CN100526457C - L-丝氨酸的抑制性被降低的3-磷酸甘油酸脱氢酶变体及其编码基因 - Google Patents
L-丝氨酸的抑制性被降低的3-磷酸甘油酸脱氢酶变体及其编码基因 Download PDFInfo
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- CN100526457C CN100526457C CNB2004100635275A CN200410063527A CN100526457C CN 100526457 C CN100526457 C CN 100526457C CN B2004100635275 A CNB2004100635275 A CN B2004100635275A CN 200410063527 A CN200410063527 A CN 200410063527A CN 100526457 C CN100526457 C CN 100526457C
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Abstract
3-磷酸甘油酸脱氢酶(PGD),其与大肠杆菌野生型PGD相比具有对丝氨酸抑制的降低的易感性,且其具有不同于大肠杆菌野生型PGD氨基酸序列(SEQ ID NO:2)的氨基酸序列,且其不同之处在于第349位的氨基酸不是甘氨酸或第372位的氨基酸不是苏氨酸。
Description
技术领域
本发明涉及L-丝氨酸的抑制性被降低的3-磷酸甘油酸脱氢酶变体及其编码基因。
背景技术
目前,主要采用微生物发酵来制备20种天然的、形成蛋白质的氨基酸。就此而言,此应用是基于微生物具有合成天然氨基酸的适当的生物合成途径。
然而,在野生株中,此类生物合成途径收到严格控制,以确保氨基酸的产生仅用来以满足细胞的内部需求。在许多生物合成中的一个重要控制机制的实例为反馈抑制(或终产物抑制)现象。在反馈抑制中,通常是在一种生物合成途径中催化该生物合成途径的最初酶促反应的酶被该生物合成途径的终产物所抑制。此抑制常由终产物与酶发生别构结合而产生的构象变化所致,该构象变化使得酶转为失活状态。由此确保当终产物在细胞中累积时,通过抑制起始的步骤而终止进一步的合成。
因此仅在消除源自代谢途径的反馈抑制的限制的情况下,才有可能以工业规模有效生产代谢产物(如氨基酸),由此得到那些相对于野生型生物体而言其产生所需代谢产物的能力大幅增加的微生物。
磷酸甘油酸族的氨基酸是由3-磷酸甘油酸(3-phosphoglycerate)通过生物合成衍生而来的氨基酸。就此而言,天然的代谢途径经由中间物3-磷酸羟基丙酮酸(3-phosphohydroxypyruvate)和3-磷酸-L-丝氨酸最初得到L-丝氨酸。L-丝氨酸其后可经转化成甘氨酸或者经由O-乙酰丝氨酸转化为L-半胱氨酸。L-色氨酸亦包括在此类氨基酸中,因为其同样地衍生自L-丝氨酸的生物合成。在相同的方式下,使用述于美国专利申请第2002/0039767A1号的方法制备的非天然氨基酸也被归类为磷酸甘油酸族。
衍生自C1代谢的化合物同样地依赖磷酸甘油酸族氨基酸的生物合成。这是由于,在L-丝氨酸转化为甘氨酸时,四氢叶酸作为C1基的受体,而装载的四氢叶酸作为C1代谢中的中心甲基供体(central methyl groupdonor),参与多种生物合成(例如L-甲硫氨酸、核苷酸、泛酸等)。根据本发明,衍生自C1代谢的化合物因此为优选的化合物,其生物合成依赖通过四氢叶酸的C1基转移。
属于磷酸甘油酸族的氨基酸的生物合成的最初步骤为D-3-磷酸甘油酸经氧化成3-磷酸羟基丙酮酸,由3-磷酸甘油酸脱氢酶(PGD)[EC 1.1.1.95]所催化。NAD+,其经转化成NADH/H+,作为在反应中形成的还原当量的受体。
已知PGD酶来自多种生物体(例如褐家鼠(Rattus norvegicus)、拟南芥(Arabidopsis thaliana)、大肠杆茵(E.coli)、枯草芽孢杆菌(Bacillussubtilis))。此类酶中了解较深入的代表微生物受制于L-丝氨酸的反馈抑制。
