CN107267561A - 通过在低pH下发酵生产二羧酸 - Google Patents

通过在低pH下发酵生产二羧酸 Download PDF

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CN107267561A
CN107267561A CN201710243972.7A CN201710243972A CN107267561A CN 107267561 A CN107267561 A CN 107267561A CN 201710243972 A CN201710243972 A CN 201710243972A CN 107267561 A CN107267561 A CN 107267561A
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米克尔·莱纳德斯·奥古斯特·詹森
瑞内·维尔瓦尔
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Abstract

本发明涉及通过在低pH下发酵生产二羧酸。本发明涉及用于生产二羧酸的方法。所述方法包括在下述pH下在存在含碳水化合物的底物和少量氧时发酵酵母，所述pH下二羧酸中的至少50％是酸性形式。本发明的方法提供了二羧酸产物的高产率并且比现有的方法更具成本效益，现有方法中生产的盐在回收期间必须被转化成酸。本发明的方法还导致更简单和更便利的下游加工。

Description

通过在低pH下发酵生产二羧酸
本申请是申请号为200980126943.6的中国专利申请的分案申请，原申请是申请日为2009年5月20日的国际申请PCT/EP2009/056181的中国国家阶段申请。
技术领域
本发明涉及生产二羧酸的方法。本发明特别涉及通过酵母的发酵来生产二羧酸。
背景技术
二羧酸(例如延胡索酸和琥珀酸)是重要的化合物，其在食品工业中用于食物制备和防腐，在医药工业中用于配制药物制品，以及用于其他工业用途，例如(生物)聚合物的单体。为了满足对二羧酸的日益增加的需要，正在开发更高效和更具成本效益的生产方法。传统上通过发酵能够生产大量二羧酸的细菌来制造二羧酸。这例如描述于US 5,573,931中，其中描述了通过使用细菌菌株生产高浓度的琥珀酸的方法。然而，与用细菌生产二羧酸的用途相关的一个主要的缺点是二羧酸盐的形成。如果使用细菌，则发酵期间的pH需要被维持在pH 6-7的范围内，这高于所有二羧酸的pKa值。因此，大部分酸将以其盐的形式被生产，这些盐必须被转化成酸。在大规模生产方法中这不实用不高效，并且提高生产成本。此外，并非细菌的微生物也已被用于生产有机酸。EP 0 424 384公开了在含有碳酸钙的培养基中通过Rhizopus生产有机酸的需氧方法。EP 1 183 385公开了经遗传操纵的酵母细胞，所述酵母细胞具有Crabtree阴性表型并且含有用于生产乳酸的外源核酸分子。
发明内容
本发明涉及用于生产二羧酸的方法。所述方法包括在低于二羧酸pKa的pH值下，在存在含碳水化合物的底物和少量氧时发酵酵母。本发明的方法提供了二羧酸产物的高产率，允许更简单的下游加工，并且比现有方法更具成本效益，现有方法中生产的是盐，其必须随后被转化成酸。因为二羧酸具有多于一个的pKa值，所以pH应当低于二羧酸的最低pKa。对大部分酸而言，pH应典型地在pH 1.0到pH 5.5的范围内，优选地在pH2.0和pH 4.0之间。在一个实施方案中，在3.0的pH值下生产琥珀酸。另一优点是由于pH低，所以污染的风险被降低。
酸生产阶段之前优选是针对最优生物质生产的生物质形成阶段。在生物质形成阶段中，pH在pH 2到pH 7的范围内。优选地，pH在pH 3到pH 6的范围内，更优选地，pH在pH 4到pH 5的范围内。
根据本发明的方法更具成本效益，并可导致低30％的成本价格。原因之一是滴定剂成本被显著降低。
本发明的方法可被用于生产任何二羧酸。合适的例子包括己二酸、延胡索酸、衣康酸(itaconic acid)、琥珀酸、苹果酸、草酸。优选地，二羧酸是琥珀酸、延胡索酸或苹果酸。
在本发明方法中使用的酵母可以是任何合适的酵母。酵母的合适例子包括Saccharomyces、Schizosaccharomyces、Kluyveromyces、Candida、Pichia和Yarrowia，例如Saccharomyces cerevisiae、Schizosaccharomyces pombe、Kluyvermoces lactis、Candida sonorensis、Pichia stipidis和Yarrowia lipolytica的物种。在一个实施方案中，本发明方法中使用的真核微生物是Saccharomyces cerevisiae——一种广泛使用的工业上感兴趣的微生物。
在一个优选的实施方案中，根据本发明的酵母是经遗传修饰的酵母。在本文中使用时，在根据本发明的方法中经遗传修饰的酵母被定义为下述酵母细胞，其含有所述酵母细胞中天然不存在的核苷酸序列或多肽，或者经所述酵母细胞中天然不存在的核苷酸序列或多肽转化或遗传修饰过，或者其含有内源核酸序列的额外的一个或多个拷贝。野生型酵母细胞在本文中被定义为重组细胞的亲本细胞。
优选地，根据本发明的方法中的酵母是经遗传修饰的酵母，其包含编码选自下组的异源酶的核苷酸序列，所述组由磷酸烯醇式丙酮酸羧激酶、延胡索酸还原酶和延胡索酸酶组成。异源酶的优选的实施方案如下文定义。
当用于指出给定的(重组)核酸或多肽分子与给定的宿主生物或宿主细胞之间的关系时，术语“同源的”应当被理解为表示该核酸或多肽分子天然地由相同物种、优选地相同品种或株系的宿主细胞或生物生产。
在关于核酸(DNA或RNA)或蛋白质的方面，使用术语“异源的”表示下述核酸或蛋白质，其不作为其存在的生物、细胞、基因组或DNA或RNA序列的部分天然存在，或其被发现于与其天然被发现的细胞或基因组或DNA或RNA序列中的位点不同的细胞或位点。异源核酸或蛋白质对其被引入的细胞而言不是内源的，而是得自另一细胞或是以合成方式或重组方式生产的。
优选地，经遗传修饰的酵母包含编码磷酸烯醇式丙酮酸羧激酶的核苷酸序列。PEP羧激酶(EC 4.1.1.49)优选地是异源酶，优选地源于细菌，更优选地具有PEP羧激酶活性的酶源于Escherichia coli、Mannheimia sp.、Actinobacillus sp.或Anaerobiospirillumsp.，更优选地，Mannheimia succiniciproducens或Actinobacillus succinogenes。在一个实施方案中，PEP羧激酶源于Actinobacillus succinogenes(PCKa)，其中PCKa优选地已被修饰为在第120-122位上用DAF氨基酸序列代替EGY。优选地，用与SEQ ID NO:6的氨基酸序列具有至少80％、85％、90％、95％、99％或100％的序列同一性的PEP羧激酶对根据本发明的酵母细胞进行了遗传修饰。
在另一个优选的实施方案中，根据本发明的方法中经遗传修饰的酵母包含编码延胡索酸还原酶的核苷酸序列。优选地，延胡索酸还原酶是异源酶，优选地是NAD(H)-依赖型延胡索酸还原酶，其可源于任何合适的来源，例如细菌、真菌、原生动物或植物。优选地，根据本发明的方法中的酵母包含异源的NAD(H)-依赖型延胡索酸还原酶，优选地源于Trypanosoma sp.，例如源于Trypanosoma brucei。在一个优选的实施方案中，编码NAD(H)-依赖型延胡索酸还原酶的核苷酸序列在胞质溶胶中表达。在编码NAD(H)-依赖型延胡索酸还原酶的核苷酸序列包含过氧化物酶体或线粒体靶向信号的情况下，可能必须修饰或缺失大量氨基酸(和编码核苷酸序列中相应的核苷酸序列)，从而阻止酶的过氧化物酶体或线粒体靶向。过氧化物酶体靶向信号的存在可例如通过et al,Nucleic acidResearch 2007,35,D815-D822所公开的方法来确定。优选地，用与SEQ ID NO:7具有至少80％、85％、90％、95％、99％或100％的序列同一性的NAD(H)-依赖型延胡索酸还原酶来对根据本发明的酵母细胞进行遗传修饰。
在另一个优选的实施方案中，根据本发明的方法中经遗传修饰的酵母包含编码延胡索酸酶的核苷酸序列，所述延胡索酸酶可以是异源或同源的酶。编码异源延胡索酸酶的核苷酸序列可以源于任何合适的来源，优选地来自微生物来源，优选地来自酵母，例如Saccharomyces cerevisiae，或来自丝状真菌，例如Rhizopus oryzae。优选地，根据本发明方法中的酵母过表达编码与SEQ ID NO:8的氨基酸序列具有至少75％、80％、85％、90％、92％、94％、95％、96％、97％、98％或99％或100％的序列同一性的延胡索酸酶的核苷酸序列。
在另一个优选的实施方案中，根据本发明的方法中经遗传修饰的酵母还包含编码苹果酸脱氢酶(MDH)的核苷酸序列，所述苹果酸脱氢酶在所述核苷酸序列表达后在胞质溶胶中有活性。优选地，MDH缺乏过氧化物酶体或线粒体靶向信号，以将酶定位于胞质溶胶中。胞质溶胶MDH可以是任何合适的同源或异源苹果酸脱氢酶。优选地，根据本发明的酵母细胞包含编码下述苹果酸脱氢酶的核苷酸序列，所述苹果酸脱氢酶与SEQ ID NO:9的氨基酸序列具有至少70％，优选地至少75％、80％、85％、90％、92％、94％、95％、96％、97％、98％、99％的序列同一性。
在另一实施方案中，根据本发明的方法中经遗传修饰的酵母包含编码二羧酸转运蛋白(优选地，苹果酸转运蛋白(MAE))的核苷酸序列。二羧酸转运蛋白可以是同源或异源的蛋白质。优选地，二羧酸转运蛋白是异源的蛋白质。二羧酸转运蛋白可源于任何合适的生物，优选地源于Schizosaccharomyces pombe。优选地，二羧酸转运蛋白是下述苹果酸转运蛋白(MAE)，所述苹果酸转运蛋白与SEQ ID NO:10具有至少80％、85％、90％、95％或99％或100％的序列同一性。
优选地，根据本发明的方法中使用的酵母是包含异源PEP-羧激酶、异源NAD(P)H-依赖型延胡索酸还原酶、异源延胡索酸酶、异源苹果酸转运蛋白和胞质溶胶苹果酸脱氢酶的经遗传修饰的酵母。这些酶的优选的实施方案如本文上文中定义。
序列同一性在本文中被定义为通过比较序列测定的两条或更多条氨基酸(多肽或蛋白质)序列或两条或更多条核酸(多核苷酸)序列之间的关系。通常，在被比较的序列的整个长度上比较序列同一性或相似性。在本领域中，“同一性”也表示氨基酸或核酸序列之间的序列相关度，这根据情况通过这类序列串之间的匹配测定。
测定同一性的优选方法被设计为在测试的序列之间给予最大匹配。测定同一性和相似性的方法被编码于公众可获得的计算机程序中。测定两条序列之间同一性和相似性的优选的计算机程序方法包括BLASTP和BLASTN，公众可从NCBI和其它来源(BLAST Manual,Altschul,S.,等,NCBI NLM NIH Bethesda,MD 20894)获得。使用BLASTP的氨基酸序列比较的优选参数为缺口开放11.0，缺口延伸1，Blosum 62矩阵。
在本文中使用时，术语“核酸”包括单链或双链形式的脱氧核糖核苷酸或核糖核苷酸多聚体，即多核苷酸，除非另有限制，其包括具有天然核苷酸主要特性的已知类似物，因为它们能够以类似于天然存在的核苷酸的方式与单链核酸杂交(例如肽核酸)。多核苷酸可以是天然或异源的结构基因或调节基因的全长或亚序列。除非另有说明，该术语包括特定的序列及其互补序列。
在一个优选的实施方案中，根据本发明的方法中的酵母过表达编码本文上文定义的任何酶的核苷酸序列。本领域可获得多种手段用于在本发明方法中在酵母中过量表达编码酶的核苷酸序列。具体地，可以通过提高细胞中编码酶的基因的拷贝数，例如通过在细胞的基因组中整合额外的基因拷贝，通过表达来自着丝粒载体、来自附加体多拷贝表达载体的基因，或者通过引入包含多拷贝基因的(附加体)表达载体，来过量表达编码酶的核苷酸序列。优选地，用(强)组成型启动子实现根据本发明的酶的过量表达。
含碳水化合物的底物可以是任何含碳水化合物的底物，包括糖蜜(molasse)，甘蔗汁，戊糖和己糖，例如葡萄糖、果糖、木糖、阿拉伯糖。优选地，含碳水化合物的底物是含葡萄糖的底物，如麦芽糖、蔗糖、葡萄糖或葡萄糖浆。含碳水化合物的底物的碳水化合物含量以干物质含量为基础优选地多于50％w/w，更优选地多于55％、60％、65％、70％、75％、80％w/w，最优选地多于85％、90％、95％或99％w/w。
根据本发明的方法优选地包括在碳(C)-受限的条件下发酵酵母。C-受限的条件在本文中被定义为溶解的碳水化合物的浓度低于1g/l，优选地低于0.9g/l、0.8g/l或低于0.5g/l溶解的碳水化合物。发现在C-受限的条件下发酵酵母导致与非C-受限的条件相比提高的琥珀酸产率。
