CN116806263A - 使用微生物代谢途径生产2,4-二羟基丁酸或l-苏氨酸的方法 - Google Patents
使用微生物代谢途径生产2,4-二羟基丁酸或l-苏氨酸的方法 Download PDFInfo
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Abstract
本发明涉及使用微生物代谢途径生产2,4‑二羟基丁酸(DHB)或L‑苏氨酸的方法，其中该代谢途径在微生物生产菌株中表达，该微生物生产菌株通过将所述酶表达所需的至少一个基因引入生产菌株中而事先相对于其天然形式(野生型)被修饰。
Description
本发明涉及使用微生物代谢途径生产2,4-二羟基丁酸(DHB)(其可以以2,4-二羟基丁酸盐的形式存在，或者以酸2,4-二羟基丁酸的形式存在)或L-苏氨酸的方法。
通过实现生物基燃料和化学品的生物可持续生产，减少对化石原材料的依赖具有重要的生物经济和生态利益。在这种情况下，由于(L)-2,4-二羟基丁酸(DHB)作为合成用于动物营养的甲硫氨酸类似物的起始材料的重要性，该行业在生物合成(L)-2,4-二羟基丁酸(DHB)方面的努力也大大增加。
氨基酸甲硫氨酸和甲硫氨酸类似物(D/L)-2-羟基-4-(甲硫基)丁酸(HMTB)主要用作养鸡的饲料添加剂，在市场上的年营业额为约30亿欧元。甲硫氨酸目前仅由化石原材料石油和天然气进行生产。氨基酸苏氨酸被用作猪育肥的饲料添加剂。甲硫氨酸制造商对将其化学甲硫氨酸生产工艺转化为可持续的微生物生产工艺有着浓厚的兴趣，因为随着化学工艺CO2排放价格的上涨，预计这些工艺的成本将大幅增加。
氨基酸L-苏氨酸目前通过微生物生产工艺从糖类的葡萄糖或蔗糖在工业规模上进行制备。可以等效使用的氨基酸D/L-甲硫氨酸和类似物D/L-2-羟基-4-(甲硫基)丁酸(HMTB)目前仅由石油和天然气在工业规模上进行生产。
从石油和天然气化学合成甲硫氨酸是不可持续的，并且必须替换为使用可再生原材料的工艺。从糖类进行微生物合成苏氨酸和甲硫氨酸明显更具可持续性。然而，这些工艺与食品生产有着竞争关系。
本发明所基于的目的在于提供如下方法，该方法为制备氨基酸甲硫氨酸和苏氨酸的可持续方法开辟了道路。
本发明的目的通过具有权利要求1的特征的方法实现。在从属权利要求中陈述了进一步的发展。
解决方法是使用微生物代谢途径生产2,4-二羟基丁酸或L-苏氨酸的方法，该方法至少包括以下步骤：
使用苏糖醛缩酶将乙醇醛经酶促转化为苏糖的步骤，
使用苏糖脱氢酶将苏糖经酶促转化为苏糖酸-1,4-内酯的步骤，
使用苏糖酸-1,4-内酯酶将苏糖酸-1,4-内酯经酶促转化为苏糖酸的步骤，以及
使用苏糖酸脱水酶将苏糖酸经酶促转化为2-酮基-4-羟基丁酸(OHB)的步骤，
其中代谢途径还包括使用OHB还原酶将OHB经酶促转化为2,4-二羟基丁酸的步骤或使用L-高丝氨酸转氨酶将OHB经酶促转化为L-高丝氨酸的步骤，然后是在ATP消耗下使用高丝氨酸激酶将L-高丝氨酸经酶促转化为O-磷酸-L-高丝氨酸的步骤，以及使用L-苏氨酸合酶将O-磷酸-高丝氨酸经酶促转化为L-苏氨酸的步骤，其中代谢途径在微生物生产菌株中表达，该微生物生产菌株通过以适当的方式将所述酶表达所需的至少一个基因引入生产菌株中而事先相对于其天然形式(野生型)被修饰。根据本发明的概念，代谢途径通过乙醇醛进行，乙醇醛可以通过不同的方式获得。因此，乙醇醛可以通过合成代谢途径由木糖提供，如Cam等人/2016/ACS Synth Biol/5/607-618中所述。此外，乙醇醛也可以由乙二醇表示。这又可以容易地从合成气中回收，这与从CO2排放中回收合成气一样是现有技术。此外，乙二醇也可以通过糖的化学氢解获得，如Zheng等人/2017/ACS Catal/7/1939-1954中所述。将糖氢解为乙二醇是用于化学消化植物废物的创新方法，其可能比传统的基于酸或酶的消化方法更有效。最后但并非最不重要的是，乙二醇是塑料聚对苯二甲酸乙二醇酯(PET)的主要成分，因此根据本发明的方法也为塑料废物的回收利用开辟了道路。
根据本发明的概念，开发了如下代谢途径，其能够将乙醇醛(其又容易从乙二醇获得)保碳转化为L-苏氨酸或HMTB。
另一个实质性优点是，在基于乙二醇的生产中仅形成很少的副产物。这是可以预期的，因为用于生产的代谢途径与自然代谢更好地分离。由于基于乙二醇的工艺中存在很少的副产物，因此可以按相对简单的方式纯化所得的有价值物质苏氨酸和DHB。
在本发明中，可以实现自然界中不以这种形式存在的代谢途径。这种代谢途径部分基于迄今为止尚不清楚的酶活性。令人惊讶的是，这两种未知的酶活性可以通过筛选发现。此外，新发现的酶活性以及来自各种其他微生物的已知活性可以在单个生产菌株中一起表达。因此，构建了先前未知的反应顺序或先前未知的代谢途径。根据本发明的有利实施例，生产菌株已经具有其天然形式的代谢途径所需的一种或多种酶。有利地，大肠杆菌(Escherichia coli)种菌株可用作生产菌株，优选大肠杆菌ΔyqhDΔaldA。这种菌株有利地适合作为生产菌株，因为它具有醛脱氢酶(AldA)和乙醇醛还原酶(YqhD)的失活，这些酶与乙醇醛转化为D-苏糖竞争。此外，恶臭假单胞菌(Pseudomonas putida)种的菌株也是合适的，特别是因为该种的菌株具有非常合适的乙二醇脱氢酶。
在本发明的一个实施例中，使用D-苏糖醛缩酶将乙醇醛转化为D-苏糖。因此，然后使用D-苏糖脱氢酶将D-苏糖经酶促转化为D-苏糖酸-1,4-内酯。接下来是使用D-苏糖酸-1,4-内酯酶将D-苏糖酸-1,4-内酯经酶促转化为D-苏糖酸的步骤。在该实施例的下一步骤中，使用D-苏糖酸脱水酶将D-苏糖酸经酶促转化为2-酮基-4-羟基丁酸(OHB)。
在根据本发明的方法的一个特别优选的实施例中，将表达来自石竹副伯克霍尔德菌(Paraburkholderia caryophylli)(Pc.TadH)和/或来自油菜黄单胞菌(Xanthomonascampestris)(Xc.Fdh)的酶D-苏醛糖-1-脱氢酶的遗传信息引入生产菌株的基因组中，用于在生产菌株中表达D-苏糖脱氢酶。类似地，为了在生产菌株中提供D-苏糖脱氢酶，可以将表达来自酿酒酵母(Saccharomyces cerevisiae)(Sc.Ara1)或来自燕麦食酸菌(Acidovoraxavenae)(Aa.TadH)的酶D-阿拉伯糖脱氢酶的遗传信息或表达来自多食伯克霍尔德菌(Burkholderia Multivorans)(Bm.Fdh)的酶L-岩藻糖脱氢酶的遗传信息引入生产菌株的基因组中。因此，D-苏糖脱氢酶可以由氨基酸序列SEQ ID No.113、SEQ ID No.117、SEQ IDNo.123、SEQ ID No.125和SEQ ID No.131中的一个表示。
D-苏糖酸脱水酶在生产菌株中的表达可以有利地实现，因为将表达来自燕麦食酸菌(Aa.AraD)和/或赫氏草螺菌(Herbaspirillum huttiense)(Hh.AraD)和/或含羞草副伯克霍尔德菌(Paraburkholderia mimosarum)(Pm.AraD)和/或来自优化突变体Hh.AraDC434S的酶D-阿拉伯糖酸脱水酶的遗传信息引入生产菌株的基因组中。因此，以下氨基酸序列可以表示D-苏糖酸脱水酶：SEQ ID No.151、SEQ ID No.153、SEQ IDNo.155和SEQ IDNo.159。
为了在生产菌株中表达D-苏糖醛缩酶，优选将表达来自大肠杆菌(Ec.FsaA)的酶D-果糖-6-磷酸醛缩酶的遗传信息和/或突变变体Ec.FsaAL107Y:A129G(Ec.FsaATA)的遗传信息引入生产菌株的基因组中。因此，D-苏糖醛缩酶可以由氨基酸序列SEQ ID NO.109或SEQ ID No.111中的一个表示。
为了在生产菌株中表达苏糖酸-1,4-内酯酶，可以将表达来自thermoguttaterrifontis(Tt.Lac11)的酶葡糖酸内酯酶的遗传信息和/或特别优选地，该酶的截短变体(Tt.Lac11v1)的遗传信息(其导致表达的显著改善)引入生产菌株的基因组中。因此，苏糖酸-1,4-内酯酶可以由氨基酸序列SEQ ID No.133和SEQ ID No.135中的一个表示。
根据本发明方法的一个特别有利的设计，除了各自代谢途径的酶之外，苏糖酸导入酶在生产菌株中表达。例如，这可以通过将表达来自钩虫贪铜菌(Cupriavidus necator)(Re.kdgT)的D-苏糖酸导入渗透酶的遗传信息引入生产菌株的基因组来实现。因此，苏糖酸导入酶可以例如由氨基酸序列SEQ ID No.165表示。