在氨基酸序列水平上,微生物PGD酶在N-端部分(在大肠杆菌序列中为氨基酸1-340)上彼此极相似,而C-端部分仅具有轻微的相似性。然而,毫无疑问的是,负责丝氨酸抑制的调节结构域即位于此C-端部分中(Peter-Wendisch等人,2002,Appl.Microbiol Biotechnol.60:437-441)。
研究最为深入的PGD来自大肠杆菌。该酶在生化方面经详细探讨(Dubrow和Pizer,J.Biol.Chem.252,1527-1538),其受到L-丝氨酸的别构反馈抑制,抑制常数Ki为5μM。
此反馈抑制阻碍了属于磷酸甘油酸族的氨基酸的有效生产,因此早已为分子生物学研究的靶向。
因此,文献EP 0620853A描述了大肠杆菌PGD的变体,其较少对丝氨酸的抑制比较不敏感,且其在野生型PGD的C-端一侧25%(即氨基酸307-410)具有修饰,优选为最后50个残基区域(即氨基酸361-410)的修饰。其中所说的突变体由接头诱变(linker mutagenesis)而得,即由简单利用大肠杆菌serA基因中存在的限制性酶切位点并插入8-14个碱基对长的寡核苷酸接头。
然而,如此接头诱变常导致一些问题,因为添加或缺失一些残基大大地改变蛋白质的结构,且因此对蛋白质的总活性和稳定性有着负面的影响。事实上,EP 0620853A中所述的大部分突变体几乎不能够检出活性。
在棒杆菌微生物的serA基因上也进行了降低PGD对丝氨酸抑制的易感性的诱变:
-Peters-Wendisch等(2002,Appl.Microbiol.Biotechnol.60:437-441)描述了谷氨酸棒杆菌(Corynebacterium glutamicum)PGD的C-端缺失。在此例中,同样地,缺失导致一些酶活性的明显损失。
-专利申请EP0943687A2描述了在黄色短杆菌(Brevibacteriumflavum)PGD中位置325的谷氨酸残基的取代。在使用GCG(GCG威斯康星软件包,遗传学计算机小组(GCG),麦迪逊,威斯康星州)程序的GAP演算法形成的比对中,此残基参照大肠杆菌PGD,已经定位于蛋白质的可变C-端部分,且相应于大肠杆菌蛋白质的天冬酰胺残基364。因为此修饰位于PGD的可变C-端部分,所以不可能得到关于该大肠杆菌蛋白质的任何结论。
发明内容
本发明的目的在于制备一种大肠杆菌PGD的变体,其与大肠杆菌野生型PGD相比,对丝氨酸抑制的易感性降低。
此目的通过一种PGD而实现,该PGD的氨基酸序列与大肠杆菌野生型PGD的位置1为甲硫氨酸的氨基酸序列(SEQ ID NO:2)的不同之处在于第349位的氨基酸不是甘氨酸或者第372位的氨基酸不是苏氨酸。
本发明的PGD亦可在上述SEQ ID NO:1中的两个位置均发生突变。
本发明还涉及一种编码本发明PGD的DNA序列。此serA等位基因不同于大肠杆菌PGD基因(serA基因,SEQ ID NO:1),其中密码子349编码一种非甘氨酸的天然氨基酸或密码子372编码一种非苏氨酸的天然氨基酸。
本发明的serA等位基因亦可同时拥有上述两个密码子的突变。
在本文内,经GAP演算法(GCG威斯康星软件包,遗传学计算机小组(GCG),麦迪逊,威斯康星州)分析后发现具有大于30%的序列相同性的那些基因,也被视为本发明的serA等位基因,条件是其具有上述突变之一。特别优选的是序列相同性大于70%。
在相同方式下,具大于40%的序列相同性的蛋白质,如由使用GAP演算法测定,在本发明的意义中,被视为衍生自大肠杆菌PGD的蛋白质,条件是其具有PGD活性和上述氨基酸取代之一。特别优选的是序列相同性大于70%。
此外,由核苷酸的缺失、插入或取代而衍生自SEQ ID No:1的序列的serA基因的等位基因变体,且其基因产物的酶活性为相应的野生型基因产物的活性的10%以上,且具有甘氨酸密码子349或苏氨酸密码子372的突变、或上述两个突变的组合,此类serA基因的等位基因变体应该被视为本发明的基因。
具有第349位或第372位的氨基酸取代或其组合的PGD变体,可使用分子生物学的标准技术产生。为达到此目的,可将突变导入编码PGD的serA基因的对应密码子中。在DNA片段内的特定位置上导入突变的合适方法是已知的。