用于发酵的氧可以以任何合适的形式供应。在一个实施方案中，氧以空气的形式供应。氧应当小量供应。这反映于酵母的氧吸收速率(OUR)和/或氧吸收比速率(qO2)中。本发明中的OUR低于约8.0mmol氧/L/小时，优选地低于约5.0、4.0、3.0或2.0mmol氧/L/小时，更优选地低于约1.0或0.5mmol氧/L/小时，优选地高于0.01mmol氧/L/小时。
本发明方法中的氧吸收比速率(qO2)范围在8mmol氧/g生物质干重/小时到0.5mmol氧/g生物质干重/小时之间，优选地在5、4、3或2mmol氧/g生物质/小时到约0.4、0.3或0.2mmol/氧/g生物质/小时之间。
根据本发明的方法可以以分批、补料分批或连续模式进行。这些发酵模式是本领域技术人员已知的。取决于发酵模式，发酵期间的生物质浓度可在发酵期间或多或少地变化。在分批和补料分批模式中，生物质浓度通常提高。因此，在分批和补料分批模式中，氧吸收比速率通常降低。
本发明方法的温度典型地在10℃和40℃之间，优选地在20℃和35℃之间，更优选地在30℃和35℃之间。
在根据本发明方法的一个实施方案中，除了含碳水化合物的底物之外还存在额外的电子供体。额外的电子供体优选地是有机电子供体。有机电子供体的合适例子包括甘油、甲酸盐/酯和多元醇例如甘露醇、山梨醇和木糖醇。
附图说明
图1.应用的OUR对pH 3下进行90小时后的琥珀酸生产的影响。
具体实施方式
实施例
实施例1.通过Saccharomyces cerevisiae生产琥珀酸
1.1.构建酵母菌株
1.1.1.构建表达构建体
对S.cerevisiae表达载体pRS414(Sirkoski R.S.and Hieter P,Genetics,1989,122(1):19-27)进行BamHI/NotI限制性消化，随后在该载体中连接由磷酸烯醇式丙酮酸羧激酶(来源Actinobacillus succinogenes)合成基因构建体(SEQ ID NO:1)构成的BamHI/NotI限制性消化片段之后，制造了表达构建体pGBS414PPK-3。使用连接混合物转化E.coliTOP10(Invitrogen)，得到酵母表达构建体pGBS414PPK-1。随后用AscI和NotI限制性消化pGBK414PPK-1。为了制造pGBS414PPK-3，将由来自T.brucei(FRDg)合成基因构建体(SEQ IDNO:2)的酵解酶体延胡索酸还原酶构成的AscI/NotI限制性消化片段连接进经限制性消化的pGBS414PPK-1载体中。连接混合物被用于转化E.coli TOP10(Invitrogen)，得到酵母表达构建体pGBS414PPK-3。
对S.cerevisiae表达载体pRS415(Sirkoski R.S.and Hieter P,Genetics,1989,122(1):19-27)进行BamHI/NotI限制性消化，随后在该载体中连接由延胡索酸酶(来源Rhizopus oryzae)合成基因构建体(SEQ ID NO:3)构成的BamHI/NotI限制性消化片段之后，制造了表达构建体pGBS415FUM-3。使用连接混合物转化E.coli TOP10(Invitrogen)，得到酵母表达构建体pGBS415FUM-1。随后用AscI和NotI限制性消化pGBK415FUM-1。为了制造pGBS415FUM-3，将由来自S.cerevisiae(MDH3)合成基因构建体(SEQ ID NO:4)的过氧化物酶体苹果酸脱氢酶构成的AscI/NotI限制性消化片段连接进经限制性消化的pGBS415FUM-1载体中。连接混合物被用于转化E.coli TOP10(Invitrogen)，得到酵母表达构建体pGBS415FUM-3。
对S.cerevisiae表达载体pRS416(Sirkoski R.S.and Hieter P,Genetics,1989,122(1):19-27)进行BamHI/NotI限制性消化，随后在该载体中连接由Schizosaccharomycespombe苹果酸转运蛋白合成基因构建体(SEQ ID NO:5)构成的BamHI/NotI限制性消化片段之后，制造了表达构建体pGBS416MAE-1。使用连接混合物转化E.coli TOP10(Invitrogen)，得到酵母表达构建体pGBS416MAE-1。
1.1.2.构建S.cerevisiae菌株
通过电穿孔将质粒pGBS414PPK-3、pGBS415FUM-3和pGBS416MAE-1(在1.1.下描述)转化进S.cerevisiae菌株RWB064(MATA ura3-52leu2-112trp1-289adh1::lox adh2::loxgpd1::Kanlox)中，制造过表达PCKa、MDH3、FUMR、FRDg和SpMAEI的菌株SUC-200。根据WO2008/000632，针对在S.cerevisiae中的表达对所有基因都进行了密码子对优化。
1.2.在低pH和氧受限条件下的S.cerevisiae琥珀酸生产
在30℃和220rpm下，在摇瓶(2x 300ml)中将酵母菌株SUC-200(过表达PCKa、MDH3、FUMR、FRDg和SpMAEI的MATA ura3-52leu2-112trp1-289adh1::lox adh2::lox gpd1::Kanlox)培养3天。培养基以Verduyn(Verduyn et.al.,1992,Yeast 8,501-517)为基础，但是如表1中所示对碳源和碳源进行了改动。
表1.预培养摇瓶培养基组成
a维生素溶液
组分 通式 浓度(g/kg)
生物素(D-) C10H16N2O3S 0.05
Ca D(+)泛酸盐 C18H32CaN2O10 1.00
烟酸 C6H5NO2 1.00
Myo-肌醇 C6H12O6 25.00
盐酸氯化硫胺 C12H18Cl2N4OsxH2O 1.00
盐酸吡哆醇 C8H12ClNO3 1.00
对氨基苯甲酸 C7H7NO2 0.20
b微量元素溶液
通式 浓度(g/kg)
C10H14N2Na2O8·2H2O(EDTA) 15.00
ZnSO4·7H2O 1.50
MnCl2·2H2O 0.84
CoCl2·6H2O 0.30
CuSO4·5H2O 0.30
Na2MoO4·2H2O 0.40
CaCl2·2H2O 4.50
FeSO4·7H2O 3.00
H3BO3 1.00
KI 0.10
随后，将摇瓶内容物转移至含有以下培养基的10L发酵罐(初始重量6kg)中：
表2.主要发酵培养基组成
通过添加6N KOH将pH控制在3.0。将温度控制在30℃。通过控制对发酵罐的补料添加，使葡萄糖浓度保持受限(<1g/l)。对发酵应用导致氧限制的不同的氧吸收速率(OUR)(图1)。
应用0.33vvm的包括10％CO2的总气流，从而为高效的琥珀酸生产提供足够的CO2。
对琥珀酸生产应用的不同OUR的结果展示于图1中。在pH 3下需要最小量的通气维持琥珀酸生产。高于5mmol/L/h的OUR导致更低的琥珀酸生产。
在90小时的培养期间，生长发生至8g干重/L的典型生物质浓度。结果，在发酵期间氧吸收比速率(qO2)持续降低。在一次发酵中应用的10mmol/L/h的OUR与从10降低至1.25mmol/g生物质干重/h的qO2相关，1mmol/L/h的OUR与从1降至0.1mmol/g生物质干重/h的qO2相关。
以下内容对应于母案申请中的原始权利要求书，现作为说明书的一部分并入此处：
1.用于制备二羧酸的方法，所述方法包括在低于有机酸最低pKa的pH值下，在存在含碳水化合物的底物和少量氧时发酵酵母。
2.根据项1的方法，其中所述二羧酸是延胡索酸、苹果酸或琥珀酸。
3.根据项1或2的方法，其中所述pH在pH 1.0到pH 5.5的范围内。
4.根据项1到3中任一项的方法，其中所述氧以低于约8mmol氧/L/小时的氧吸收速率被供应。
5.根据项1到3中任一项的方法，其中所述氧以8到0.5mmol/g生物质干重/小时之间的范围内的氧吸收比速率被供应。
6.根据项1到5中任一项的方法，所述方法包括在碳受限的条件下发酵酵母。
7.根据项1到6中任一项的方法，所述方法在存在额外的电子供体时进行。
8.根据项1到7中任一项的方法，其中所述酵母是Saccharomyces cerevisiae。
9.根据项1到8中任一项的方法，其中所述酵母是经遗传修饰的酵母。
10.根据项9的方法，其中所述经遗传修饰的酵母包含编码选自下组的异源酶的核苷酸序列，所述组由磷酸烯醇式丙酮酸羧激酶、延胡索酸还原酶和延胡索酸酶组成。
序列表
<110> 帝斯曼知识产权资产管理有限公司
米克尔·莱纳德斯·奥古斯特·詹森
瑞内·维尔瓦尔
<120> 通过在低ph下发酵生产二羧酸
<130> 26725-WO-PCT
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 3148
<212> DNA
<213> 人工序列
<220>
<223> 用于在S. cerevisiae中表达的合成构建体TDH1p-PCKa-TDH1t
<400> 1
ggatcccttc ccttttacag tgcttcggaa aagcacagcg ttgtccaagg gaacaatttt 60
tcttcaagtt aatgcataag aaatatcttt ttttatgttt agctaagtaa aagcagcttg 120
gagtaaaaaa aaaaatgagt aaatttctcg atggattagt ttctcacagg taacataaca 180
aaaaccaaga aaagcccgct tctgaaaact acagttgact tgtatgctaa agggccagac 240
taatgggagg agaaaaagaa acgaatgtat atgctcattt acactctata tcaccatatg 300
gaggataagt tgggctgagc ttctgatcca atttattcta tccattagtt gctgatatgt 360
cccaccagcc aacacttgat agtatctact cgccattcac ttccagcagc gccagtaggg 420
ttgttgagct tagtaaaaat gtgcgcacca caagcctaca tgactccacg tcacatgaaa 480
ccacaccgtg gggccttgtt gcgctaggaa taggatatgc gacgaagacg cttctgctta 540
gtaaccacac cacattttca gggggtcgat ctgcttgctt cctttactgt cacgagcggc 600
ccataatcgc gctttttttt taaaaggcgc gagacagcaa acaggaagct cgggtttcaa 660
ccttcggagt ggtcgcagat ctggagactg gatctttaca atacagtaag gcaagccacc 720
atctgcttct taggtgcatg cgacggtatc cacgtgcaga acaacatagt ctgaagaagg 780
gggggaggag catgttcatt ctctgtagca gtaagagctt ggtgataatg accaaaactg 840
gagtctcgaa atcatataaa tagacaatat attttcacac aatgagattt gtagtacagt 900
tctattctct ctcttgcata aataagaaat tcatcaagaa cttggtttga tatttcacca 960
acacacacaa aaaacagtac ttcactaaat ttacacacaa aacaaaatga ccgatttgaa 1020
ccaattgact caagaattgg gtgctttggg tattcacgat gtccaagaag ttgtctacaa 1080
cccatcttac gaattgttgt ttgctgaaga aaccaagcca ggtttggaag gttacgaaaa 1140
gggtactgtt accaaccaag gtgctgttgc tgtcaacacc ggtatcttca ccggtcgttc 1200
tccaaaggac aaatacattg tcttggatga caagaccaag gacactgtct ggtggacttc 1260