根据本发明，OHB还原酶用于将OHB转化为DHB。在本发明的一个实施例变体中，从C.J.R.；Topham,C.M.；Malbert,Y.；/>J.M.；Walther,T.RationalEngineering of a Malate Dehydrogenase for Microbial Production of2,4-Dihydroxybutyric Acid via Homoserine Pathway[通过高丝氨酸途径微生物生产2,4-二羟基丁酸的苹果酸脱氢酶的合理工程化].Biochem.J.[生物化学杂志]2018,475(23),3887–3901已知的NADH依赖性OHB还原酶Ec.Mdh5Q用作OHB还原酶。OHB还原酶可以由氨基酸序列SEQ ID No.163表示。
令人惊讶的是，在OHB还原为DHB的过程中，可以通过使用辅助因子NADPH而不是NADH来改善OHB还原为DHB。通过将突变引入NADH依赖性Ec.Mdh5Q酶中，可以改变其辅助因子特异性以有利于NADPH。值得注意的是，到目前为止还不存在NADPH依赖性OHB还原酶。
在本发明的有利实施例中，Ec.Mdh5Q酶的NADPH依赖性变体在DHB生物合成中在生产菌株中表达，该变体在位置D34或I35的至少一个中具有突变。也就是说，为了在生产菌株中表达偏好NADPH的OHB还原酶，将表达来自大肠杆菌的酶L-苹果酸脱氢酶(Ec.Mdh)的突变变体的遗传信息引入生产菌株的基因组中，其中除了相对于野生型酶Ec.Mdh突变的变体Ec.Mdh5Q(Ec.Mdh I12V:R81A:M85Q:D86S:G179D)的五个点突变之外，其中在位置12处的异亮氨酸被缬氨酸取代(I12V)，在位置81处的精氨酸被丙氨酸取代(R81A)，在位置85处的甲硫氨酸被谷氨酰胺取代(M85Q)，在位置86处的天冬氨酸被丝氨酸取代(D86S)以及在位置179处的甘氨酸被天冬氨酸取代(G179D)，该突变酶在位置D34和I35的至少一个中具有另一个突变。这样，D34表示对应于野生型酶中被天冬氨酸占据的位置34的位置，并且I35表示对应于野生型酶中被异亮氨酸占据的位置的位置。优选地，为了在生产菌株中表达偏好NADPH的OHB还原酶，将表达以下酶中一种的遗传信息引入生产菌株的基因组中：
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G(Ec.Mdh5Q D34G)，其中位置34的天冬氨酸被甘氨酸取代，由氨基酸序列SEQ ID No.173表示，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:I35S(Ec.Mdh5Q I35S)，其中位置35的异亮氨酸被丝氨酸取代，由氨基酸序列SEQ ID No.175表示，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35K(Ec.Mdh5Q D34G I35K)，其中位置34的天冬氨酸被甘氨酸取代，位置35的异亮氨酸被赖氨酸取代，由氨基酸序列SEQ IDNo.177表示，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35R(Ec.Mdh5Q D34G I35R＝Ec.Mdh7Q)，其中位置34的天冬氨酸被甘氨酸取代，位置35的异亮氨酸被精氨酸取代，由氨基酸序列SEQ ID No.179表示，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35S(Ec.Mdh5Q D34G I35S)，其中位置34的天冬氨酸被甘氨酸取代，位置35的异亮氨酸被丝氨酸取代，由氨基酸序列SEQ IDNo.181表示，以及
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35T(Ec.Mdh5Q D34G I35T)，其中位置34的天冬氨酸被甘氨酸取代，位置35的异亮氨酸被苏氨酸取代，由氨基酸序列SEQ IDNo.183表示。
在本发明的一个特别有利的实施例中，酶Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35R(Ec.Mdh7Q)被表达为NADPH依赖性OHB还原酶。
具有2-酮基-4-羟基丁酸(OHB)还原酶活性的Ec.Mdh5Q酶的相应NADPH依赖性变体也代表了具有上述实施例的本发明中的一个独立目的，该变体催化2-酮基-4-羟基丁酸(OHB)转化为2,4-二羟基丁酸(DHB)并代表来自大肠杆菌的L-苹果酸脱氢酶(Ec.Mdh)的突变体，并且与野生型酶相比，除了五个点突变I12V、R81A、M85Q、D86S和G179D之外，在位置D34和I35的至少一个中具有另一个突变。这同样适用于使用这种酶将OHB转化为2,4-DHB，其中该酶的用途也包括在使用除上述以外的产生DHB的代谢途径的方法中，但像这些一样，还包括使用OHB还原酶将2-酮基-4-羟基丁酸(OHB)经酶促转化为2,4-二羟基丁酸(DHB)，例如，根据WO 2014/009435A1的方法。通常，相应修饰的微生物将催化所选DHB产生代谢途径的步骤所需的基因表达为制备2,4-二羟基丁酸(DHB)的生产菌株。由于这些代谢途径中的每一个都包括使用OHB还原酶将2-酮基-4-羟基丁酸(OHB)经酶促转化为2,4-二羟基丁酸(DHB)，因此可以将表达上述酶中每一种的遗传信息引入微生物的基因组中以表达OHB还原酶。
替代地，从乙醇醛开始，也可以将乙醇醛转化为L-苏糖而不是D-苏糖。为此，可以使用选自已知酶类D-苏氨酸醛缩酶(酶类4.1.2.42)、L-别苏氨酸醛缩酶(4.1.2.49)、L-苏氨酸醛缩酶(4.1.2.5)、4-羟基-2-氧戊二酸醛缩酶(4.1.3.16)和2-脱氢-3-脱氧-D-戊酸醛缩酶(4.1.2.28)的醛缩酶活性。直至2-酮基-4-羟基丁酸的上述代谢途径的所有进一步阶段都可以与L-形式类似。所以，Kim,Suk Min,Hyun Seung Lim,和Sun Bok Lee.“Discoveryof a RuBisCO-like Protein that Functions as an Oxygenase in the Novel D-Hamamelose Pathway[发现在新型D-金缕梅糖途径中作为加氧酶的RuBisCO样蛋白].”Biotechnology and bioprocess engineering[生物技术和生物工艺工程]23.5(2018):490-499已经可以证明L-苏糖脱氢酶活性与来自人苍白杆菌(Ochrobactrum anthropi)(Oa.HamH)的金缕梅糖脱氢酶。例如，在Westlake,A.“Thermostable Enzymes ImportantFor Industrial Biotechnology[对工业生物技术重要的耐热酶].”(2019)中描述了对L-苏糖酸-1,4-内酯具有活性的内酯酶。在那里，来自Thermogutta terrifontis(Tt.Ara11)的葡糖酸内酯酶显示出相应的活性。对L-苏糖酸具有活性的脱水酶是已知的，例如来自硫化叶菌(Sulfolobus solfataricus)的二羟基酸脱水酶，如出版物Kim,S.；Lee,S.B.Catalytic Promiscuity in Dihydroxy-Acid Dehydratase from theThermoacidophilic Archaeon Sulfolobus Solfataricus[来自嗜热嗜酸古菌硫化叶菌的二羟基酸脱水酶的催化混杂性].J.Biochem[生物化学杂志].2006,139(3),591–596中所述。
