trp Ile Ala Leu Ala Cys Thr Ile Gly Ala Phe Val Met Arg Gly Ala
85 90 95
Gly Cys Thr Trp Asn Asp Ile Thr Asp Arg Arg Ile Asp Ala Gln Val
100 105 110
Ala Arg Thr Arg Ser Arg Pro Leu Pro Ser Gly Gln Val Thr Leu Arg
115 120 125
Gly Ala Tyr Gly Trp Leu Ile Ala Gln Gly Leu Ile Gly Leu Ala Ile
130 135 140
Leu Leu Thr Leu Gly Gln Ala Ala Ile Trp Met Gly Val Ala Ser Leu
145 150 155 160
Ala Leu Val Ala Ile Tyr Pro Phe Ala Lys Arg Phe Thr Trp Trp Pro
165 170 175
Gln Ile Phe Leu Gly Leu Ala Phe Asn Trp Gly Val Met Leu Ala Tyr
180 185 190
Ala Ala His Ala Gly Arg Val Asp Ala Ala Pro Val Val Ala Trp Leu
195 200 205
Gly Ala Ile Ala Trp Thr Ile Phe Tyr Asp Thr Ile Tyr Ala His Gln
210 215 220
Asp Ala Glu Asp Asp Ala Leu Ile Gly Val Lys Ser Thr Ala Arg Leu
225 230 235 240
Phe Gly Asp Lys Ser Pro Arg Ile Leu Ala Gly Phe Ala Leu Gly Ala
245 250 255
Val Leu Val Leu Met Leu Ala Thr Ala Leu pro Gly Arg Asn Leu Leu
260 265 270
Ile Ala Trp Ala Gly Val Ala Gly Phe Gly Leu His Leu Gly Trp Gln
275 280 285
Leu Arg Lys Phe Gln Pro Asp Gln Gly Asp Thr Cys Leu Arg Leu Phe
290 295 300
Arg Ser Asn Arg Asp Ala Gly Leu Ile Leu Ala Leu Phe Leu Ala Val
305 310 315 320
Ala Gly Leu Ala
<210>21
<211>30
<212>DNA
<213>人工
<220>
<221>misc_feature
<222>(1)..(30)
<223>PCR引物ubiA-Nde
<400>21
aaggcctcat atgaacaatc gtatcttcgc 30
<210>22
<211>28
<212>DNA
<213>人工
<220>
<221>misc_feature
<222>(1)..(28)
<223>PCR引物ubiA-Bam
<400>22
cgggatcctc atgcgaggcc cgccacgg 28
<210>23
<211>975
<212>DNA
<213>Paracoccus zeaxanthinifaciens
<220>
<221>CDS
<222>(1)..(975)
<223>
<400>23
atg aac aat cgt atc ttc gcc atg atg ggc aac gct atg caa agc agc 48
Met Asn Asn Arg Ile Phe Ala Met Met Gly Asn Ala Met Gln Ser Ser
1 5 10 15
acg gaa aga cca gac gcg gtc gtc gac gcg ccg aag gga aac tgg gtc 96
Thr Glu Arg Pro Asp Ala Val Val Asp Ala Pro Lys Gly Asn Trp Val
20 25 30
gat gag atc gcc ccg cca tgg tcg cgc ccc tgg ctg cgg ctc agc cgc 144
Asp Glu Ile Ala Pro Pro Trp Ser Arg Pro Trp Leu Arg Leu Ser Arg
35 40 45
gcg gac cgg ccc atc ggg aca tgg ctg ctg ctg ctg ccc tgc tgg tgg 192
Ala Asp Arg Pro Ile Gly Thr Trp Leu Leu Leu Leu Pro Cys Trp Trp
50 55 60
ggg atc ggg ctg gcg atg atg gca gac ggg ccg cgc tgg ttc gat gcg 240
Gly Ile Gly Leu Ala Met Met Ala Asp Gly Pro Arg Trp Phe Asp Ala
65 70 75 80
tgg atc gcg ctg gcc tgc acc atc ggc gcc ttc gtc atg cgg ggc gcg 288
Trp Ile Ala Leu Ala Cys Thr Ile Gly Ala Phe Val Met Arg Gly Ala
85 90 95