tgaaaaggtc aagaacgaca acaaaccaat gtcccaagac acttggaact ctttaaaggg 1320
tttagtcgct gaccaattgt ctggtaagag attattcgtt gtcgatgctt tctgtggtgc 1380
caacaaggac accagattag ctgtcagagt tgtcactgaa gttgcttggc aagctcactt 1440
cgttaccaac atgttcatca gaccatctgc tgaagaattg aaaggtttca agccagattt 1500
cgttgtcatg aacggtgcca aatgtaccaa cccaaactgg aaggaacaag gtttgaactc 1560
tgaaaacttt gttgctttca acatcactga aggtgttcaa ttgattggtg gtacctggta 1620
cggtggtgaa atgaagaagg gtatgttctc catgatgaac tacttcttgc cattgagagg 1680
tattgcttcc atgcactgtt ctgccaatgt cggtaaggac ggtgacactg ccatcttctt 1740
cggtctatcc ggtaccggta agaccacttt gtccactgac ccaaagagac aattgattgg 1800
tgatgacgaa cacggttggg atgacgaagg tgttttcaac tttgaaggtg gttgttacgc 1860
caagaccatc aacttatctg ctgaaaatga accagatatc tacggtgcca tcaagcgtga 1920
cgctctattg gaaaacgttg ttgttttgga caatggtgac gtcgattatg ctgacggttc 1980
caagactgaa aacaccagag tttcttaccc aatctaccat attcaaaaca ttgtcaagcc 2040
agtttccaag gctggtccag ctaccaaagt tatcttcttg tctgctgatg ctttcggtgt 2100
tttgcctcct gtttccaagt tgactccaga acaaaccaag tactacttct tgtctggttt 2160
caccgccaag ttggctggta ctgaaagagg tatcactgaa ccaactccaa ctttctctgc 2220
ttgtttcggt gctgcctttt tgtctttgca cccaactcaa tacgctgaag ttttggtcaa 2280
gagaatgcaa gaatctggtg ctgaagctta cttggtcaac actggttgga acggtaccgg 2340
taagagaatc tccatcaaag ataccagagg tatcatcgat gccatcttgg atggttccat 2400
tgacaaggct gaaatgggtt ctttgccaat tttcgatttc tccattccaa aggctttgcc 2460
aggtgtcaac ccagccatct tagacccaag agacacctac gctgacaaag ctcaatggga 2520
agaaaaggct caagacttgg ctggtagatt cgtcaagaac ttcgaaaaat acactggtac 2580
tgctgaaggt caagctttgg ttgctgctgg tccaaaggcc taaggcccgg gcataaagca 2640
atcttgatga ggataatgat ttttttttga atatacataa atactaccgt ttttctgcta 2700
gattttgtga agacgtaaat aagtacatat tactttttaa gccaagacaa gattaagcat 2760
taactttacc cttttctctt ctaagtttca atactagtta tcactgttta aaagttatgg 2820
cgagaacgtc ggcggttaaa atatattacc ctgaacgtgg tgaattgaag ttctaggatg 2880
gtttaaagat ttttcctttt tgggaaataa gtaaacaata tattgctgcc tttgcaaaac 2940
gcacataccc acaatatgtg actattggca aagaacgcat tatcctttga agaggtggat 3000
actgatacta agagagtctc tattccggct ccacttttag tccagagatt acttgtcttc 3060
ttacgtatca gaacaagaaa gcatttccaa agtaattgca tttgcccttg agcagtatat 3120
atatactaag aaggcgcgcc gcggccgc 3148
<210> 2
<211> 4959
<212> DNA
<213> 人工序列
<220>
<223> 用于在S. cerevisiae中表达的合成构建体TDH3p-FRDg-TDH3t
<400> 2
ggatccggcg cgccctattt tcgaggacct tgtcaccttg agcccaagag agccaagatt 60
taaattttcc tatgacttga tgcaaattcc caaagctaat aacatgcaag acacgtacgg 120
tcaagaagac atatttgacc tcttaacagg ttcagacgcg actgcctcat cagtaagacc 180
cgttgaaaag aacttacctg aaaaaaacga atatatacta gcgttgaatg ttagcgtcaa 240
caacaagaag tttaatgacg cggaggccaa ggcaaaaaga ttccttgatt acgtaaggga 300
gttagaatca ttttgaataa aaaacacgct ttttcagttc gagtttatca ttatcaatac 360
tgccatttca aagaatacgt aaataattaa tagtagtgat tttcctaact ttatttagtc 420
aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta 480
cacagaatat ataacatcgt aggtgtctgg gtgaacagtt tattcctggc atccactaaa 540
tataatggag cccgcttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa 600
tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacagaga 660
acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcctg 720
gagtaaatga tgacacaagg caattgaccc acgcatgtat ctatctcatt ttcttacacc 780
ttctattacc ttctgctctc tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt 840
tccctgaaat tattccccta cttgactaat aagtatataa agacggtagg tattgattgt 900
aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt 960
tagttttaaa acaccaagaa cttagtttcg aataaacaca cataaacaaa caaaatggtt 1020
gatggtagat cttctgcttc cattgttgcc gttgacccag aaagagctgc cagagaaaga 1080
gatgctgctg ccagagcttt gttgcaagac tctccattgc acaccaccat gcaatacgct 1140
acctctggtt tggaattgac tgttccatac gctttgaagg ttgttgcttc tgctgacact 1200
ttcgacagag ccaaggaagt tgctgatgaa gtcttgagat gtgcctggca attggctgac 1260
accgttttga actctttcaa cccaaactct gaagtctctt tagtcggtag attaccagtc 1320
ggtcaaaagc atcaaatgtc tgctccattg aaacgtgtca tggcttgttg tcaaagagtc 1380
tacaactcct ctgctggttg tttcgaccca tccactgctc cagttgccaa ggctttgaga 1440
gaaattgctt tgggtaagga aagaaacaat gcttgtttgg aagctttgac tcaagcttgt 1500
accttgccaa actctttcgt cattgatttc gaagctggta ctatctccag aaagcacgaa 1560
cacgcttctt tggatttggg tggtgtttcc aagggttaca tcgtcgatta cgtcattgac 1620
aacatcaatg ctgctggttt ccaaaacgtt ttctttgact ggggtggtga ctgtcgtgcc 1680
tccggtatga acgccagaaa cactccatgg gttgtcggta tcactagacc tccttccttg 1740
gacatgttgc caaaccctcc aaaggaagct tcttacatct ccgtcatctc tttggacaat 1800
gaagctttgg ctacctctgg tgattacgaa aacttgatct acactgctga cgataaacca 1860
ttgacctgta cctacgattg gaaaggtaag gaattgatga agccatctca atccaatatc 1920
gctcaagttt ccgtcaagtg ttactctgcc atgtacgctg acgctttggc taccgcttgt 1980
ttcatcaagc gtgacccagc caaggtcaga caattgttgg atggttggag atacgttaga 2040
gacaccgtca gagattaccg tgtctacgtc agagaaaacg aaagagttgc caagatgttc 2100
gaaattgcca ctgaagatgc tgaaatgaga aagagaagaa tttccaacac tttaccagct 2160
cgtgtcattg ttgttggtgg tggtttggct ggtttgtccg ctgccattga agctgctggt 2220
tgtggtgctc aagttgtttt gatggaaaag gaagccaagt tgggtggtaa ctctgccaag 2280
gctacctctg gtatcaacgg ttggggtact agagctcaag ctaaggcttc cattgtcgat 2340
ggtggtaagt acttcgaaag agatacctac aagtctggta tcggtggtaa caccgatcca 2400
gctttggtta agactttgtc catgaaatct gctgacgcta tcggttggtt gacttctcta 2460
ggtgttccat tgactgtttt gtcccaatta ggtggtcact ccagaaagag aactcacaga 2520
gctccagaca agaaggatgg tactccattg ccaattggtt tcaccatcat gaaaacttta 2580
gaagatcatg ttagaggtaa cttgtccggt agaatcacca tcatggaaaa ctgttccgtt 2640