根据本发明的进一步设计，上述方法包括至少一个进一步的先前步骤，用于例如从乙二醇、甲醇或木糖微生物生产乙醇醛。这样，乙醇醛可以通过代谢途径从乙二醇衍生，对于乙二醇的转化，该代谢途径使用吡咯喹啉醌(PQQ)依赖性乙二醇脱氢酶(膜结合)的酶活性，由Mückschel,B.；Simon,O.；Klebensberger,J.；Graf,N.；Rosche,B.；Altenbuchner,J.；Pfannstiel,J.；Huber,A.；Hauer,B.Ethylene Glycol Metabolism by PseudomonasPutida[恶臭假单胞菌的乙二醇代谢].Appl.Environ.Microbiol[应用与环境微生物学].2012,78(24),8531–8539报道，或NAD(P)依赖性乙二醇脱氢酶(胞质)，已知来自Lu,Z.；Cabiscol,E.；Obradors,N.；Tamarit,J.；Ros,J.；Aguilar,J.；Lin,E.C.Evolution of anEscherichia Coli Protein with Increased Resistance to Oxidative Stress[具有增加的对氧化应激的抗性的大肠杆菌蛋白的进化].J.Biol.Chem[生物化学杂志].1998,273(14),8308–8316，和Zhang,X.；Zhang,B.；Lin,J.；Wei,D.Oxidation of Ethylene Glycolto Glycolaldehyde Using a Highly Selectivealcohol Dehydrogenase fromGluconobacter Oxydans[使用来自氧化葡糖杆菌的高选择性醇脱氢酶将乙二醇氧化为乙醇醛].J.Mol.Catalysis B[分子催化杂志B]2015,112,69–75。
乙醇醛也可以通过代谢途径从甲醇衍生，该代谢途径依次使用甲醇脱氢酶的酶活性以将甲醇转化为甲醛，并使用乙醇醛合酶以将甲醛转化为乙醇醛，如出版物Lu,X.；Liu,Y.；Yang,Y.；Wang,S.；Wang,Q.；Wang,X.；Yan,Z.；Cheng,J.；Liu,C.；Yang,X.；等人Constructing a Synthetic Pathway for Acetyl-Coenzyme Afrom One-Carbon throughEnzyme Design[通过酶设计从单碳构建乙酰辅酶A的合成途径].Nat Commun[自然通讯]2019,10(1),1378中所述。
乙醇醛也可以通过多阶段代谢途径从木糖衍生，该多阶段代谢途径依次使用木糖异构酶的酶活性以将D-木糖转化为D-木酮糖，使用木酮糖-1-激酶以将D-木酮糖转化为D-木酮糖-1P，以及使用木酮糖-1P-醛缩酶以将木糖-1P-醛缩酶转化为乙醇醛，从Cam等人/2016/ACS Synth Biol[ACS合成生物学]/5/607-61中得知。
使用甲醇的一个优点是，就像乙二醇一样，它可以容易地从合成气衍生。因此，从乙二醇通过DHB生物合成苏氨酸或HMTB可以被恰当地描述为一种特别可持续的生产方法。
本发明实施例的进一步细节、特征和优点源于附图和以下示例性实施例的描述。其中
图1：显示了用于将乙醇醛转化为DHB的五阶段代谢途径草案的示意图，
图2：显示了筛选候选酶的NAD(P)依赖性D-苏糖脱氢酶活性的结果的柱状图，
图3：显示了筛选候选酶的D-苏糖酸脱水酶活性的结果的柱状图，
图4：显示了表示表达各种乙二醇脱氢酶的大肠杆菌菌株的生长模式的图表，以及
图5：显示了基于13C的代谢流分析结果的柱状图，显示了通过合成代谢途径从乙醇醛(GA)生物合成L-苏氨酸。
在图1中，示意性地表示了使用微生物代谢途径从乙醇醛制备2,4-二羟基丁酸(DHB)或L-苏氨酸的各种方法，其中所有这些微生物代谢途径共有四个反应阶段，它们相继进行并由苏糖醛缩酶、苏糖脱氢酶、苏糖酸-1,4-内酯酶和苏糖酸脱水酶催化。
代谢途径在微生物生产菌株中表达，优选大肠杆菌型，其通过将所述酶表达所需的至少一个基因引入生产菌株中而事先相对于其天然形式(野生型)被修饰。
在生产L-2,4-二羟基丁酸的情况下，两个乙醇醛分子可以通过上述四个连续反应阶段转化为2-酮基-4-羟基丁酸(OHB)，最后通过随后的第五反应阶段转化为L-2,4-二羟基丁酸(DHB)，而不损失碳。
在生产L-苏氨酸的情况下，乙醇醛首先通过这四个连续反应阶段转化为2-酮基-4-羟基丁酸(OHB)，然后是将2-酮基-4-羟基丁酸经酶促转化为L-高丝氨酸的步骤，将L-高丝氨酸经酶促转化为O-磷酸-L-高丝氨酸(O-P-L-高丝氨酸)的步骤和将O-磷酸-L-高丝氨酸经酶促转化为L-苏氨酸的步骤。
两种代谢途径都与使用乙二醇、甲醇或D-木糖作为起始材料相容。产生乙醇醛的反应在图1中显示为虚线箭头。代谢途径的不同酶或酶活性在图1中用罗马数字表示。
乙醇醛可以通过多阶段代谢途径从木糖衍生，该多阶段代谢途径连续使用木糖异构酶(I)的酶活性以将D-木糖转化为D-木酮糖，使用木酮糖-1-激酶(II)以将D-木酮糖转化为D-木酮糖-1P以及使用木酮糖-1P-醛缩酶(III)以将D-木酮糖-1P转化为乙醇醛。
乙醇醛可以通过代谢途径从乙二醇衍生，该代谢途径使用PQQ依赖性乙二醇脱氢酶(膜结合)(IV)或NAD(P)依赖性乙二醇脱氢酶(胞质)(V)的酶活性来转化乙二醇。
乙醇醛可以通过代谢途径从甲醇衍生，该代谢途径连续使用甲醇脱氢酶(VI)的酶活性以将甲醇转化为甲醛，以及使用乙醇醛合酶(VII)以将甲醛转化为乙醇醛。
通过设计具有五个连续反应阶段的代谢途径，可以在大肠杆菌中从乙醇醛生产代谢产物DHB，这五个连续反应阶段由D-苏糖醛缩酶(VIII)、D-苏糖脱氢酶(IX)、D-苏糖酸-1,4-内酯酶(X)、D-苏糖酸脱水酶(XI)和OHB还原酶(XV)的酶活性催化。在第一阶段，两个乙醇醛(GA)分子结合形成分子D-苏糖。然后，所得的四碳糖被D-苏糖脱氢酶(IX)氧化为D-苏糖酸-1,4-内酯，其在D-苏糖酸-1,4-内酯酶(X)催化的反应中被转化为相应的糖酸或D-苏糖酸。在最后两个酶促步骤中，D-苏糖酸通过D-苏糖酸脱水酶(XI)脱水为OHB，其最终在OHB还原酶(XV)催化的反应中还原为DHB。
在L-苏氨酸的生产中，乙醇醛首先通过上述连续的四个反应阶段转化为2-酮基-4-羟基丁酸(OHB)，然后是使用L-高丝氨酸转氨酶(XII)将2-酮基-4-羟基丁酸经酶促转化为L-高丝氨酸的步骤，然后是通过ATP消耗并使用L-高丝氨酸激酶(XIII)将L-高丝氨酸经酶促转化为O-磷酸-L-高丝氨酸的步骤，以及使用L-苏氨酸合酶(XIV)将O-磷酸高丝氨酸经酶促转化为L-苏氨酸的步骤。
所提到的大多数酶活性都是已知的，可以将必要的基因(如果尚未包含在生产菌株中)以适当的方式引入生产菌株中，但其他基因必须通过筛选来鉴定。
D-苏糖醛缩酶和OHB还原酶活性已经在文献中描述过。特别地，根据出版物Szekrenyi,A.；Soler,A.；Garrabou,X.；Guérard-Hélaine,C.；Parella,T.；Joglar,J.；Lemaire,M.；Bujons,J.；Clapés,P.Engineering the Donor Selectivity of D-Fructose-6-Phosphate Aldolase for Biocatalytic Asymmetric Cross-AldolAdditions of Glycolaldehyde[工程化D-果糖-6-磷酸醛缩酶的供体选择性以用于乙醇醛的生物催化不对称交叉羟醛加成].Chemistry[化学]2014,20(39),12572-12583，在来自大肠杆菌的D-果糖-6-磷酸醛缩酶(Ec.FsaA)的情况下，已经显示了乙醇醛可逆酶促同羟醛加成到D-苏糖的体外催化。此外，已知突变变体Ec.FsaAL107Y:A129G(Ec.FsaATA)具有与野生型相比增加三个数量级的用于生产D-苏糖的活性。因此，这种突变酶可以有利地用于上述代谢途径。此外，通过在大肠杆菌的L-苹果酸脱氢酶中引入5点突变(Ec.Mdh I12V:R81A:M85Q:D86S:G179D)而获得的突变苹果酸脱氢酶Ec.Mdh5Q在出版物C.J.R.；Topham,C.M.；Malbert,Y.；/>J.M.；Walther,T.Rational Engineering of a MalateDehydrogenase for Microbial Production of2,4-Dihydroxybutyric Acid viaHomoserine Pathway[通过高丝氨酸途径微生物生产2,4-二羟基丁酸的苹果酸脱氢酶的合理工程化].Biochem.J[生物化学杂志].2018,475(23),3887–3901中描述为具有高度活性。因此，可以选择该酶作为OHB还原酶，以催化DHB合成途径的最后一个转化步骤。