ggc tgc acc tgg aac gac atc acc gac cgc cgg atc gac gcg cag gtc 336
Gly Cys Thr Trp Asn Asp Ile Thr Asp Arg Arg Ile Asp Ala Gln Val
100 105 110
gca cgc acc cgc tcg cgc ccg ctg cca agc gga cag gtc acg ctg cgg 384
Ala Arg Thr Arg Ser Arg Pro Leu Pro Ser Gly Gln Val Thr Leu Arg
115 120 125
ggc gcc tat ggc tgg ctg atc gcg cag ggg ctg atc ggg ctg gcg atc 432
Gly Ala Tyr Gly Trp Leu Ile Ala Gln Gly Leu Ile Gly Leu Ala Ile
130 135 140
ctg ctg acc ctg ggg cag gcc gcg atc tgg atg ggc gtc gcc tcg ctg 480
Leu Leu Thr Leu Gly Gln Ala Ala Ile Trp Met Gly Val Ala Ser Leu
145 150 155 160
gcg ctg gtc gcg atc tat ccc ttc gcg aaa cgc ttc acc tgg tgg ccg 528
Ala Leu Val Ala Ile Tyr Pro Phe Ala Lys Arg Phe Thr Trp Trp Pro
165 170 175
cag atc ttc ctg ggg ctg gcc ttc aac tgg ggc gtc atg ctg gcc tat 576
Gln Ile Phe Leu Gly Leu Ala Phe Asn Trp Gly Val Met Leu Ala Tyr
180 185 190
gcc gcg cat gcg ggc cgt gtc gat gcg gcc cct gtc gtg gca tgg ctg 624
Ala Ala His Ala Gly Arg Val Asp Ala Ala Pro Val Val Ala Trp Leu
195 200 205
ggg gcc atc gcc tgg acg atc ttc tac gac acc acc tat gcc cac cag 672
Gly Ala Ile Ala Trp Thr Ile Phe Tyr Asp Thr Thr Tyr Ala His Gln
210 215 220
gac gcc gag gac gac gcc ctg atc ggg gtg aaa tcc acc gcg cgg ctg 720
Asp Ala Glu Asp Asp Ala Leu Ile Gly Val Lys Ser Thr Ala Arg Leu
225 230 235 240
ttc ggc gac aaa agc ccg cgc atc ctt gcg gga ttc gcc ctg ggc gcg 768
Phe Gly Asp Lys Ser Pro Arg Ile Leu Ala Gly Phe Ala Leu Gly Ala
245 250 255
gtc ctg gtg ctg atg ctg gcc acc gcg ctg ccc ggt cgc aat ctg ttg 816
Val Leu Val Leu Met Leu Ala Thr Ala Leu Pro Gly Arg Asn Leu Leu
260 265 270
att gcc tgg gcg ggc gtc gcg ggt ttc ggc ctg cac cta ggc tgg cag 864
Ile Ala Trp Ala Gly Val Ala Gly Phe Gly Leu Hig Leu Gly Trp Gln
275 280 285
ctt cgc aaa ttc cag ccg gat cag ggc gat acc tgc ctg cgc ctg ttc 912
Leu Arg Lys Phe Gln Pro Asp Gln Gly Asp Thr Cys Leu Arg Leu Phe
290 295 300
cgg tcc aac cgc gat gcg ggg ctg atc ctt gcg ctg ttt ctt gcc gtg 960
Arg Ser Asn Arg Asp Ala Gly Leu Ile Leu Ala Leu Phe Leu Ala Val
305 310 315 320
gcg ggc ctc gca tga 975
Ala Gly Leu Ala
<210>24
<211>324
<212>PRT
<213>Paracoccus zeaxanthinifaciens
<400>24
Met Asn Asn Arg Ile Phe Ala Met Met Gly Asn Ala Met Gln Ser Ser
1 5 10 15
Thr Glu Arg Pro Asp Ala Val Val Asp Ala Pro Lys Gly Asn Trp Val
20 25 30
Asp Glu Ile Ala Pro Pro Trp Ser Arg Pro Trp Leu Arg Leu Ser Arg
35 40 45
Ala Asp Arg Pro Ile Gly Thr Trp Leu Leu Leu Leu Pro Cys Trp Trp