acctctttgt tgtctgaaac caaggaaaga ccagacggta ccaagcaaat cagagttacc 2700
ggtgtcgaat tcactcaagc tggttctggt aagaccacca ttttggctga tgctgttatc 2760
ttggccaccg gtggtttctc caacgacaag actgctgatt ctttgttgag agaacatgcc 2820
ccacacttgg ttaacttccc aaccaccaac ggtccatggg ctactggtga tggtgtcaag 2880
ttggctcaaa gattaggtgc tcaattggtc gatatggaca aggttcaatt gcacccaact 2940
ggtttgatca acccaaagga cccagccaac ccaaccaaat tcttgggtcc agaagctcta 3000
agaggttctg gtggtgtttt gttgaacaaa caaggtaaga gatttgtcaa cgaattggat 3060
ttgagatctg ttgtttccaa ggccatcatg gaacaaggtg ctgaataccc aggttctggt 3120
ggttccatgt ttgcttactg tgtcttgaac gctgctgctc aaaaattgtt tggtgtttcc 3180
tctcacgaat tctactggaa gaagatgggt ttgttcgtca aggctgacac catgagagac 3240
ttggctgctt tgattggttg tccagttgaa tccgttcaac aaactttaga agaatacgaa 3300
agattatcca tctctcaaag atcttgtcca attaccagaa aatctgttta cccatgtgtt 3360
ttgggtacca aaggtccata ctatgtcgcc tttgtcactc catctatcca ctacaccatg 3420
ggtggttgtt tgatttctcc atctgctgaa atccaaatga agaacacttc ttccagagct 3480
ccattgtccc actccaaccc aatcttgggt ttattcggtg ctggtgaagt caccggtggt 3540
gtccacggtg gtaacagatt aggtggtaac tctttgttgg aatgtgttgt tttcggtaga 3600
attgccggtg acagagcttc taccattttg caaagaaagt cctctgcttt gtctttcaag 3660
gtctggacca ctgttgtttt gagagaagtc agagaaggtg gtgtctacgg tgctggttcc 3720
cgtgtcttga gattcaactt accaggtgct ctacaaagat ctggtctatc cttgggtcaa 3780
ttcattgcca tcagaggtga ctgggacggt caacaattga ttggttacta ctctccaatc 3840
actttgccag acgatttggg tatgattgac attttggcca gatctgacaa gggtacttta 3900
cgtgaatgga tctctgcttt ggaaccaggt gacgctgtcg aaatgaaggc ttgtggtggt 3960
ttggtcatcg aaagaagatt atctgacaag cacttcgttt tcatgggtca cattatcaac 4020
aagctatgtt tgattgctgg tggtaccggt gttgctccaa tgttgcaaat catcaaggcc 4080
gctttcatga agccattcat cgacactttg gaatccgtcc acttgatcta cgctgctgaa 4140
gatgtcactg aattgactta cagagaagtt ttggaagaac gtcgtcgtga atccagaggt 4200
aaattcaaga aaactttcgt tttgaacaga cctcctccat tatggactga cggtgtcggt 4260
ttcatcgacc gtggtatctt gaccaaccac gttcaaccac catctgacaa cttattggtt 4320
gccatctgtg gtccaccagt tatgcaaaga attgtcaagg ccactttaaa gactttaggt 4380
tacaacatga acttggtcag aaccgttgac gaaactgaac catctggaag ttaaggcccg 4440
ggcgtgaatt tactttaaat cttgcattta aataaatttt ctttttatag ctttatgact 4500
tagtttcaat ttatatacta ttttaatgac attttcgatt cattgattga aagctttgtg 4560
ttttttcttg atgcgctatt gcattgttct tgtctttttc gccacatgta atatctgtag 4620
tagatacctg atacattgtg gatgctgagt gaaattttag ttaataatgg aggcgctctt 4680
aataattttg gggatattgg cttttttttt taaagtttac aaatgaattt tttccgccag 4740
gataacgatt ctgaagttac tcttagcgtt cctatcggta cagccatcaa atcatgccta 4800
taaatcatgc ctatatttgc gtgcagtcag tatcatctac atgaaaaaaa ctcccgcaat 4860
ttcttataga atacgttgaa aattaaatgt acgcgccaag ataagataac atatatctag 4920
atgcagtaat atacacagat tccggccggc cgcggccgc 4959
<210> 3
<211> 2950
<212> DNA
<213> 人工序列
<220>
<223> 用于在S. cerevisiae中表达的合成构建体TDH1p-FUMR-TDH1t
<400> 3
ggatcccttc ccttttacag tgcttcggaa aagcacagcg ttgtccaagg gaacaatttt 60
tcttcaagtt aatgcataag aaatatcttt ttttatgttt agctaagtaa aagcagcttg 120
gagtaaaaaa aaaaatgagt aaatttctcg atggattagt ttctcacagg taacataaca 180
aaaaccaaga aaagcccgct tctgaaaact acagttgact tgtatgctaa agggccagac 240
taatgggagg agaaaaagaa acgaatgtat atgctcattt acactctata tcaccatatg 300
gaggataagt tgggctgagc ttctgatcca atttattcta tccattagtt gctgatatgt 360
cccaccagcc aacacttgat agtatctact cgccattcac ttccagcagc gccagtaggg 420
ttgttgagct tagtaaaaat gtgcgcacca caagcctaca tgactccacg tcacatgaaa 480
ccacaccgtg gggccttgtt gcgctaggaa taggatatgc gacgaagacg cttctgctta 540
gtaaccacac cacattttca gggggtcgat ctgcttgctt cctttactgt cacgagcggc 600
ccataatcgc gctttttttt taaaaggcgc gagacagcaa acaggaagct cgggtttcaa 660
ccttcggagt ggtcgcagat ctggagactg gatctttaca atacagtaag gcaagccacc 720
atctgcttct taggtgcatg cgacggtatc cacgtgcaga acaacatagt ctgaagaagg 780
gggggaggag catgttcatt ctctgtagca gtaagagctt ggtgataatg accaaaactg 840
gagtctcgaa atcatataaa tagacaatat attttcacac aatgagattt gtagtacagt 900
tctattctct ctcttgcata aataagaaat tcatcaagaa cttggtttga tatttcacca 960
acacacacaa aaaacagtac ttcactaaat ttacacacaa aacaaaatgt cctctgcttc 1020
tgctgctttg caaaaattca gagctgaaag agataccttc ggtgacttgc aagttccagc 1080
tgaccgttac tggggtgctc aaactcaaag atctttgcaa aactttgaca ttggtggtcc 1140
aactgaaaga atgccagaac cattaatcag agctttcggt gttttgaaga aggctgctgc 1200
caccgtcaac atgacctacg gtttggaccc aaaggttggt gaagccatcc aaaaggctgc 1260
tgacgaagtt atcgatggtt ctttgattga ccatttccca ttggttgtct ggcaaaccgg 1320
ttctggtact caaaccaaga tgaacgtcaa tgaagtcatc tccaacagag ccattgaatt 1380
gttgggtggt gaattaggtt ccaaggctcc agtccaccca aacgatcatg tcaacatgtc 1440
tcaatcttcc aacgacactt tcccaactgc catgcacgtt gctgccgttg ttgaaattca 1500
cggtagattg attccagctt tgaccacttt gagagatgct ttgcaagcca aatctgctga 1560
attcgaacac atcatcaaga ttggtagaac ccacttgcaa gatgctaccc cattgacttt 1620
aggtcaagaa ttctccggtt acactcaaca attgacctac ggtattgctc gtgttcaagg 1680
tactttggaa agattataca acttggctca aggtggtact gctgtcggta ctggtttgaa 1740
caccagaaag ggtttcgatg ccaaggttgc tgaagccatt gcttccatca ctggtttacc 1800
attcaagacc gctccaaaca aattcgaagc tttggctgct cacgacgctt tggttgaagc 1860
tcacggtgct ttgaacaccg ttgcttgttc tttgatgaag attgccaacg atatccgtta 1920
cttgggttct ggtccaagat gtggtttagg tgaattgtct ctaccagaaa acgaaccagg 1980
ttcttccatc atgccaggta aggtcaaccc aactcaatgt gaagctatga ccatggtttg 2040
tgctcaagtc atgggtaaca acactgccat ctctgttgct ggttccaacg gtcaattcga 2100
attgaatgtc tttaaaccag tcatgatcaa gaacttgatc caatccatca gattaatctc 2160