大肠杆菌serA基因的DNA优选作为诱变的材料。待突变的serA基因可在染色体或染色体外编码。然而,优选地以聚合酶链反应扩增serA基因并克隆入载体。用前述的诱变方法改变DNA序列中的一或多个核苷酸,以使得编码的PGD在第349位或第372位的氨基酸发生取代,而第1位为SEQ ID NO:1的起始甲硫氨酸。
此类突变产生对L-丝氨酸抑制较不敏感(=反馈抗性)的编码的PGD。就此,特别有利地,本发明的PGD变体的活性在无L-丝氨酸的情况下为野生型PGD活性的10%以上,且优选为活性未改变。
能在无L-丝氨酸情况下测定酶活性的任何方法可用以测定由本发明的PGD变体所具有的反馈抗性程度。例如,PGD活性可采用类似于McKitrick和Pizer所述的方法(1980,J.Bacteriol.141:235-245)测定。用逆反应来测定含有磷酸羟基丙酮酸和NADH/H+的分析样品中的酶活性。加入酶开始反应,因NADH/H+氧化引起吸光值的降低,在分光光度计中监测在340纳米下吸光值的降低。PGD活性的抑制在不同浓度的L-丝氨酸下在反应混合液中测试。不同PGD变体的催化活性在无和有L-丝氨酸下测定,并自此类数值计算抑制剂常数Ki。Ki表示其活性为在无抑制剂下测定的活性的50%的抑制剂浓度。
因为具有反馈抗性,本发明的PGD酶可用以产生磷酸甘油酸或衍生自C1代谢的化合物的氨基酸。为此目的,本发明的serA等位基因在宿主株中表达。
本发明的serA等位基因可在其自身启动子的控制下表达,其位于serA基因的上游,或由使用本领域人员所知的其他合适启动子系统。就此,对应的等位基因可例如在此类启动子的控制下,以一或以多个拷贝存在于宿主生物体的染色体中。整合基因至染色体的策略为本领域的现有技术。然而,优选地将要表达的serA等位基因克隆入载体,优选为质粒。
本发明因此还涉及含有置于启动子的功能控制下的本发明的serA等位基因的载体。
为克隆本发明的serA等位基因,可使用己含有遗传元件(例如组成性或可调节的启动子和终止子)的载体,其可实现编码PGD基因的连续表达或受控的、可诱导的表达。此外,其他调节元件,如核糖体结合位点和终止序列,以及编码选择性标记和/或受体基因的序列亦优选位于表达载体中。此类选择性标记的表达促进转化体的鉴定。合适的选择性标记为编码对例如氨苄青霉素、四环素、氯霉素或卡那霉素或其他抗生素的抗性基因。若本发明的serA等位基因在染色体外复制时,质粒载体应优选含有复制起点。特别优选地为一些质粒载体如大肠杆菌载体pACYC184、pUC18、pQE-70、pSC322和pSC101及其衍生物。合适的可诱导启动子的实例为lac、tac、trc、λPL、ara和tet启动子或自其衍生的序列。
此外,特别优选的是已含有基因/等位基因的质粒载体,所述基因/等位基因同样地导致过度产生磷酸甘油酸族的氨基酸或衍生自C1代谢的化合物的氨基酸,如产生以下的基因/等位基因:
-L-丝氨酸(例如述于DE10044831A1的serB基因、serC基因或输出载体基因(export carrier gene))
-N-乙酰丝氨酸、O-乙酰丝氨酸、胱氨酸、半胱氨酸或半胱氨酸衍生物(例如述于WO97/15673的cysE等位基因、如述于EP0885962A1的外排基因(efflux gene)、如述于DE19949579C1的cysB基因或如述于DE10232930A的yfiK基因)
-L-色氨酸(例如述于EP0662143A的trpE等位基因)
-泛酸(例如述于WO02061108)
此类载体可直接自任何任意微生物株制备具有高产量的本发明的微生物株,因为此类质粒还可中和微生物中代谢途径中的其他限制。
可用传统转化方法(例如电穿孔法)导入含serA等位基因的本发明的质粒,并例如通过抗生素抗性选择携带质粒的克隆。
本发明因此还涉及一种产生本发明的微生物株的方法,其包括将本发明的载体导入微生物株中。
还可以将具有serA等位基因的本发明载体导入微生物中,所述微生物例如已自染色体表达一或多个上述基因/等位基因且己可过度产生一种代谢产物。在此种情况下,本发明的serA等位基因的导入可进一步增加产量。