50 55 60
Gly Ile Gly Leu Ala Met Met Ala Asp Gly Pro Arg Trp Phe Asp Ala
65 70 75 80
Trp Ile Ala Leu Ala Cys Thr Ile Gly Ala Phe Val Met Arg Gly Ala
85 90 95
Gly Cys Thr Trp Asn Asp Ile Thr Asp Arg Arg Ile Asp Ala Gln Val
100 105 110
Ala Arg Thr Arg Ser Arg Pro Leu Pro Ser Gly Gln Val Thr Leu Arg
115 120 125
Gly Ala Tyr Gly Trp Leu Ile Ala Gln Gly Leu Ile Gly Leu Ala Ile
130 135 140
Leu Leu Thr Leu Gly Gln Ala Ala Ile Trp Met Gly Val Ala Ser Leu
145 150 155 160
Ala Leu Val Ala Ile Tyr Pro Phe Ala Lys Arg Phe Thr Trp Trp Pro
165 170 175
Gln Ile Phe Leu Gly Leu Ala Phe Asn Trp Gly Val Met Leu Ala Tyr
180 185 190
Ala Ala His Ala Gly Arg Val Asp Ala Ala Pro Val Val Ala Trp Leu
195 200 205
Gly Ala Ile Ala Trp Thr Ile Phe Tyr Asp Thr Thr Tyr Ala His Gln
210 215 220
Asp Ala Glu Asp Asp Ala Leu Ile Gly Val Lys Ser Thr Ala Arg Leu
225 230 235 240
Phe Gly Asp Lys Ser Pro Arg Ile Leu Ala Gly Phe Ala Leu Gly Ala
245 250 255
Val Leu Val Leu Met Leu Ala Thr Ala Leu Pro Gly Arg Asn Leu Leu
260 265 270
Ile Ala Trp Ala Gly Val Ala Gly Phe Gly Leu His Leu Gly Trp Gln
275 280 285
Leu Arg Lys Phe Gln Pro Asp Gln Gly Asp Thr Cys Leu Arg Leu Phe
290 295 300
Arg Ser Asn Arg Asp Ala Gly Leu Ile Leu Ala Leu Phe Leu Ala Val
305 310 315 320
Ala Gly Leu Ala
Claims (9)
1.一种经分离的DNA，其中包含(1)编码癸异戊二烯基二磷酸酯(DPP)合酶的核苷酸序列，所述核苷酸序列选自由以下序列构成的组：
(a)SEQ ID NO：16所示的DNA序列或其互补链；
(b)一种DNA序列，其在标准条件下能与(a)中定义的DNA序列或其片段杂交，并编码具有DPP合酶活性的多肽；
(c)一种DNA序列，其与编码SEQ ID NO：17所示的多肽的DNA至少80％相同，并且其编码具有DPP合酶活性的多肽；和
(d)一种DNA序列，其编码与SEQ ID NO：17所示的氨基酸序列至少80％相同的多肽，并编码具有DPP合酶活性的多肽；以及
(2)编码4-羟基苯甲酸聚异戊二烯基转移酶的核苷酸序列，所述核苷酸序列选自由以下序列构成的组：
(a’)SEQ ID NO：23所示的DNA序列或其互补链；
(b’)一种DNA序列，其在标准条件下能与(a’)中定义的DNA序列或其片段杂交，并编码具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽；
(c’)一种DNA序列，其与编码SEQ ID NO：24所示的多肽的DNA至少80％相同，并且其编码具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽；和
(d’)一种DNA序列，其编码与SEQ ID NO：24所示的氨基酸序列至少80％相同的多肽，并编码具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽。
2.一种多肽，包含(1)具有氨基酸序列SEQ ID NO：17，或较之SEQ ID NO：17有一个或几个氨基酸缺失、添加或插入的氨基酸序列，并具有DPP合酶活性的多肽，和(2)具有氨基酸序列SEQ ID NO：24，或较之SEQ ID NO：24有一个或几个氨基酸缺失、添加或插入的氨基酸序列，并具有4-羟基苯甲酸聚异戊二烯基转移酶活性的多肽。
3.包含如权利要求1所述的DNA的构建体。
4.如权利要求3所述的构建体，进一步地包含调控序列。
5.包含如权利要求1所述的DNA的微生物。
6.包含如权利要求3或4所述的构建体的微生物。
7.一种在微生物中生产辅酶Q-10的方法，所述方法包括如下步骤：
(a)增加具有DPP合酶的蛋白质和/或具有4-羟基苯甲酸聚异戊二烯基转移酶的蛋白质的活性，以及
(b)在生产辅酶Q-10的条件下，于培养基中培养所述的微生物。
8.如权利要求7所述的方法，其中编码DPP合酶的ddsA的表达，和/或编码4-羟基苯甲酸聚异戊二烯基转移酶的ubiA的表达被增加。