通过文献中的参考，还可以确定如下酶，除其他外，该酶还具有D-苏糖酸-1,4-内酯酶活性。Westlake,A.Thermostable Enzymes Important For IndustrialBiotechnology[对工业生物技术重要的耐热酶].日期:2019年6月10日报道，来自Thermogutta terrifontis的葡糖酸内酯酶，此处缩写为Tt.Lac11，对大量内酯具有活性。这些酶也被描述为对D-苏糖酸-1,4-内酯具有活性，尽管仅报道了低反应速率。发明人最近分析了动力学特性，并对天然底物L-岩藻糖酸-1,4-内酯和D-苏糖酸-1,4-内酯都测定了令人惊讶的可比催化活性。由于该酶对D-苏糖酸-1,4-内酯具有高亲和力(Km＝2.92mM)，因此适用于根据本发明使用的代谢途径。
已知几种酶催化乙二醇NAD依赖性氧化为乙醇醛。这些包括来自大肠杆菌的1,2-丙二醇脱氢酶(Ec.FucO)，参见Boronat,A.；Caballero,E.；Aguilar,J.ExperimentalEvolution of a Metabolic Pathway for Ethylene Glycol Utilization byEscherichia Coli[大肠杆菌利用乙二醇代谢途径的实验进化].J.Bacteriol[细菌学杂志].1983,153(1),134–139，和来自氧化葡糖杆菌的醇脱氢酶GOX0313(Go.Adh)，参见Zhang,X.；Zhang,B.；Lin,J.；Wei,D.Oxidation of Ethylene Glycol to GlycolaldehydeUsing aHighly Selectivealcohol Dehydrogenase from Gluconobacter Oxydans[使用来自氧化葡糖杆菌的高选择性醇脱氢酶将乙二醇氧化为乙醇醛].J.Mol.Catalysis B[分子催化杂志B]2015,112,69–75。此外，已知通过Ec.FucO在位置Ile6Leu和Leu7Val的突变，可以实现所得酶(Ec.FucO I6L:L7V)的更高耐氧性，参见Lu,Z.；Cabiscol,E.；Obradors,N.；Tamarit,J.；Ros,J.；Aguilar,J.；Lin,E.C.Evolution of an Escherichia ColiProtein with Increased Resistance to Oxidative Stress[具有增加的对氧化应激的抗性的大肠杆菌蛋白的进化].J.Biol.Chem[生物化学杂志].1998,273(14),8308–8316。所得酶也被称为Ec.FucOOR，其中OR是耐氧性的缩写。
作为具有L-高丝氨酸转氨酶活性的酶，用于将2-酮基-4-羟基丁酸经酶促转化为L-高丝氨酸的步骤(XII)，已知来自大肠杆菌的天冬氨酸转氨酶(Ec.AspC)和突变变体Ec.AlaC A142P:Y275D的谷氨酸-丙酮酸转氨酶，参见Bouzon,M.；Perret,A.；Loreau,O.；Delmas,V.；Perchat,N.；Weissenbach,J.；Taran,F.；Marlière,P.ASyntheticAlternative to Canonical One-Carbon Metabolism[典型单碳代谢的合成替代方案].ACS Synth Biol[ACS合成生物学]2017,6(8),1520–1533。用于将L-高丝氨酸转化为O-磷酸-L-高丝氨酸的步骤(XIII)的具有L-高丝氨酸激酶活性的酶也是已知的，特别是来自大肠杆菌的高丝氨酸激酶(EcThrB)。来自大肠杆菌的苏氨酸合酶(Ec.ThrC)具有L-苏氨酸合酶活性，用于将O-磷酸-L-高丝氨酸经酶促转化为L-苏氨酸的步骤(XIV)。
示意性地表示上述代谢途径的酶活性并在图1中表示，具有D-苏糖脱氢酶活性(IX)和D-苏糖酸脱水酶活性(XI)的那些尚未被描述。然而，通过筛选候选酶的选择，可以鉴定出这些活性。
关于所使用的材料和方法，应注意以下：
所有化学品和溶剂均购自西格玛奥德里奇公司(Sigma-Aldrich)，除非说明其他公司。限制性核酸内切酶和DNA修饰酶是从新英格兰生物实验室(New England Biolabs(NEB))公司获得的，并按照制造商的说明书使用。DNA质粒分离通过NEB公司的质粒小型制备试剂盒进行。从琼脂糖凝胶中提取DNA和聚合酶链式反应(PCR)(一种体外扩增遗传物质(DNA)的方法)产物的纯化通过来自NEB公司的/>DNA凝胶提取试剂盒进行。DNA测序由Eurofins SAS公司(德国埃伯斯贝格(Ebersberg))公司进行。
表1中列出了构建并用于研究的所有质粒和宿主菌株。表2和表12中列出了引物。
表1：使用的菌株和质粒
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表2：所用引物的序列
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在表2中，引物序列中的限制性位点加下划线，编码起始/终止序列以粗体表示，RBS序列以斜体表示。通过分别使用引物对224/223、222/223和315/226对Ec.fucO野生型、Ec.fucOI6L:L7V和密码子优化的Go.adh基因进行PCR扩增来构建质粒pEXT20-Ec.fucO、pEXT20-Ec.fucOI6L:L7V和pEXT20-Go.adh。来自大肠杆菌MG1655的基因组DNA和合成基因作为衍生自Ec.fucO和Go.gox0313的基因的模板DNA。所有引物都引入了位于各自基因两侧的某些限制性位点。此外，引物紧邻编码序列之前引入了核糖体结合序列(RBS)。
大肠杆菌K-12底物MG1655ΔyqhDΔaldA被用作构建苏氨酸生产菌株的起始菌株。内源性thrBC和rhtB基因的表达通过用合成组成型和分离启动子proD Davis,J.H.；Rubin,A.J.；Sauer,R.T.:Design,construction and characterization of a set ofinsulated bacterial promoters[一组绝缘细菌启动子的设计、构建和表征].于:NucleicAcid Res[核酸研究].,2011,3,第1131-1141页取代每个操纵子或基因的天然染色体5'-UTR而制成组合型的。proD序列之前是氯霉素抗性盒(FRT-cat-FRT-PproD)，其元件首先用表2中列出的引物从质粒pTOPO-proD和pKD扩增。PCR产物用DpnI消化，使用与基因组靶基因座侧翼区具有约50bp同源性的引物通过融合PCR进行纯化和组装。将所得DNA片段转化到各自的靶菌株中，其表达来自pKD46质粒的λ-红色重组酶，以取代这些菌株中的天然基因启动子。在富含抗生素的LB琼脂平板上选择氯霉素抗性克隆，并通过PCR分析(引物参见表2)证实它们含有相应的插入片段大小。通过DNA测序检查整合的启动子序列的正确测序。通过表达来自pCP20质粒Cherepanov P.P.；Wackernagel,W.:Gene disruption in Escherichiacoli:TcR and KmR cassettes with the option of Flp-catalysed excision of theantibiotic-resistance determinant[大肠杆菌中的基因破坏：TcR和KmR盒，选择Flp催化切除抗生素抗性决定簇].于:Gene[基因],1995,158(1),第9-14页的FLP重组酶从基因组中去除cat盒，并使用基因座特异性引物通过PCR检查盒的正确切除(表2)。在标准方案的帮助下，将质粒转化到靶大肠杆菌菌株中。
使用引物对209/284扩增高拷贝质粒pEXT20。用Xhol/Xbal限制酶消化PCR产物，并使用T4 DNA连接酶(NEB公司)连接到载体骨架中。
通过使用引物对326/327扩增Ec.fsaA野生型和Ec.fsaAL107Y:A129G基因来构建质粒pEXT20-Ec.fsaA和pEXT20-Ec.fsaATA。大肠杆菌MG1655的基因组DNA和合成基因作为模板DNA。所得PCR产物和pEXT20表达载体用BamHI/XbaI消化并连接。通过分别使用引物对326/328和303/304对Ec-fsaAL107Y:A129G和密码子优化的合成Pc.tadH基因进行PCR扩增来构建质粒Ec.fsaATA-Pc.tadH。所得PCR产物分别用BamHI/SwaI和SwaI/Xbai限制酶消化，并连接到用BamHI/XbaI消化的pEXT20载体中。