一般而言,具有生物合成磷酸甘油酸族氨基酸的途径的、可自重组方法获得的、且可通过发酵而培养的所有生物体,均适合作为本发明的载体的宿主生物体。此类微生物可为真菌、酵母或细菌。优选地使用真细菌系统发生群的细菌。特别优选地为肠细菌科的微生物,且特别是大肠杆菌。
本发明因此还涉及一种微生物株,其适合用于发酵产生磷酸甘油酸族的氨基酸或其衍生物、或衍生自C1代谢的化合物,该菌株具有本发明的PGD。
本发明还涉及通过培养本发明的微生物株来生产磷酸甘油酸族的氨基酸或衍生自C1代谢的化合物。
为此目的,将本发明的微生物株在例如发酵罐的营养培养基中培养,所述培养基含有合适的碳源和合适的能量源以及其他添加物。
在发酵期间形成的物质,如L-磷酸丝氨酸、L-丝氨酸、O-乙酰基-L-丝氨酸、L-半胱氨酸、甘氨酸、L-色氨酸、1,2,4-三唑-2-基-L-丙氨酸、L-甲硫氨酸或泛酸,可随后被纯化。
以下的实施例用以阐明本发明。采用的全部分子生物学方法,如聚合酶链反应、DNA的分离和纯化、DNA的限制酶修饰、Klenow片段和连接酶、转化等是以本领域人员已知的方式、以述于文献中的方式或以相应的厂商建议的方式进行。
实施例
实施例1:克隆serA基因
以聚合酶链反应自大肠杆菌菌株W3110(美国典型培养物保藏中心,ATCC27325)中扩增serA基因。以核苷酸
serA-fw:(SEQ ID NO:3)
5’-GAA TTC CAT ATG GCAAAG GTA TCG CTG GAG-3’
NdeI
和
serA-rev:(SEQ ID NO:4)
5’-AGA AAG CTT TTA TTA GTA CAG CAG ACG GGC-3’
HindIII
作为特定的引物。
生成的DNA片段以限制酶NdeI和HindIII消化,并将5’突出端用Klenow酶填充。DNA片段其后通过琼脂糖凝胶电泳法纯化和使用GeneClean法(GeneClean试剂盒BIO101,邮政信箱2284,La Jolla,加利福尼亚,92038-2284)分离。以此方式得到的serA片段经克隆入表达载体pQE-70(Qiagen,Hilden,德国)。为达成此目的,载体先以SphI和BamHI酶切,将3’突出端使用Klenow酶消化掉,并将5’突出端使用Klenow酶填充。载体片段然后经纯化并连结至serA片段上。生成的载体命名为pFL209。在构建体由测序确认后,转化大肠杆菌株JM109(Stratagene,阿姆斯特丹,荷兰),相应的转化株以氨苄青霉素筛选。根据布达佩斯条约,大肠杆菌菌株JM109/pFL209保藏于DSMZ(德国微生物保藏中心,D-38142 Braunschweig),保藏号为DSM 15628。
实施例2:serA基因的定点诱变
采用逆转录聚合酶链反应以进行serA基因的密码子349和372上的定点诱变。使用实施例1所述的载体pFL209作为模板。引物
serA40-mut(SEQ ID NO:5)
5’-GAA AAC CGT CCG NNN GTG CTA ACT GCG-3’
N=G、A、T或C
及
serA40-rev(SEQ ID NO:6)
5’-GTG GAT GTG CAT CAG ACG-3’
用于诱变密码子349。
将生成的PCR产物连接成环状并转化入大肠杆菌株JM109。最后,测序以确定密码子349上的突变,并核对其余序列的正确性。
原则上,采用相同的程序用以诱变密码子372,但使用的引物为
serA20-mut2(SEQ ID NO:7)
5’-CAA TAT CTG CAA NNN TCC GCC CAG ATG GG-3’
N=G、A、T或C
及
serA20-rev(SEQ ID NO:8)
5’-CGC GGC GAT GTT GAC GCC-3’。
实施例3:确定PGD活性及抑制剂常数Ki