tgacgcttcc atctctttca ccaagaactg tgttgtcggt attgaagcta acgaaaagaa 2220
gatctcctcc atcatgaacg aatctttgat gttggtcact gctttgaacc ctcacattgg 2280
ttacgacaag gctgccaagt gtgccaagaa ggctcacaag gaaggtacca ctttgaaaga 2340
agctgctcta tctttgggtt acttgacctc tgaagaattc gaccaatggg ttagacctga 2400
ggacatgatt tctgccaagg attaaggccc gggcataaag caatcttgat gaggataatg 2460
attttttttt gaatatacat aaatactacc gtttttctgc tagattttgt gaagacgtaa 2520
ataagtacat attacttttt aagccaagac aagattaagc attaacttta cccttttctc 2580
ttctaagttt caatactagt tatcactgtt taaaagttat ggcgagaacg tcggcggtta 2640
aaatatatta ccctgaacgt ggtgaattga agttctagga tggtttaaag atttttcctt 2700
tttgggaaat aagtaaacaa tatattgctg cctttgcaaa acgcacatac ccacaatatg 2760
tgactattgg caaagaacgc attatccttt gaagaggtgg atactgatac taagagagtc 2820
tctattccgg ctccactttt agtccagaga ttacttgtct tcttacgtat cagaacaaga 2880
aagcatttcc aaagtaattg catttgccct tgagcagtat atatatacta agaaggcgcg 2940
ccgcggccgc 2950
<210> 4
<211> 1966
<212> DNA
<213> 人工序列
<220>
<223> 用于在S. cerevisiae中表达的合成构建体TDH3p-MDH3-TDH3t
<400> 4
ggatccggcg cgccacgcgt ggccggcctt agtcaaaaaa ttagcctttt aattctgctg 60
taacccgtac atgcccaaaa tagggggcgg gttacacaga atatataaca tcgtaggtgt 120
ctgggtgaac agtttattcc tggcatccac taaatataat ggagcccgct ttttaagctg 180
gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc aaccatcagt 240
tcataggtcc attctcttag cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg 300
ggcacaacct caatggagtg atgcaacctg cctggagtaa atgatgacac aaggcaattg 360
acccacgcat gtatctatct cattttctta caccttctat taccttctgc tctctctgat 420
ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg aaattattcc cctacttgac 480
taataagtat ataaagacgg taggtattga ttgtaattct gtaaatctat ttcttaaact 540
tcttaaattc tacttttata gttagtcttt tttttagttt taaaacacca agaacttagt 600
ttcgaataaa cacacataaa caaacaaaat ggttaaggtt gccatcttag gtgcttctgg 660
tggtgtcggt caaccattat ctctattatt gaaattgtct ccatacgttt ctgaattggc 720
tttgtacgat atcagagctg ctgaaggtat tggtaaggat ttgtcccaca tcaacaccaa 780
ctcctcttgt gttggttacg acaaggattc catcgaaaac actttgtcca atgctcaagt 840
tgtcttgatt ccagctggtg ttccaagaaa gccaggtttg accagagatg atttgttcaa 900
gatgaacgct ggtatcgtta agtctttggt tactgctgtc ggtaaatttg ccccaaacgc 960
tcgtatctta gtcatctcca accctgttaa ctctttggtt ccaattgccg ttgaaacttt 1020
gaagaagatg ggtaagttca agccaggtaa cgttatgggt gtcaccaact tggatttggt 1080
cagagctgaa actttcttgg ttgactactt gatgttgaag aacccaaaga tcggtcaaga 1140
acaagacaag accaccatgc acagaaaggt caccgtcatc ggtggtcact ctggtgaaac 1200
catcattcca atcatcactg acaaatcctt ggttttccaa ttggacaagc aatacgaaca 1260
tttcatccac agagtccaat tcggtggtga cgaaattgtc aaggccaagc aaggtgccgg 1320
ttctgctacc ttgtccatgg ctttcgctgg tgccaaattt gctgaagaag tcttacgttc 1380
tttccacaac gaaaagccag aaactgaatc tttgtctgct ttcgtctact tgccaggttt 1440
gaagaacggt aagaaggctc aacaattagt cggtgacaac tccattgaat acttctcttt 1500
gccaattgtt ttgagaaacg gttccgttgt ttccattgac acttctgttt tggaaaaatt 1560
gtctccaaga gaagaacaat tggtcaacac tgctgtcaag gaattgagaa agaacattga 1620
aaagggtaag tctttcatct tggacagtta aggtgaattt actttaaatc ttgcatttaa 1680
ataaattttc tttttatagc tttatgactt agtttcaatt tatatactat tttaatgaca 1740
ttttcgattc attgattgaa agctttgtgt tttttcttga tgcgctattg cattgttctt 1800
gtctttttcg ccacatgtaa tatctgtagt agatacctga tacattgtgg atgctgagtg 1860
aaattttagt taataatgga ggcgctctta ataattttgg ggatattggc tttttttttt 1920
aaagtttaca aatgaatttt ttccgccagg atgggcccgc ggccgc 1966
<210> 5
<211> 2240
<212> DNA
<213> 人工序列
<220>
<223> 用于在S. cerevisiae中表达的合成构建体Eno1p-SpMAE1-ENOt
<400> 5
ggatccggcg cgccccgcgg aaccgccaga tattcattac ttgacgcaaa agcgtttgaa 60
ataatgacga aaaagaagga agaaaaaaaa agaaaaatac cgcttctagg cgggttatct 120
actgatccga gcttccacta ggatagcacc caaacacctg catatttgga cgacctttac 180
ttacaccacc aaaaaccact ttcgcctctc ccgcccctga taacgtccac taattgagcg 240
attacctgag cggtcctctt ttgtttgcag catgagactt gcatactgca aatcgtaagt 300
agcaacgtct caaggtcaaa actgtatgga aaccttgtca cctcacttaa ttctagctag 360
cctaccctgc aagtcaagag gtctccgtga ttcctagcca cctcaaggta tgcctctccc 420
cggaaactgt ggccttttct ggcacacatg atctccacga tttcaacata taaatagctt 480
ttgataatgg caatattaat caaatttatt ttacttcttt cttgtaacat ctctcttgta 540
atcccttatt ccttctagct atttttcata aaaaaccaag caactgctta tcaacacaca 600
aacactaaaa caaaatgggt gaattgaagg aaatcttgaa gcaacgttac catgaattgt 660
tggactggaa cgtcaaggct ccacacgttc cattgtctca aagattgaag catttcacct 720
ggtcctggtt tgcttgtacc atggccactg gtggtgtcgg tttgatcatt ggttctttcc 780
cattcagatt ctacggtttg aacaccattg gtaagattgt ctacatctta caaatcttct 840
tattctcttt gtttggttct tgtatgttgt tcagattcat caaataccca tctaccatca 900
aggactcctg gaaccaccac ttggaaaaat tattcattgc tacctgtttg ctatccatct 960
ccactttcat tgacatgttg gccatctacg cttacccaga cactggtgaa tggatggtct 1020
gggttatcag aatcttatac tacatctacg ttgctgtctc tttcatctac tgtgtcatgg 1080
ctttcttcac cattttcaac aaccacgttt acaccattga aactgcttct ccagcttgga 1140
tcttaccaat tttcccacca atgatctgtg gtgtcattgc tggtgctgtc aactccactc 1200
aaccagctca ccaattgaag aacatggtta tcttcggtat cttattccaa ggtttgggtt 1260
tctgggttta cttgttgttg tttgctgtca acgttttgag attcttcacc gttggtttgg 1320
ccaagcctca agacagacca ggtatgttca tgtttgttgg tccaccagct ttctccggtt 1380
tggctttgat caacattgcc cgtggtgcta tgggttccag accatacatt ttcgtcggtg 1440
ccaattcttc tgaatacttg ggtttcgttt ccactttcat ggccattttc atctggggtt 1500
tggctgcttg gtgttactgt ttggccatgg tttctttctt ggctggtttc ttcaccagag 1560
ctccattgaa atttgcttgt ggttggtttg ctttcatctt cccaaacgtc ggtttcgtta 1620
actgtaccat tgaaattggt aagatgattg actccaaggc cttccaaatg ttcggtcaca 1680