类似的程序用于pACT3-Ec.fsaATA-Pc.tadH的构建，但其中中拷贝质粒pACT3用作骨架。通过使用引物对438/439扩增密码子优化的合成Tt.lac11基因构建质粒pACT3-Ec.fsaATA-Pc.tadH-Tt.lac11。然后用XbaI/SalI限制酶消化PCR产物和pACT3-Ec.fsaATA-Pc.tadH载体并连接。Tt.thte1497op基因的较短版本也用PCR扩增，其中N末端周质输出的信号序列(1:115-1,068nt；2:154-1.068nt)缺失。
为了构建质粒pACT3-Ec.fsaATA-Aa.tadH-Tt.lac11v1、pACT3-Ec.fsaATA-Xc.fdh-Tt.lac11v1和pACT3-Ec.fsaATA-Ppi.tadH-Tt.lac11v1，在引物对667/668、671/672或716/717的帮助下对基因Aa.tadH、Xc.fdh和Ppi.tadH进行PCR扩增。菌株燕麦食酸菌DSM7227、油菜黄单胞菌DSM3586的基因组DNA和Ppi.tadH的合成基因作为模板。将所得PCR产物和载体pACT3-Ec.fsaATA-Pc.tadH-Tt.lac11v1用SwaI/XbaI消化，然后进行连接。
为了构建质粒pACT3-Go.adh-Ec.fsaATA-Pc.tadH-Tt.lac11v1，在引物对313/314、326/328、303/304和600/439的帮助下扩增基因Go.adh、Ec.fsaATA、Pc.tadH和Tt.lac11v1。将所得PCR产物用KpnI/BamHI、BamhI/SwaI、SwaI/XbaI或XbaI/SalI消化，并连接到在KpnI/SalI中消化的pACT3载体中。
质粒pEXT22-Ec.mdh5Q通过使用引物对305/258对OBH还原酶编码基因Ec.mdh5Q(合成基因)进行PCR扩增来构建。然后用SacI/BamhI消化所得PCR产物和低拷贝载体pEXT22并连接。
质粒pEXT22-Ec.mdh7Q通过使用引物对305/258对编码OHB还原酶的基因Ec.mdh7Q进行PCR扩增而构建，该基因由如下所述的Ec.mdh5Q的突变产生。质粒pET28-Ec.mdh7Q作为模板DNA。然后用SacI/BamhI消化所得PCR产物和低拷贝载体pEXT22并连接。
质粒pEXT22-Ec.mdh5Q-Aa.araD、pEXT22-Ec.mdh5Q-Hh.araD和pEXT22-Ec.mdh7Q-Hh.araD分别通过使用引物对551/552和553/554扩增Aa.araD和Hh.araD基因而构建。燕麦食酸菌DSM7227和赫氏草螺菌DSM10281的基因组DNA作为各自的模板DNA。将所得PCR产物用BamHI/XbaI消化，并单独连接到用BamHI/XbaI消化的载体pEXT22-Ec.mdh5Q或pEXT22-Ec.mdh7Q中的相应位点。质粒pEXT21-Re.kdgT通过使用引物对454/455扩增来自钩虫贪铜菌H16 DSM428的基因组DNA的Re.kdgT基因而构建。将PCR产物和pEXT21载体骨架用BamHI/HindIII限制酶消化并连接。
为了构建质粒pEXT22-Ec.aspC-Hh.araD，首先用引物对805/806或553/554对基因Ec.aspC和Hh.araD进行PCR扩增。使用的引物列于表1中。大肠杆菌MG1655和赫氏草螺菌DSM10281的基因组DNA作为相应的模板。将所得PCR产物用SacI/BamHI或BamHI/XbaI消化，并单独连接到用SacI/XbaI消化的载体pEXT22中的相应位点。
质粒pEXT22-Ec.mdh5Q-Hh.araDC434S通过使用引物对718/719在模板pEXT22-Ec.mdh5Q-Hh.araD(在位置434产生从半胱氨酸到丝氨酸的突变)上进行反向PCR来构建。为了构建质粒pEXT22-Ec.mdh5Q-Ca.araD和pEXT22-Ec.mdh5Q-Pm.araD，分别使用引物对724/725和732/733对基因Ca.araD和Pm.araD进行PCR扩增。丙酮丁醇梭菌(Clostridiumacetobutylicum)DSM1731和含羞草副伯克霍尔德菌DSM21841的基因组DNA作为模板。将所得PCR产物和质粒pEXT22-Ec.mdh5Q-Hh.araD用BamHI/XbaI消化，纯化并连接。
将所有所得构建体转移到化学感受态大肠杆菌细胞(DH5α，NEB)中，并通过DNA测序验证靶基因的正确掺入。然后使用标准方法将质粒转化到选择为生产菌株的相应大肠杆菌菌株中，如从Sambrook,J.；Fritsch,E.F.；Maniatis,T.Molecular Cloning:ALaboratory Manual.[分子克隆：实验室手册]Mol.cloning a Lab.Manual[分子克隆实验室手册].1989,第2版中已知。
下面描述了酶促试验：
在酶促试验(测定)之前，通过Bradfort(-Quant,Roth)方法测定蛋白质浓度。除非另有说明，否则所有酶促试验均在37℃下在96孔微量滴定板中进行20分钟，总体积为250μL。最大反应速率(vmax)和米氏常数(Km)的值通过使至少五个不同底物浓度的动力学数据适应米氏方程来确定。适应通过/>R2015a中的非线性回归进行。
乙二醇脱氢酶活性的测定：
通过在乙二醇氧化期间在340nm(ε＝6.22mM-1cm-1)下测量NAD+的还原，在氧化方向上测定酶活性。用于活性测定的反应混合物含有100mM甘氨酸钠(pH9.5)、0.5mM NAD+和相应量的纯化酶或粗蛋白提取物。通过加入相应浓度的底物开始反应。乙二醇脱氢酶活性的单位U定义为每分钟催化1.0μmol NAD+转化的酶量。
D-苏糖醛缩酶活性的测定：
通过将乙醇醛的同羟醛加成与D-苏糖的NAD依赖性氧化偶联来测定酶活性，由来自石竹副伯克霍尔德菌(Pc.TadH)的纯化D-苏醛糖-1-脱氢酶催化。用于活性测定的反应混合物含有60mM 2-(4-(2-羟乙基)-1-哌嗪基)-乙磺酸(HEPES)pH 8，10mM NAD+，100μg mL-1辅助酶和适量的纯化酶或粗蛋白提取物。通过加入相应浓度的底物开始反应。D-苏糖醛缩酶活性的单位U定义为每分钟催化1.0μmol D-苏糖转化的酶量。
糖脱氢酶活性的测定：
通过在候选糖氧化期间在340nm下测量NAD(P)+的还原，在氧化方向上测定酶活性。用于活性测定的反应混合物含有50mM HEPES(pH 8)、10mM NAD(P)+和适量的纯化酶或粗蛋白提取物。通过添加各种浓度的(D)-阿拉伯糖或(D)-苏糖(英国卡博森斯公司(Carbosynth))开始反应。糖脱氢酶活性的单位U定义为每分钟催化1.0μmol糖转化的酶量。
内酯酶活性的测定：
酶活性通过使用比色pH指示剂溴百里酚蓝(ε＝1.14mM-1cm-1)在616nm下测量内酯水解期间从羧酸产物释放的质子的浓度来进行。用于活性测定的反应混合物含有2.5mMHEPES(pH 7.1)、200mM NaCl、1％(v/v)DMSO、0.1mM溴百里酚蓝和适量的纯化酶。通过加入不同量的(L)-岩藻糖酸-1,4-内酯或(D)-苏糖酸-1,4-内酯开始反应。内酯酶活性的单位U定义为每分钟催化1.0μmol内酯水解的酶量。
糖酸脱水酶活性的测定：
通过将2-酮酸反应产物转化为在250nm下测量浓度的缩氨基脲来测定酶活性。使用丙酮酸作为2-酮酸(ε＝2.24mM-1cm-1)获得校准曲线。用于活性测定的反应混合物含有60mM HEPES(pH 7.3)、50mM KCl、10mM MgCl2和适量的纯化酶或粗蛋白提取物。通过加入各种糖酸(L-岩藻酸、2R-二羟基戊酸、D-阿卓糖酸、D-酒石酸、D-阿拉伯糖酸或D-苏糖酸)开始1mL反应，并在37℃下孵育。在0、10、20和40分钟后在反应过程中除去200μl等分试样，并与100μl 2M HCl混合。然后向样品补充300μl氨基脲溶液(10g*L-1氨基脲盐酸盐和15g*L-1乙酸钠)，并在30℃下孵育10分钟。最后，向衍生化产物中加入500μL蒸馏水，其中立即使用石英比色皿测量吸收。糖酸脱水酶活性(U)的单位U定义为每分钟催化1.0μmol 2-酮酸形成的酶量。
D-苏糖酸脱水酶活性的测定：