为确定PGD酶活性及L-丝氨酸对其活性的影响,将100毫升体积的LB培养基(10克/升的胰蛋白胨,5克/升的酵母提取物,10克/升的NaCl),其中额外含有100毫克/升的氨苄青霉素,在各例中接种了2毫升的携带编码serA等位基因的质粒的过夜培养菌株,并在30℃和150rpm下在振荡器中培养。在光学密度1.0时,在各例中加入0.4mM异丙基-β-硫代半乳糖苷以诱导serA表达,并将培养物再培养3小时。其后离心收集细胞、洗涤及再悬浮于2毫升缓冲液(100mM磷酸钾,pH7.0;10mMMgCl2;1mM二硫苏糖醇)中。细胞使用French press(Spectronic仪器公司,洛彻斯特,纽约州,美国)在18000psi的压力下裂解。30000g离心以使粗提取物澄清,使用McKitrick和Pizer试验法(1980,J.Bacteriol.141:235-245)测定PGD活性。
以下的各表显示不同突变株的PGD活性和相应的抑制剂常数Ki。
表1:密码子349的突变
等位基因 | 突变 | 活性[单位/毫克] | K<sub>i</sub>[mM] |
serA | 野生型 | 0.05 | <0.1 |
serA40 | G349D | 0.05 | 25 |
serA45 | G349I | 0.05 | 5 |
serA46 | G349M | 0.05 | 1 |
serA47 | G349E | 0.05 | 20 |
serA49 | G349P | 0.05 | 6 |
serA410 | G349S | 0.04 | 2 |
serA411 | G349T | 0.04 | 3 |
serA412 | G349V | 0.05 | 5 |
serA413 | G349L | 0.05 | 5 |
serA414 | G349A | 0.05 | 1 |
serA415 | G349K | 0.03 | 15 |
serA416 | G349R | 0.04 | 15 |
serA417 | G349W | 0.02 | 8 |
serA418 | G349Y | 0.05 | 6 |
serA419 | G349F | 0.05 | 10 |
serA420 | G349H | 0.05 | 10 |
serA421 | G349N | 0.05 | 15 |
serA422 | G349Q | 0.05 | 15 |
serA45 | G349C | 0.04 | 5 |
表2:密码子372的突变
等位基因 | 突变 | 活性[单位/毫克] | K<sub>i</sub>[mM] |
serA | 野生型 | 0.05 | <0.1 |
serA20 | T372I | 0.05 | 40 |
serA21 | T372D | 0.05 | 120 |
serA211 | T372Y | 0.05 | 35 |
serA219 | T372G | 0.05 | 8 |
serA220 | T372S | 0.05 | 1 |
serA223 | T372E | 0.05 | 150 |
serA229 | T372R | 0.05 | 120 |
serA234 | T372K | 0.05 | 110 |
serA206 | T372P | 0.05 | 120 |
serA208 | T372H | 0.05 | 80 |
serA210 | T372W | 0.04 | 60 |
serA212 | T372F | 0.05 | 60 |
serA214 | T372A | 0.04 | 10 |
serA218 | T372N | 0.05 | 100 |
serA221 | T372Q | 0.05 | 100 |
serA222 | T372V | 0.05 | 40 |
serA226 | T372L | 0.04 | 40 |
serA228 | T372M | 0.03 | 60 |
serA231 | T372C | 0.02 | 3 |
实施例4:等位基因serA20和serA40同时突变
密码子349和372同时突变可显示出是否取代在反馈抑制上具有协同作用。为此使用了两个突变位点间的一个独特HindIII限制性酶切位点。因此,以HindII/HindIII限制性酶切含有serA20等位基因的载体以分离一个183bp片段,其对应着serA基因的3’端并含有在密码子372上的突变。将此片段克隆入一载体,其含有serA40等位基因且已经用HindII/BamHI消化,由此生成具有双突变的克隆。下表显示了从属酶数据(appurtenantenzyme data)。