tcatcggtgt catcctatgt atccaatgga tcttgttgat gtacttgatg gtcagagctt 1740
tcttggtcaa cgatttgtgt tacccaggta aggatgaaga tgctcaccca cctccaaagc 1800
caaacactgg tgttttgaac ccaactttcc caccagaaaa ggctccagct tctttggaaa 1860
aggttgacac ccacgttact tccactggtg gtgaatctga tcctccatct tctgaacacg 1920
aaagcgttta agagcttttg attaagcctt ctagtccaaa aaacacgttt ttttgtcatt 1980
tatttcattt tcttagaata gtttagttta ttcattttat agtcacgaat gttttatgat 2040
tctatatagg gttgcaaaca agcatttttc attttatgtt aaaacaattt caggtttacc 2100
ttttattctg cttgtggtga cgcgggtatc cgcccgctct tttggtcacc catgtattta 2160
attgcataaa taattcttaa aagtggagct agtctatttc tatttacata cctctcattt 2220
ctcatttcct ccgcggccgc 2240
<210> 6
<211> 538
<212> PRT
<213> 人工序列
<220>
<223> Actinobacillus succinogenes磷酸烯醇式丙酮酸羧激酶氨基酸序列，第
120-122位具有EGY到DAF的修饰
<400> 6
Met Thr Asp Leu Asn Lys Leu Val Lys Glu Leu Asn Asp Leu Gly Leu
1 5 10 15
Thr Asp Val Lys Glu Ile Val Tyr Asn Pro Ser Tyr Glu Gln Leu Phe
20 25 30
Glu Glu Glu Thr Lys Pro Gly Leu Glu Gly Phe Asp Lys Gly Thr Leu
35 40 45
Thr Thr Leu Gly Ala Val Ala Val Asp Thr Gly Ile Phe Thr Gly Arg
50 55 60
Ser Pro Lys Asp Lys Tyr Ile Val Cys Asp Glu Thr Thr Lys Asp Thr
65 70 75 80
Val Trp Trp Asn Ser Glu Ala Ala Lys Asn Asp Asn Lys Pro Met Thr
85 90 95
Gln Glu Thr Trp Lys Ser Leu Arg Glu Leu Val Ala Lys Gln Leu Ser
100 105 110
Gly Lys Arg Leu Phe Val Val Asp Ala Phe Cys Gly Ala Ser Glu Lys
115 120 125
His Arg Ile Gly Val Arg Met Val Thr Glu Val Ala Trp Gln Ala His
130 135 140
Phe Val Lys Asn Met Phe Ile Arg Pro Thr Asp Glu Glu Leu Lys Asn
145 150 155 160
Phe Lys Ala Asp Phe Thr Val Leu Asn Gly Ala Lys Cys Thr Asn Pro
165 170 175
Asn Trp Lys Glu Gln Gly Leu Asn Ser Glu Asn Phe Val Ala Phe Asn
180 185 190
Ile Thr Glu Gly Ile Gln Leu Ile Gly Gly Thr Trp Tyr Gly Gly Glu
195 200 205
Met Lys Lys Gly Met Phe Ser Met Met Asn Tyr Phe Leu Pro Leu Lys
210 215 220
Gly Val Ala Ser Met His Cys Ser Ala Asn Val Gly Lys Asp Gly Asp
225 230 235 240
Val Ala Ile Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser
245 250 255
Thr Asp Pro Lys Arg Gln Leu Ile Gly Asp Asp Glu His Gly Trp Asp
260 265 270
Glu Ser Gly Val Phe Asn Phe Glu Gly Gly Cys Tyr Ala Lys Thr Ile
275 280 285
Asn Leu Ser Gln Glu Asn Glu Pro Asp Ile Tyr Gly Ala Ile Arg Arg
290 295 300
Asp Ala Leu Leu Glu Asn Val Val Val Arg Ala Asp Gly Ser Val Asp
305 310 315 320
Phe Asp Asp Gly Ser Lys Thr Glu Asn Thr Arg Val Ser Tyr Pro Ile
325 330 335
Tyr His Ile Asp Asn Ile Val Arg Pro Val Ser Lys Ala Gly His Ala
340 345 350
Thr Lys Val Ile Phe Leu Thr Ala Asp Ala Phe Gly Val Leu Pro Pro
355 360 365
Val Ser Lys Leu Thr Pro Glu Gln Thr Glu Tyr Tyr Phe Leu Ser Gly
370 375 380
Phe Thr Ala Lys Leu Ala Gly Thr Glu Arg Gly Val Thr Glu Pro Thr
385 390 395 400
Pro Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Leu Ser Leu His Pro
405 410 415
Ile Gln Tyr Ala Asp Val Leu Val Glu Arg Met Lys Ala Ser Gly Ala
420 425 430
Glu Ala Tyr Leu Val Asn Thr Gly Trp Asn Gly Thr Gly Lys Arg Ile
435 440 445
Ser Ile Lys Asp Thr Arg Gly Ile Ile Asp Ala Ile Leu Asp Gly Ser
450 455 460
Ile Glu Lys Ala Glu Met Gly Glu Leu Pro Ile Phe Asn Leu Ala Ile
465 470 475 480
Pro Lys Ala Leu Pro Gly Val Asp Pro Ala Ile Leu Asp Pro Arg Asp
485 490 495
Thr Tyr Ala Asp Lys Ala Gln Trp Gln Val Lys Ala Glu Asp Leu Ala
500 505 510
Asn Arg Phe Val Lys Asn Phe Val Lys Tyr Thr Ala Asn Pro Glu Ala
515 520 525
Ala Lys Leu Val Gly Ala Gly Pro Lys Ala
530 535
<210> 7
<211> 1139
<212> PRT
<213> 人工序列
<220>
<223> Glycosomal Trypanosoma brucei延胡索酸还原酶(FRDg)氨基酸
序列，缺乏3 aa C-端靶向信号。
<400> 7
Met Val Asp Gly Arg Ser Ser Ala Ser Ile Val Ala Val Asp Pro Glu
1 5 10 15
Arg Ala Ala Arg Glu Arg Asp Ala Ala Ala Arg Ala Leu Leu Gln Asp
20 25 30
Ser Pro Leu His Thr Thr Met Gln Tyr Ala Thr Ser Gly Leu Glu Leu
35 40 45
Thr Val Pro Tyr Ala Leu Lys Val Val Ala Ser Ala Asp Thr Phe Asp
50 55 60
Arg Ala Lys Glu Val Ala Asp Glu Val Leu Arg Cys Ala Trp Gln Leu
65 70 75 80
Ala Asp Thr Val Leu Asn Ser Phe Asn Pro Asn Ser Glu Val Ser Leu
85 90 95
Val Gly Arg Leu Pro Val Gly Gln Lys His Gln Met Ser Ala Pro Leu
100 105 110
Lys Arg Val Met Ala Cys Cys Gln Arg Val Tyr Asn Ser Ser Ala Gly
115 120 125
Cys Phe Asp Pro Ser Thr Ala Pro Val Ala Lys Ala Leu Arg Glu Ile
130 135 140
Ala Leu Gly Lys Glu Arg Asn Asn Ala Cys Leu Glu Ala Leu Thr Gln
145 150 155 160
Ala Cys Thr Leu Pro Asn Ser Phe Val Ile Asp Phe Glu Ala Gly Thr
165 170 175
Ile Ser Arg Lys His Glu His Ala Ser Leu Asp Leu Gly Gly Val Ser
180 185 190
Lys Gly Tyr Ile Val Asp Tyr Val Ile Asp Asn Ile Asn Ala Ala Gly
195 200 205
Phe Gln Asn Val Phe Phe Asp Trp Gly Gly Asp Cys Arg Ala Ser Gly
210 215 220
Met Asn Ala Arg Asn Thr Pro Trp Val Val Gly Ile Thr Arg Pro Pro
225 230 235 240
Ser Leu Asp Met Leu Pro Asn Pro Pro Lys Glu Ala Ser Tyr Ile Ser
245 250 255
Val Ile Ser Leu Asp Asn Glu Ala Leu Ala Thr Ser Gly Asp Tyr Glu
260 265 270
Asn Leu Ile Tyr Thr Ala Asp Asp Lys Pro Leu Thr Cys Thr Tyr Asp
275 280 285
Trp Lys Gly Lys Glu Leu Met Lys Pro Ser Gln Ser Asn Ile Ala Gln
290 295 300
Val Ser Val Lys Cys Tyr Ser Ala Met Tyr Ala Asp Ala Leu Ala Thr
305 310 315 320
Ala Cys Phe Ile Lys Arg Asp Pro Ala Lys Val Arg Gln Leu Leu Asp
325 330 335
Gly Trp Arg Tyr Val Arg Asp Thr Val Arg Asp Tyr Arg Val Tyr Val
340 345 350
Arg Glu Asn Glu Arg Val Ala Lys Met Phe Glu Ile Ala Thr Glu Asp
355 360 365
Ala Glu Met Arg Lys Arg Arg Ile Ser Asn Thr Leu Pro Ala Arg Val
370 375 380
Ile Val Val Gly Gly Gly Leu Ala Gly Leu Ser Ala Ala Ile Glu Ala
385 390 395 400
Ala Gly Cys Gly Ala Gln Val Val Leu Met Glu Lys Glu Ala Lys Leu