酶活性通过将D-苏糖酸的脱水与纯化的OHB还原酶Ec.Mdh5Q对2-酮基-4-羟基丁酸(OHB)的NADH依赖性还原偶联来测定，其从出版物C.J.R.；Topham,C.M.；Malbert,Y.；/>J.M.；Walther,T.Rational Engineering of a Malate Dehydrogenase forMicrobial Production of 2,4-Dihydroxybutyric Acid via Homoserine Pathway[通过高丝氨酸途径微生物生产2,4-二羟基丁酸的苹果酸脱氢酶的合理工程化].Biochem.J[生物化学杂志].2018,475(23),3887–3901中已知。反应混合物含有60mM HEPES(pH 7.3)、50mM KCl、10mM MgCl2、0.25mM NADH、100μg mL-1辅助酶和适量的纯化酶。通过加入不同量的底物开始反应。D-苏糖酸脱水酶活性的单位U定义为每分钟催化1.0μmol OHB形成的酶量。
表3中列出了测试D-苏糖脱氢酶或D-苏糖酸脱水酶活性的候选酶。
OHB还原酶活性的测定：
通过在OHB还原为DHB期间在340nm下测量NAD(P)H的氧化，在还原方向上测定酶活性。用于活性测定的反应混合物含有60mM HEPES(pH 7)、5mM MgCl2、50mM KCl、0.25mMNADH或NADPH、2mM OHB和适量的纯化酶。通过加入OHB开始反应。OHB还原酶活性的单位U定义为每分钟催化1.0μmol NAD(P)H转化的酶量。为了确定辅助因子和OHB的Km值，适当改变一种底物的初始浓度，而另一种底物的初始浓度保持不变。
表3：测试D-苏糖脱氢酶或D-苏糖酸脱水酶活性的候选酶
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表3还说明是否进行密码子优化。
下面描述了候选酶的克隆、表达和纯化：
使用表4中列出的克隆技术和引物对，通过PCR扩增相应的编码基因并将其克隆到表达载体pET28a(Novagen公司)的相应位点中，其中N末端六His标签连接到靶序列上。将所得质粒转化到感受态大肠杆菌DH5α细胞(NEB)中。在将由此获得的质粒转化到表达菌株大肠杆菌BL21(DE3)(NEB)之前，通过分离质粒和DNA测序来验证正确的插入。根据其适用性，蛋白质在50mL LB培养基或自动诱导培养基中表达(Studier F.W./2005/Prot ExprPurif/41/207-234/Protein production by auto-induction in high density shakingcultures[在高密度振荡培养物中通过自动诱导产生蛋白质]))。在足够的孵育时间后，通过离心将细胞从培养基中分离，离心在1700×g和4℃下进行15分钟。将由此获得的细胞沉淀储存在-20℃下直到进一步处理。通过将细胞沉淀溶于1mL HEPES缓冲液(50mM，pH 7)中并随后以四个间隔(每个间隔20秒)进行超声处理(UDS 751，Topas GmbH，输出40％)来纯化酶。通过在13000×g和4℃下离心15分钟来分离细胞碎片，然后将澄清的上清液转移到新的反应容器中。根据钴树脂(talon)的制造商说明书，通过亲和层析从由此获得的粗蛋白质提取物中纯化靶蛋白质。然后就纯化的酶对天然底物(阳性对照)和靶底物的活性对其进行表征。蛋白质纯化从冷冻细胞球开始进行。
表4：用于将靶基因克隆到pET28表达载体中的引物序列、技术和限制性位点
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具有D-苏糖脱氢酶活性的酶的鉴定如下：
为了鉴定D-苏糖脱氢酶活性，来自石竹副伯克霍尔德菌(Pc.TadH)的酶D-苏醛糖-1-脱氢酶，来自酿酒酵母Sc.Ara1和Sc.Ara2的D-阿拉伯糖脱氢酶，来自Paracoccuslaeviglucosivorans(Pl.LgdA)的鲨肌醇-2-脱氢酶，来自假单胞菌属1143(Ps.Fdh)的D-苏醛糖-1-脱氢酶，来自硫化叶菌(Ss.Adh4)的D-阿拉伯糖脱氢酶和来自多食伯克霍尔德菌(Bm.BmulJ04919)(这里称为Bm.Fdh)的L-岩藻糖脱氢酶使用表4中列出的基因组DNA的引物或从合成基因开始从基因组DNA扩增(参见表3)。使用表4中指定的方法克隆到表达载体pET28a中。在表4中，限制性位点加下划线，密码子起始/终止序列以粗体表示。
图2以柱状图的形式示意性地显示了筛选候选酶的NAD(P)依赖性D-苏糖脱氢酶活性的结果。对于筛选，设定10mM辅助因子和糖的底物浓度。在没有活性的情况下，图2中的酶标有星号(*)。酶活性在柱状图中以对数标度表示。结果是至少两个独立生物学实验的平均值。误差条对应于与平均值的标准偏差。确切的值如表5所示。表5显示了适合作为D-苏糖脱氢酶的N-His标签酶的比活性，以U/mg纯化酶表示，用固定量(10mM)的底物D-阿拉伯糖或D-苏糖以及辅助因子NAD+或NADP+测量。缩写“n.d.”的意思是“未检测到”。
表5：候选酶对D-阿拉伯糖和D-苏糖的脱氢酶活性
在总共七种候选酶中，Sc.Ara1、Pc.TadH和Bm.Fdh与辅助因子NAD+或NADP+中的一种一起对D-苏糖显示出可测量的活性，这也从图2和表5中显而易见。由于Pc.TadH具有0.27U*mg-1的最高比活性，因此使用该酶进行进一步的动力学分析。测定底物D-苏糖的km值为26.63mM。由于Pc.TadH对D-苏糖具有最高的活性，并且在大肠杆菌中具有明确的表达，因此该酶优选用于构建合成的代谢途径。
如所示，在体外测试的帮助下，可以鉴定具有D-苏糖酸脱水酶活性的酶，其结果如下所述。虽然如前所述，对L-苏糖酸具有活性的脱水酶是充分已知的，但到目前为止，在相应的D-立体异构体上还没有报道这种酶活性。因此，类似于用于证明D-苏糖脱氢酶活性的策略，选择对具有(2S,3R)构型的糖酸具有已知活性的候选酶。候选酶的选择包括来自油菜黄单胞菌(Xc.FucD)和恶臭假单胞菌(Pp.FucD)的L-岩藻酸脱水酶、来自燕麦食酸菌(Aa.AraD)和赫氏草螺菌(Hh.AraD)的D-阿拉伯糖酸脱水酶、来自大豆慢生根瘤菌(Bj.TarD)的D-酒石酸脱水酶和来自大肠杆菌(Ec.UxaA)的D-阿卓糖酸脱水酶。此外，来自大肠杆菌(Ec.IlvD)和硫化叶菌(Ss.IlvD)的D-羟基酸脱水酶也被纳入研究。通过表3和表4中所述的引物和技术将相应的基因克隆到pET28a载体中。在根据上述方法表达和纯化酶后，如上所述测试它们对D-苏糖酸和其他糖酸的活性。
图3显示了筛选上述候选酶的D-苏糖酸脱水酶活性的结果的柱状图。使用上述识别2-酮酸的氨基脲测试，在D-苏糖酸和相应的天然底物上测试所有纯化的候选酶。将底物浓度调节至10mM，但Aa.AraD和Hh.AraD除外，其中使用1mM天然底物。在没有活性的情况下，酶标有星号(*)。酶活性在柱状图中以对数标度表示。活性的确切值如表6所示。表6显示了测试的N-His标签酶的比活性。结果表示为至少两个生物复制的平均值(+/-标准偏差)。缩写“n.d.”的意思是“未检测到”。
表6：各种脱水酶对D-苏糖酸及其天然底物的活性
在测试的酶中，Hh.Arad和Aa.AraD对D-苏糖酸显示出显著的活性，其中它们分别具有0.30U mg-1和0.18U mg-1的比活性，如表6和图3所示。
以下描述了在大肠杆菌中具有改进表达的来自T.terrifontis的苏糖酸-1,4-内酯酶Tt.Lac11的变体的构建。来自T.terrifontis的内酯酶Tt.Lac11只能在大肠杆菌中非常地困难表达，这导致用于动力学表征的纯化酶的产量低，并且可以预期该酶在生产菌株中的低活性。对内酯酶氨基酸序列的分析确定了N末端信号序列，该信号序列可能影响酶输出到周质中。为了提高Tt.Lac11的胞质表达，制备了该蛋白的N末端截短变体，并测试了其在大肠杆菌中的表达能力。可以表明，使用截短了38个氨基酸的酶的变体(Δ1-38)，可以实现表达增加34倍，如表7所示。该变体在下文中称为Tt.Lac11v1，而截短了51个氨基酸的变体称为Tt.Lac11v2，截短了76个氨基酸的变体称为Tt.Lac11v3。表7显示了多His标签的Tt.Lac11-内酯酶的截短变体对用pET28在大肠杆菌BL21(DE3)中表达的产量和活性。结果对应于两个独立生物重复的平均值和标准偏差。