表3:密码子349和372的突变
等位基因 | 突变 | 活性[单位/毫克] | K<sub>i</sub>[mM] |
serA | 野生型 | 0.05 | <0.1 |
serA2040 | G349D、T372I | 0.05 | 120 |
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Claims (9)
1.一种3-磷酸甘油酸脱氢酶,其与大肠杆菌野生型3-磷酸甘油酸脱氢酶相比对丝氨酸抑制的易感性降低,且其具有的氨基酸序列与SEQID NO:2所示的大肠杆菌野生型3-磷酸甘油酸脱氢酶的氨基酸序列的不同之处在于第349位的氨基酸不是甘氨酸或第372位的氨基酸不是苏氨酸。
2.如权利要求1的3-磷酸甘油酸脱氢酶,其中第349位的氨基酸不是甘氨酸且第372位的氨基酸不是苏氨酸。
3.一种编码如权利要求1的3-磷酸甘油酸脱氢酶的serA等位基因,其与大肠杆菌野生型3-磷酸甘油酸脱氢酶基因的不同之处在于密码子349编码一种非甘氨酸的天然氨基酸或密码子372编码一种非苏氨酸的天然氨基酸,所述野生型3-磷酸甘油酸脱氢酶基因是SEQ ID NO:1所示的serA基因。
4.一种编码如权利要求2的3-磷酸甘油酸脱氢酶的serA等位基因,其与大肠杆菌野生型3-磷酸甘油酸脱氢酶基因的不同之处在于密码子349编码一种非甘氨酸的天然氨基酸且密码子372编码非苏氨酸的天然氨基酸,所述野生型3-磷酸甘油酸脱氢酶基因是SEQ ID NO:1所示的serA基因。
5.一种载体,其含有置于一种启动子的功能性控制下的如权利要求3或4的serA等位基因。
6.一种产生微生物株的方法,其包括将如权利要求5的载体导入一种微生物株中。
7.一种微生物株,其适合用于发酵生产磷酸甘油酸族氨基酸或衍生自C1代谢的化合物,该微生物株含有如权利要求1或2的3-磷酸甘油酸脱氢酶。
8.一种通过培养如权利要求7的微生物株生产磷酸甘油酸族氨基酸或衍生自C1代谢的化合物的方法。
9.如权利要求1或2的3-磷酸甘油酸脱氢酶在生产磷酸甘油酸族氨基酸或衍生自C1代谢的化合物中的用途。
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DE602009000714D1 (de) * | 2008-03-06 | 2011-03-24 | Ajinomoto Kk | L-Zystein-produzierendes Bakterium und Verfahren zur Herstellung von L-Zystein |
JP5332237B2 (ja) * | 2008-03-06 | 2013-11-06 | 味の素株式会社 | L−システイン生産菌及びl−システインの製造法 |
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WO2011065469A1 (ja) | 2009-11-30 | 2011-06-03 | 味の素株式会社 | L-システイン生産菌及びl-システインの製造法 |
CN103119154B (zh) | 2010-09-14 | 2015-11-25 | 味之素株式会社 | 含硫氨基酸生产菌以及含硫氨基酸的制造方法 |
JP2014087259A (ja) | 2011-02-22 | 2014-05-15 | Ajinomoto Co Inc | L−システイン生産菌及びl−システインの製造法 |
CN110016484A (zh) | 2011-04-01 | 2019-07-16 | 味之素株式会社 | 用于产生l-半胱氨酸的方法 |
US9234223B2 (en) | 2011-04-01 | 2016-01-12 | Ajinomoto Co., Inc. | Method for producing L-cysteine |