405 410 415
Gly Gly Asn Ser Ala Lys Ala Thr Ser Gly Ile Asn Gly Trp Gly Thr
420 425 430
Arg Ala Gln Ala Lys Ala Ser Ile Val Asp Gly Gly Lys Tyr Phe Glu
435 440 445
Arg Asp Thr Tyr Lys Ser Gly Ile Gly Gly Asn Thr Asp Pro Ala Leu
450 455 460
Val Lys Thr Leu Ser Met Lys Ser Ala Asp Ala Ile Gly Trp Leu Thr
465 470 475 480
Ser Leu Gly Val Pro Leu Thr Val Leu Ser Gln Leu Gly Gly His Ser
485 490 495
Arg Lys Arg Thr His Arg Ala Pro Asp Lys Lys Asp Gly Thr Pro Leu
500 505 510
Pro Ile Gly Phe Thr Ile Met Lys Thr Leu Glu Asp His Val Arg Gly
515 520 525
Asn Leu Ser Gly Arg Ile Thr Ile Met Glu Asn Cys Ser Val Thr Ser
530 535 540
Leu Leu Ser Glu Thr Lys Glu Arg Pro Asp Gly Thr Lys Gln Ile Arg
545 550 555 560
Val Thr Gly Val Glu Phe Thr Gln Ala Gly Ser Gly Lys Thr Thr Ile
565 570 575
Leu Ala Asp Ala Val Ile Leu Ala Thr Gly Gly Phe Ser Asn Asp Lys
580 585 590
Thr Ala Asp Ser Leu Leu Arg Glu His Ala Pro His Leu Val Asn Phe
595 600 605
Pro Thr Thr Asn Gly Pro Trp Ala Thr Gly Asp Gly Val Lys Leu Ala
610 615 620
Gln Arg Leu Gly Ala Gln Leu Val Asp Met Asp Lys Val Gln Leu His
625 630 635 640
Pro Thr Gly Leu Ile Asn Pro Lys Asp Pro Ala Asn Pro Thr Lys Phe
645 650 655
Leu Gly Pro Glu Ala Leu Arg Gly Ser Gly Gly Val Leu Leu Asn Lys
660 665 670
Gln Gly Lys Arg Phe Val Asn Glu Leu Asp Leu Arg Ser Val Val Ser
675 680 685
Lys Ala Ile Met Glu Gln Gly Ala Glu Tyr Pro Gly Ser Gly Gly Ser
690 695 700
Met Phe Ala Tyr Cys Val Leu Asn Ala Ala Ala Gln Lys Leu Phe Gly
705 710 715 720
Val Ser Ser His Glu Phe Tyr Trp Lys Lys Met Gly Leu Phe Val Lys
725 730 735
Ala Asp Thr Met Arg Asp Leu Ala Ala Leu Ile Gly Cys Pro Val Glu
740 745 750
Ser Val Gln Gln Thr Leu Glu Glu Tyr Glu Arg Leu Ser Ile Ser Gln
755 760 765
Arg Ser Cys Pro Ile Thr Arg Lys Ser Val Tyr Pro Cys Val Leu Gly
770 775 780
Thr Lys Gly Pro Tyr Tyr Val Ala Phe Val Thr Pro Ser Ile His Tyr
785 790 795 800
Thr Met Gly Gly Cys Leu Ile Ser Pro Ser Ala Glu Ile Gln Met Lys
805 810 815
Asn Thr Ser Ser Arg Ala Pro Leu Ser His Ser Asn Pro Ile Leu Gly
820 825 830
Leu Phe Gly Ala Gly Glu Val Thr Gly Gly Val His Gly Gly Asn Arg
835 840 845
Leu Gly Gly Asn Ser Leu Leu Glu Cys Val Val Phe Gly Arg Ile Ala
850 855 860
Gly Asp Arg Ala Ser Thr Ile Leu Gln Arg Lys Ser Ser Ala Leu Ser
865 870 875 880
Phe Lys Val Trp Thr Thr Val Val Leu Arg Glu Val Arg Glu Gly Gly
885 890 895
Val Tyr Gly Ala Gly Ser Arg Val Leu Arg Phe Asn Leu Pro Gly Ala
900 905 910
Leu Gln Arg Ser Gly Leu Ser Leu Gly Gln Phe Ile Ala Ile Arg Gly
915 920 925
Asp Trp Asp Gly Gln Gln Leu Ile Gly Tyr Tyr Ser Pro Ile Thr Leu
930 935 940
Pro Asp Asp Leu Gly Met Ile Asp Ile Leu Ala Arg Ser Asp Lys Gly
945 950 955 960
Thr Leu Arg Glu Trp Ile Ser Ala Leu Glu Pro Gly Asp Ala Val Glu
965 970 975
Met Lys Ala Cys Gly Gly Leu Val Ile Glu Arg Arg Leu Ser Asp Lys
980 985 990
His Phe Val Phe Met Gly His Ile Ile Asn Lys Leu Cys Leu Ile Ala
995 1000 1005
Gly Gly Thr Gly Val Ala Pro Met Leu Gln Ile Ile Lys Ala Ala
1010 1015 1020
Phe Met Lys Pro Phe Ile Asp Thr Leu Glu Ser Val His Leu Ile
1025 1030 1035
Tyr Ala Ala Glu Asp Val Thr Glu Leu Thr Tyr Arg Glu Val Leu
1040 1045 1050
Glu Glu Arg Arg Arg Glu Ser Arg Gly Lys Phe Lys Lys Thr Phe
1055 1060 1065
Val Leu Asn Arg Pro Pro Pro Leu Trp Thr Asp Gly Val Gly Phe
1070 1075 1080
Ile Asp Arg Gly Ile Leu Thr Asn His Val Gln Pro Pro Ser Asp
1085 1090 1095
Asn Leu Leu Val Ala Ile Cys Gly Pro Pro Val Met Gln Arg Ile
1100 1105 1110
Val Lys Ala Thr Leu Lys Thr Leu Gly Tyr Asn Met Asn Leu Val
1115 1120 1125
Arg Thr Val Asp Glu Thr Glu Pro Ser Gly Ser
1130 1135
<210> 8
<211> 472
<212> PRT
<213> 人工序列
<220>
<223> Rhizopus oryzae延胡索酸酶氨基酸序列，缺乏最初23个N-端氨基酸。
<400> 8
Met Ser Ser Ala Ser Ala Ala Leu Gln Lys Phe Arg Ala Glu Arg Asp
1 5 10 15
Thr Phe Gly Asp Leu Gln Val Pro Ala Asp Arg Tyr Trp Gly Ala Gln
20 25 30
Thr Gln Arg Ser Leu Gln Asn Phe Asp Ile Gly Gly Pro Thr Glu Arg
35 40 45
Met Pro Glu Pro Leu Ile Arg Ala Phe Gly Val Leu Lys Lys Ala Ala
50 55 60
Ala Thr Val Asn Met Thr Tyr Gly Leu Asp Pro Lys Val Gly Glu Ala
65 70 75 80
Ile Gln Lys Ala Ala Asp Glu Val Ile Asp Gly Ser Leu Ile Asp His
85 90 95
Phe Pro Leu Val Val Trp Gln Thr Gly Ser Gly Thr Gln Thr Lys Met
100 105 110
Asn Val Asn Glu Val Ile Ser Asn Arg Ala Ile Glu Leu Leu Gly Gly
115 120 125
Glu Leu Gly Ser Lys Ala Pro Val His Pro Asn Asp His Val Asn Met
130 135 140
Ser Gln Ser Ser Asn Asp Thr Phe Pro Thr Ala Met His Val Ala Ala
145 150 155 160
Val Val Glu Ile His Gly Arg Leu Ile Pro Ala Leu Thr Thr Leu Arg
165 170 175
Asp Ala Leu Gln Ala Lys Ser Ala Glu Phe Glu His Ile Ile Lys Ile
180 185 190
Gly Arg Thr His Leu Gln Asp Ala Thr Pro Leu Thr Leu Gly Gln Glu
195 200 205
Phe Ser Gly Tyr Thr Gln Gln Leu Thr Tyr Gly Ile Ala Arg Val Gln
210 215 220
Gly Thr Leu Glu Arg Leu Tyr Asn Leu Ala Gln Gly Gly Thr Ala Val
225 230 235 240
Gly Thr Gly Leu Asn Thr Arg Lys Gly Phe Asp Ala Lys Val Ala Glu
245 250 255
Ala Ile Ala Ser Ile Thr Gly Leu Pro Phe Lys Thr Ala Pro Asn Lys
260 265 270
Phe Glu Ala Leu Ala Ala His Asp Ala Leu Val Glu Ala His Gly Ala
275 280 285
Leu Asn Thr Val Ala Cys Ser Leu Met Lys Ile Ala Asn Asp Ile Arg
290 295 300
Tyr Leu Gly Ser Gly Pro Arg Cys Gly Leu Gly Glu Leu Ser Leu Pro
305 310 315 320
Glu Asn Glu Pro Gly Ser Ser Ile Met Pro Gly Lys Val Asn Pro Thr
325 330 335
Gln Cys Glu Ala Met Thr Met Val Cys Ala Gln Val Met Gly Asn Asn
340 345 350
Thr Ala Ile Ser Val Ala Gly Ser Asn Gly Gln Phe Glu Leu Asn Val
355 360 365
Phe Lys Pro Val Met Ile Lys Asn Leu Ile Gln Ser Ile Arg Leu Ile