表7：多His标签的Tt.Lac11-内酯酶的截短变体对用pET28在大肠杆菌BL21(DE3)中表达的产量和活性
通过在生产菌株中同时表达整个代谢途径，证明了DHB从乙醇醛的生物合成。为此，在所有实验中使用起始菌株大肠杆菌TW64(MG1655ΔyqhDΔaldA)，并用质粒转化，确保合成代谢途径的全部或部分表达。将细胞在250mL摇瓶中在补充有10％(v/v)LB培养基的矿物盐培养基上，在37℃和220rpm下在培养箱(Infors)中培养。在培养物达到约0.6的OD600值后加入IPTG(0.5mM)。当培养物的OD600值为约2.0时，将乙醇醛(20mM)添加到培养物中。孵育时间为48小时。结果表示为至少两个生物重复的平均值(+/-标准偏差)。DHB和代谢中间体以及乙醇醛的浓度在配备UV-Vis检测器(HP 1047A，Hewlett-Packard，美国)的HPLC系统(K-2600，Knaur)上测定。注射体积为20μL，在配备SecurityGuard柱(Phenomenex，美国)的Rezex RoA-有机酸H+柱上分离物质，使用0.5mM H2SO4作为流动相，流速为0.5mL/min。D-葡萄糖、D-苏糖、乙醇醛、D-苏糖酸和乙酸盐在35℃下测定，DHB和乙二醇在80℃下测定。由于D-苏糖酸内酯和D-苏糖酸不能用这种方法溶解，因此这些代谢物的浓度作为“D-苏糖酸/内酯”的合并值给出。
乙醇醛(GA)生物转化为DHB的实验结果如表8所示。
表8：20mM乙醇醛生物转化为DHB的结果
在菌株TW293中，表达了所有酶活性，这些酶活性根据所提出的代谢途径是将乙醇醛转化为DHB所必需的。令人惊讶的是，在培养48小时后可以检测到中间阶段D-苏糖和D-苏糖酸/内酯，但没有DHB(表8)。
由于内酯酶Tt-Lac11具有可能影响该酶转运到周质中的信号序列，因此怀疑内酯向苏糖酸的环裂解发生在周质中，因此需要重新导入所得的D-苏糖酸。为了验证这一假设，除了代谢途径的所有酶外，来自钩虫贪铜菌的D-苏糖酸导入渗透酶(Re.kdgT)也在菌株TW304中表达。事实上，该菌株可以从乙醇醛产生0.08mM的DHB。这提供了根据图1所提出的代谢途径的功能证明。
为了消除代谢中间体输出或输入需求，产生了三种截短形式的TtLac11，从中去除了不同长度的N末端序列部分(表7)。内酯酶变体Tt.Lac11v1(Δ1-38aa)在大肠杆菌中的表达提高了34倍，D-苏糖酸-1,4-内酯的比活性提高了10倍。因此选择该构建体是为了在该示例性实施例中在合成途径中提供所需的细胞质内酯酶活性。表达改进的内酯酶的菌株(TW354)能够积累0.16mM DHB。
在乙醇醛向DHB的全细胞生物转化的帮助下鉴定具有D-苏糖脱氢酶活性的酶
在前面的示例性实施例中，表明在所提出的代谢途径的帮助下，在全细胞生物转化中将乙醇醛转化为DHB是可能的。因此，用于所述代谢途径中的单个反应步骤的替代候选酶的表达并同时测量所得DHB浓度可以用于鉴定具有所需活性的额外的或更合适的酶。根据该策略，首先构建不表达D-苏糖脱氢酶的菌株，但以其他方式包含整个代谢途径，包括苏糖酸渗透酶。此外，还构建了TW 354、TW444、TW445和TW446菌株，这些菌株还表达了用于D-苏糖脱氢酶活性的各种候选酶。如上述示例性实施例培养菌株，并在孵育48小时后测量DHB和其他中间体的浓度。
如预期的那样，没有D-苏糖脱氢酶的大肠杆菌TW559菌株不能将乙醇醛合成的D-苏糖转化为DHB或代谢产物D-苏糖酸/内酯，如表9所示的结果所示。如果另外表达候选酶Pc.TadH、Xc.Fdh或Aa.TadH，则可以检测到DHB的产生，这表明这些酶具有D-苏糖脱氢酶活性。相比之下，表达Ppi.TadH时不能测量到DHB，这表明该酶没有D-苏糖脱氢酶活性，或不能在大肠杆菌中充分表达。
表9：10mM乙醇醛生物转化为DHB的结果，取决于用于D-苏糖脱氢酶活性的各种候选酶
(*)括号中显示的值对应于24小时后这些物质的浓度
在乙醇醛向DHB的全细胞生物转化的帮助下鉴定具有D-苏糖酸脱水酶活性的酶
类似于前面的实例，通过定量生产菌株中Hh.AraD被其他候选酶交换后乙醇醛上的DHB形成，可以鉴定D-苏糖酸脱水酶。鉴定改进的D-苏糖酸脱水酶的另一个标准是D-苏糖酸降解速率。根据该策略，在生产菌株TW452、TW453和TW454中分别表达突变体Hh.AraDC434S、Ca.AraD和酶Pm.AraD而不是先前使用的Hh.AraD，并研究它们对苏糖酸降解速率和DHB形成速率的影响。从表10中所示的结果可以看出，酶Pm.AraD不提供任何D-苏糖酸脱水酶活性或不能在大肠杆菌中充分表达，因为使用该酶时在生产菌株中不能检测到DHB产生。相比之下，突变体Hh.AraD C434S和Pm.AraD允许产生DHB，这证明了它们的D-苏糖酸脱水酶活性。此外，结果表明突变体Hh.AraD C434S更快地降解D-苏糖酸，从而导致DHB的产生增加。
表10：20mM乙醇醛生物转化为DHB的结果，取决于用于D-苏糖酸脱水酶活性的各种候选酶
合适的NAD依赖性乙二醇脱氢酶的鉴定
为了在所述代谢途径的帮助下实现乙二醇(EG)转化为DHB或苏氨酸，必须通过允许乙二醇氧化为乙醇醛的反应来扩展代谢途径。如前所述，已知有几种酶催化乙二醇向乙醇醛的NAD依赖性氧化。为了测试哪种酶最适合补充所述代谢途径，采用了生长依赖性测试系统，其中测试菌株的生长速率取决于乙二醇脱氢酶的体内活性。众所周知，大肠杆菌不能天然表达乙二醇脱氢酶，因此不能生长乙二醇作为底物上的唯一碳源。然而，乙二醇脱氢酶的表达允许乙二醇转化为乙醇醛，从而在该底物上生长。因此，在大肠杆菌菌株大肠杆菌ΔyqhD和大肠杆菌ΔyqhDΔaldA中借助pEXT20载体表达三种候选酶Go.Adh、Ec.FucO和Ec.FucO I6L:L7V。基于双突变体大肠杆菌ΔyqhDΔaldA的构建体作为对照，因为这些菌株由于乙醇醛脱氢酶AldA的缺失而不能在乙二醇上生长。将测试菌株在与上述实验具有相同组成的矿物盐培养基上孵育。在这种培养基中，只有葡萄糖作为唯一的碳源被100mM乙二醇替代。将测试菌株在微量滴定板(每孔250μL培养基)中以880rpm的振荡频率和37℃的温度在微量滴定板读数仪(Tecan)中孵育。生长速率通过定期测量OD600来确定。如图4所示，乙二醇脱氢酶Go.Adh使生长最快，其次是Ec.FucOI6L:L7V和Ec.FucO。因此，Go.Adh随后被用作乙二醇脱氢酶，用于在所述代谢途径的帮助下将乙二醇转化为DHB。
从乙二醇开始合成DHB的证明
通过在生产菌株中同时表达包括乙二醇脱氢酶的整个代谢途径来证明从乙二醇生物合成DHB。为此，所有实验均使用起始菌株大肠杆菌TW64(MG1655ΔyqhDΔaldA)，并用质粒转化，确保包括乙二醇脱氢酶Go.Adh的合成代谢途径的表达。以这种方式构建的生产菌株TW363的培养以及底物和产物浓度的测量如上述从乙醇醛合成DHB的示例性实施例所述进行。与上述实例相反，没有向培养物中添加乙醇醛，但在OD达到0.6后，以各种初始浓度向培养物添加乙二醇。这些实验的结果如表11所示。
表11：TW363菌株将乙二醇(EG)生物转化为DHB的结果
对NADPH比NADH具有更高特异性的OHB还原酶的构建
为了构建偏爱辅助因子NADPH的OHB还原酶，在已经描述的NADH依赖性OHB还原酶Ec.Mdh5Q中分别或同时在位置D34和I35进行氨基酸交换。模板质粒pET28-Ec.Mdh5Q的构建及其使用的方法在C.J.R.；Topham,C.M.；Malbert,Y.；/>J.M.；Walther,T.Rational Engineering of a Malate Dehydrogenase for Microbial Production of2,4-Dihydroxybutyric Acid via Homoserine Pathway[通过高丝氨酸途径微生物生产2,4-二羟基丁酸的苹果酸脱氢酶的合理工程化].Biochem.J[生物化学杂志].2018,475(23),3887–3901中描述。使用表12中列出的引物，使用相同的方法通过反向PCR引入额外的突变。PCR产物用DpnI消化以去除模板质粒并转化到大肠杆菌DH5α细胞中。分离所得质粒，并通过测序验证DNA序列的正确性。
表12：用于将突变引入模板酶Ec.Mdh5Q的所用引物