9.如权利要求7或8所述的方法，进一步包含通过在所述微生物中对crtE基因进行突变来去除香叶基香叶基二磷酸酯(GGPP)合酶活性的步骤。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US44862603P | 2003-02-19 | 2003-02-19 | |
| US60/448,626 | 2003-02-19 | ||
| PCT/EP2004/001380 WO2004074487A1 (en) | 2003-02-19 | 2004-02-13 | Microbial production of coq10 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1751124A true CN1751124A (zh) | 2006-03-22 |
| CN1751124B CN1751124B (zh) | 2010-05-12 |
Family
ID=32908618
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200480004590XA Expired - Fee Related CN1751124B (zh) | 2003-02-19 | 2004-02-13 | CoQ10的微生物生产方法 |
Country Status (7)
| Country | Link |
|---|---|
| US (1) | US7435571B2 (zh) |
| EP (1) | EP1594967A1 (zh) |
| JP (1) | JP4511517B2 (zh) |
| KR (1) | KR20050108359A (zh) |
| CN (1) | CN1751124B (zh) |
| BR (1) | BRPI0407691A (zh) |
| WO (1) | WO2004074487A1 (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20080047144A (ko) * | 2006-11-24 | 2008-05-28 | 에스케이에너지 주식회사 | 높은 조효소 ｑ１０ 함량을 갖는 로도박터 스패로이데스ｓｋ２ｈ２ 균주 및 이를 이용한 조효소 ｑ１０의 생산방법 |
| KR100738681B1 (ko) * | 2007-04-11 | 2007-07-12 | 대상 주식회사 | 코엔자임 ｑ10의 생산성이 우수한 변이주 및 코엔자임ｑ10의 생산방법 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3891504A (en) * | 1970-07-31 | 1975-06-24 | Hoffmann La Roche | Process for the manufacture of zeaxanthin |
| CA2718652C (en) * | 1999-10-14 | 2014-07-29 | Kyowa Hakko Bio Co., Ltd. | Process for producing ubiquinone-10 |
-
2004
- 2004-02-13 KR KR1020057015169A patent/KR20050108359A/ko not_active Application Discontinuation
- 2004-02-13 WO PCT/EP2004/001380 patent/WO2004074487A1/en active Application Filing
- 2004-02-13 US US10/546,109 patent/US7435571B2/en active Active
- 2004-02-13 JP JP2006501840A patent/JP4511517B2/ja not_active Expired - Fee Related
- 2004-02-13 EP EP04710850A patent/EP1594967A1/en not_active Withdrawn
- 2004-02-13 CN CN200480004590XA patent/CN1751124B/zh not_active Expired - Fee Related
- 2004-02-13 BR BRPI0407691-5A patent/BRPI0407691A/pt not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| WO2004074487A1 (en) | 2004-09-02 |
| KR20050108359A (ko) | 2005-11-16 |
| BRPI0407691A (pt) | 2006-03-01 |
| US20060088920A1 (en) | 2006-04-27 |
| EP1594967A1 (en) | 2005-11-16 |
| CN1751124B (zh) | 2010-05-12 |
| JP4511517B2 (ja) | 2010-07-28 |
| JP2006517794A (ja) | 2006-08-03 |
| US7435571B2 (en) | 2008-10-14 |
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