370 375 380
Ser Asp Ala Ser Ile Ser Phe Thr Lys Asn Cys Val Val Gly Ile Glu
385 390 395 400
Ala Asn Glu Lys Lys Ile Ser Ser Ile Met Asn Glu Ser Leu Met Leu
405 410 415
Val Thr Ala Leu Asn Pro His Ile Gly Tyr Asp Lys Ala Ala Lys Cys
420 425 430
Ala Lys Lys Ala His Lys Glu Gly Thr Thr Leu Lys Glu Ala Ala Leu
435 440 445
Ser Leu Gly Tyr Leu Thr Ser Glu Glu Phe Asp Gln Trp Val Arg Pro
450 455 460
Glu Asp Met Ile Ser Ala Lys Asp
465 470
<210> 9
<211> 340
<212> PRT
<213> 人工序列
<220>
<223> Saccharomyces cerevisiae过氧化物酶体苹果酸脱氢酶(Mdh3)氨基酸序列，
缺乏3 C-端过氧化物酶体靶向序列(SKL)
<400> 9
Met Val Lys Val Ala Ile Leu Gly Ala Ser Gly Gly Val Gly Gln Pro
1 5 10 15
Leu Ser Leu Leu Leu Lys Leu Ser Pro Tyr Val Ser Glu Leu Ala Leu
20 25 30
Tyr Asp Ile Arg Ala Ala Glu Gly Ile Gly Lys Asp Leu Ser His Ile
35 40 45
Asn Thr Asn Ser Ser Cys Val Gly Tyr Asp Lys Asp Ser Ile Glu Asn
50 55 60
Thr Leu Ser Asn Ala Gln Val Val Leu Ile Pro Ala Gly Val Pro Arg
65 70 75 80
Lys Pro Gly Leu Thr Arg Asp Asp Leu Phe Lys Met Asn Ala Gly Ile
85 90 95
Val Lys Ser Leu Val Thr Ala Val Gly Lys Phe Ala Pro Asn Ala Arg
100 105 110
Ile Leu Val Ile Ser Asn Pro Val Asn Ser Leu Val Pro Ile Ala Val
115 120 125
Glu Thr Leu Lys Lys Met Gly Lys Phe Lys Pro Gly Asn Val Met Gly
130 135 140
Val Thr Asn Leu Asp Leu Val Arg Ala Glu Thr Phe Leu Val Asp Tyr
145 150 155 160
Leu Met Leu Lys Asn Pro Lys Ile Gly Gln Glu Gln Asp Lys Thr Thr
165 170 175
Met His Arg Lys Val Thr Val Ile Gly Gly His Ser Gly Glu Thr Ile
180 185 190
Ile Pro Ile Ile Thr Asp Lys Ser Leu Val Phe Gln Leu Asp Lys Gln
195 200 205
Tyr Glu His Phe Ile His Arg Val Gln Phe Gly Gly Asp Glu Ile Val
210 215 220
Lys Ala Lys Gln Gly Ala Gly Ser Ala Thr Leu Ser Met Ala Phe Ala
225 230 235 240
Gly Ala Lys Phe Ala Glu Glu Val Leu Arg Ser Phe His Asn Glu Lys
245 250 255
Pro Glu Thr Glu Ser Leu Ser Ala Phe Val Tyr Leu Pro Gly Leu Lys
260 265 270
Asn Gly Lys Lys Ala Gln Gln Leu Val Gly Asp Asn Ser Ile Glu Tyr
275 280 285
Phe Ser Leu Pro Ile Val Leu Arg Asn Gly Ser Val Val Ser Ile Asp
290 295 300
Thr Ser Val Leu Glu Lys Leu Ser Pro Arg Glu Glu Gln Leu Val Asn
305 310 315 320
Thr Ala Val Lys Glu Leu Arg Lys Asn Ile Glu Lys Gly Lys Ser Phe
325 330 335
Ile Leu Asp Ser
340
<210> 10
<211> 438
<212> PRT
<213> Schizosaccharomyces pombe苹果酸通透酶氨基酸序列。
<400> 10
Met Gly Glu Leu Lys Glu Ile Leu Lys Gln Arg Tyr His Glu Leu Leu
1 5 10 15
Asp Trp Asn Val Lys Ala Pro His Val Pro Leu Ser Gln Arg Leu Lys
20 25 30
His Phe Thr Trp Ser Trp Phe Ala Cys Thr Met Ala Thr Gly Gly Val
35 40 45
Gly Leu Ile Ile Gly Ser Phe Pro Phe Arg Phe Tyr Gly Leu Asn Thr
50 55 60
Ile Gly Lys Ile Val Tyr Ile Leu Gln Ile Phe Leu Phe Ser Leu Phe
65 70 75 80
Gly Ser Cys Met Leu Phe Arg Phe Ile Lys Tyr Pro Ser Thr Ile Lys
85 90 95
Asp Ser Trp Asn His His Leu Glu Lys Leu Phe Ile Ala Thr Cys Leu
100 105 110
Leu Ser Ile Ser Thr Phe Ile Asp Met Leu Ala Ile Tyr Ala Tyr Pro
115 120 125
Asp Thr Gly Glu Trp Met Val Trp Val Ile Arg Ile Leu Tyr Tyr Ile
130 135 140
Tyr Val Ala Val Ser Phe Ile Tyr Cys Val Met Ala Phe Phe Thr Ile
145 150 155 160
Phe Asn Asn His Val Tyr Thr Ile Glu Thr Ala Ser Pro Ala Trp Ile
165 170 175
Leu Pro Ile Phe Pro Pro Met Ile Cys Gly Val Ile Ala Gly Ala Val
180 185 190
Asn Ser Thr Gln Pro Ala His Gln Leu Lys Asn Met Val Ile Phe Gly
195 200 205
Ile Leu Phe Gln Gly Leu Gly Phe Trp Val Tyr Leu Leu Leu Phe Ala
210 215 220
Val Asn Val Leu Arg Phe Phe Thr Val Gly Leu Ala Lys Pro Gln Asp
225 230 235 240
Arg Pro Gly Met Phe Met Phe Val Gly Pro Pro Ala Phe Ser Gly Leu
245 250 255
Ala Leu Ile Asn Ile Ala Arg Gly Ala Met Gly Ser Arg Pro Tyr Ile
260 265 270
Phe Val Gly Ala Asn Ser Ser Glu Tyr Leu Gly Phe Val Ser Thr Phe
275 280 285
Met Ala Ile Phe Ile Trp Gly Leu Ala Ala Trp Cys Tyr Cys Leu Ala
290 295 300
Met Val Ser Phe Leu Ala Gly Phe Phe Thr Arg Ala Pro Leu Lys Phe
305 310 315 320
Ala Cys Gly Trp Phe Ala Phe Ile Phe Pro Asn Val Gly Phe Val Asn
325 330 335
Cys Thr Ile Glu Ile Gly Lys Met Ile Asp Ser Lys Ala Phe Gln Met
340 345 350
Phe Gly His Ile Ile Gly Val Ile Leu Cys Ile Gln Trp Ile Leu Leu
355 360 365
Met Tyr Leu Met Val Arg Ala Phe Leu Val Asn Asp Leu Cys Tyr Pro
370 375 380
Gly Lys Asp Glu Asp Ala His Pro Pro Pro Lys Pro Asn Thr Gly Val
385 390 395 400
Leu Asn Pro Thr Phe Pro Pro Glu Lys Ala Pro Ala Ser Leu Glu Lys
405 410 415
Val Asp Thr His Val Thr Ser Thr Gly Gly Glu Ser Asp Pro Pro Ser
420 425 430
Ser Glu His Glu Ser Val
435

Claims (8)

1.用于制备二羧酸的方法，所述方法包括在低于有机酸最低pKa的pH值下，在存在含碳水化合物的底物和少量氧时发酵酵母，其中所述氧以8到0.2mmol/g生物质干重/小时之间的范围内的氧吸收比速率被供应。
2.根据权利要求1的方法，其中所述二羧酸是延胡索酸、苹果酸或琥珀酸。
3.根据权利要求1或2的方法，其中所述pH在pH 1.0到pH 5.5的范围内。
4.根据权利要求1到3中任一项的方法，所述方法包括在碳受限的条件下发酵酵母。
5.根据权利要求1到4中任一项的方法，所述方法在存在额外的电子供体时进行。
6.根据权利要求1到5中任一项的方法，其中所述酵母是Saccharomyces cerevisiae。
7.根据权利要求1到6中任一项的方法，其中所述酵母是经遗传修饰的酵母。
8.根据权利要求7的方法，其中所述经遗传修饰的酵母包含编码选自下组的异源酶的核苷酸序列，所述组由磷酸烯醇式丙酮酸羧激酶、延胡索酸还原酶和延胡索酸酶组成。
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CN102089436A (zh) 2011-06-08
EP2297297A1 (en) 2011-03-23
EP2297297B1 (en) 2017-02-08
US9012187B2 (en) 2015-04-21
CA2730595A1 (en) 2010-01-14
CA2730595C (en) 2014-12-16
CN107267562A (zh) 2017-10-20
EA201100156A1 (ru) 2011-08-30
US20110229945A1 (en) 2011-09-22
UA103033C2 (ru) 2013-09-10
WO2010003728A4 (en) 2010-04-08
EA018463B1 (ru) 2013-08-30
ES2623932T3 (es) 2017-07-12
EP3176265A1 (en) 2017-06-07
US20150232891A1 (en) 2015-08-20
BRPI0915534B1 (pt) 2021-04-20
BRPI0915534A2 (pt) 2020-08-18
WO2010003728A1 (en) 2010-01-14

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