JP2014131487A (ja) | 2011-04-18 | 2014-07-17 | Ajinomoto Co Inc | L−システインの製造法 |
DE102011075656A1 (de) * | 2011-05-11 | 2012-03-29 | Wacker Chemie Ag | Verfahren zur fermentativen Produktion von L-Cystin |
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CN102433312A (zh) * | 2011-11-30 | 2012-05-02 | 天津科技大学 | 一种d-3-磷酸甘油酸脱氢酶及其编码基因及构建方法 |
DE102012208359A1 (de) | 2012-05-18 | 2013-11-21 | Wacker Chemie Ag | Verfahren zur fermentativen Produktion von L-Cystein und Derivaten dieser Aminosäure |
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DE102013209274A1 (de) | 2013-05-17 | 2014-11-20 | Wacker Chemie Ag | Mikroorganismus und Verfahren zur fermentativen Überproduktion von Gamma-Glutamylcystein und Derivaten dieses Dipeptids |
CN103436504A (zh) * | 2013-09-02 | 2013-12-11 | 江南大学 | 抗l-丝氨酸反馈抑制的谷氨酸棒杆菌syps-062的构建方法及应用 |
WO2021259491A1 (de) | 2020-06-26 | 2021-12-30 | Wacker Chemie Ag | Verbesserte cystein produzierende stämme |
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ATE361361T1 (de) | 2007-05-15 |
DE10331291A1 (de) | 2005-02-17 |
EP1813669B1 (de) | 2009-05-20 |
ES2283906T3 (es) | 2007-11-01 |
JP4695855B2 (ja) | 2011-06-08 |
EP1496111A3 (de) | 2005-03-09 |
CN1609208A (zh) | 2005-04-27 |
EP1813669A1 (de) | 2007-08-01 |
EP1496111B1 (de) | 2007-05-02 |
EP1950287A3 (de) | 2008-08-13 |
EP1950287A2 (de) | 2008-07-30 |
US7582460B2 (en) | 2009-09-01 |
ATE440946T1 (de) | 2009-09-15 |
DE502004003661D1 (de) | 2007-06-14 |
EP1950287B1 (de) | 2009-08-26 |
ATE431847T1 (de) | 2009-06-15 |
JP2005040134A (ja) | 2005-02-17 |
ES2325603T3 (es) | 2009-09-09 |
US20050009162A1 (en) | 2005-01-13 |
DE502004009513D1 (de) | 2009-07-02 |
DE502004009985D1 (de) | 2009-10-08 |
EP1496111A2 (de) | 2005-01-12 |
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