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如上所述，将由此获得的酶变体在大肠杆菌中表达、纯化并表征其动力学参数。如表13所示，可以表明突变D35G单独或与位置I35的突变组合使OHB还原酶的辅助因子特异性向NADPH方向移动。对NADPH具有最高活性和特异性的酶变体(Ec.Mdh5Q D35G:I35R)在下文中称为Ec-Mdh7Q。详细测定了该酶的动力学参数，并列于表14中。
表13：OHB还原酶突变体的辅助因子特异性
在恒定初始浓度的底物OHB(2mM)和NAD(P)H(0.25mM)下测定比活性。酶Ec.Mdh5Q是已经描述的NADH依赖性OHB还原酶Ec.Mdh I12V:R81A:M85Q:D86S:G179D。
已表明，Ec.Mdh7Q对NADPH的特异性比起始酶Ec.Mdh5Q高8600倍。
表14：Ec.Mdh5Q和Ec.Mdh7Q(Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35R)的OHB还原酶变体的动力学参数分析
(a)在恒定OHB浓度(2mM)和不同NAD(P)H浓度(2-0.002mM)下测定OHB还原酶活性。
(b)在恒定NAD(P)H浓度(0.25mM)和不同OHB浓度(10-0.05mM)下测定OHB还原酶活性。
动力学参数Km和Vmax通过在Matlab的帮助下将测量的初始反应速率适应Michaelis-Menten模型来确定。
通过使用NADPH依赖性OHB还原酶增加DHB产生
在下文中，研究了使用NADPH依赖性OHB还原酶提高DHB产量的适用性。为此，仅在NADH或NADPH依赖性OHB还原酶的表达上不同的菌株TW354和TW469在相同的条件下培养(起始底物10mM乙醇醛，实验和分析条件的进一步细节见上文)，并在48小时后比较DHB积累。可以表明，使用NADPH依赖性OHB还原酶显著提高DHB产生，如表15所示。
表15：表达NADH或NADPH依赖性OHB还原酶的大肠杆菌菌株将10mM乙醇醛生物转化为DHB的结果
n.d.-未检测到
从乙醇醛(GA)开始合成L-苏氨酸的证明
通过同时表达包括将OHB转化为苏氨酸的酶的整个代谢途径来实现由乙醇醛生物合成L-苏氨酸。为此，所有实验均使用起始菌株大肠杆菌TW64(MG1655ΔyqhDΔaldA)，并用质粒转化，确保包括高丝氨酸转氨酶Ec.AspC的合成代谢途径的表达。由此产生的菌株被命名为TW612。为了进一步提高苏氨酸产生，在该菌株中过表达苏氨酸输出渗透酶RhtB、高丝氨酸激酶Ec.ThrB和苏氨酸合酶Ec.ThrC。这通过用强组成型启动子proD Davis,J.H.；Rubin,A.J.；Sauer,R.T.:Design,construction and characterization of a set ofinsulated bacterial promoters[一组绝缘细菌启动子的设计、构建和表征].于:NucleicAcid Res[核酸研究].,2011,3,第1131-1141页替换染色体中相应基因的天然启动子来实现。由此获得的菌株被命名为TW613。
苏氨酸可以通过合成代谢途径和天然代谢途径在生产菌株中合成。为了明确证明苏氨酸通过本发明的合成代谢途径合成，一方面，使用仅表达合成代谢途径的不完整且因此无功能变体的菌株TW619进行对照实验。特别是，该菌株不含苏糖酸脱水酶表达的遗传信息。此外，在实验中，使用完全13C标记的乙醇醛(Omicron Biochemicals)作为底物，并将培养基中标记和未标记的苏氨酸的比例彼此比较。通过检测完全标记的苏氨酸，可以证明相应的碳衍生自乙醇醛。
茎培养在37℃下在旋转振荡器(Infors HT，德国)上以220rpm进行。将预培养物在50mL Falcon管中的5mL LB中孵育。约10小时后，使用500μL这些培养物接种第二预培养物(在50mL Falcon管中的10mL 90％v/v M9矿物培养基和10％v/vLB)，培养过夜。将生产起始OD600为0.2的主要培养物所需的生物量转移到90％(v/v)矿物M9培养基和10％(v/v)LB的培养基中。将抗生素以标准浓度添加到所有培养基中(氯霉素，35μg mL-1；卡那霉素，50μgmL-1；大观霉素，100μg mL-1)。当OD600达到约0.6时，加入IPTG(0.5mM)。在OD600达到2后，加入完全13C标记的乙醇醛。定期采集用于分析细胞外代谢物的样品。将1mL培养物样品离心(以13,000g离心5分钟)，并将上清液储存在-20℃直到进一步使用。测量前用0.2μmPTFE膜注射器过滤器过滤样品。
LC/MS分析：将无细胞上清液在10mM乙酸铵溶液(pH 9.2)中稀释100倍，该溶液溶解在60％(v/v)乙腈和40％(v/v)水中。LC-MS平台包括Xcalibur 2.1软件控制的Vanquish和Thermo ScientificTMQ ExactiveTMFocus(均来自加利福尼亚州圣何塞赛默飞世尔科技公司(ThermoFisher Scientific))。液相色谱分离使用 pHILIC(5μm聚合物150×2.1mm)柱进行，流速为0.15mL min-1。使用A(5％ ACN，10mM乙酸铵，通过NH4OH为pH9.2)和B(90％ ACN，10mM乙酸铵，通过NH4OH为pH 9.2)的梯度，以获得最佳分离效率。梯度为0min，95％ B；2min，95％ B；3min，89.4％ B；5min，89.4％ B；6min，83.8％ B；7min，83.8％ B；8min，78.2％ B；9min，78.2％ B；10min，55.9％ B；12min，55.9％ B；13min，27.9％B；16min，27.9％B；18min，0％B；23min，0％B；24min，95％B；30min，95％ B。取样器的温度保持在6℃，注射体积为5μL，烘箱温度保持在25℃。电喷雾电离的设备设置优化为0.15mL min-1的流速。进一步的参数设置如下：地幔气的流速32(设备专用单位)，辅助气体的流速8(设备专用单位)，吹扫气体的流速0(吹扫专用单位)，喷雾电压-3.5kV，毛细管温度250℃和辅助气体温度200℃。
这些实验的结果如图5所示，其中包括基于13C的代谢物质流分析结果的柱状图，其显示了通过合成代谢途径从乙醇醛(GA)生物合成L-苏氨酸。在培养基中仅发现未标记的苏氨酸(M+0)或完全标记的苏氨酸(M+4)。
对照菌株TW619仅产生少量苏氨酸。此外，在该菌株的培养基中没有检测到标记的苏氨酸，因此表明该菌株中的苏氨酸仅由葡萄糖制备。相反，在表达整个合成代谢途径的菌株TW612和TW613的培养基中发现了完全标记的苏氨酸。通过这些实验，可以毫无疑问地证明合成代谢途径适合生产苏氨酸。
表16分别列出了编码某些酶的基因的DNA序列的SEQ ID号和相应酶的氨基酸序列的SEQ ID号。
表16：酶序列
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表17列出了不同质粒的DNA序列的SEQ ID号。
表17：质粒的DNA序列
表18分别列出了编码不同OHB还原酶突变体的基因的DNA序列的SEQ ID号和相应OHB还原酶突变体的氨基酸序列的SEQ ID号。
表18：OHB还原酶突变体的序列
质粒pEXT22-Ec.mdh7Q-Hh.araD的DNA序列被分配为SEQ ID No.184。
序列表-独立文本
SEQ ID No.
独立文本
1–48: 质粒构建的引物序列
49–89: 用于将靶基因克隆到pET28表达载体中的引物序列
90–101: 用于将突变引入Ec.Mdh5Q酶的引物序列
104： Ec.FucO突变体的序列
105： Ec.FucO突变体
106： Go.Adh的密码子优化序列
110： Ec.FsaA突变体的序列
111： Ec.FsaA突变体
116： Pc.TadH的密码子优化序列
118： Pl.LgdA的密码子优化序列
120： Ps.Fdh的密码子优化序列
126： Ppi.TadH的密码子优化序列
128： Ss.Adh4的密码子优化序列
132： Tt.lac11的密码子优化序列
134,136,138： Tt.lac11截短变体的密码子优化序列
135： 截短了38个氨基酸的Tt.Lac11变体
137： 截短了51个氨基酸的Tt.Lac11变体
139： 截短了76个氨基酸的Tt.Lac11变体
142： Ss.IlvD的密码子优化序列
144： Xc.FucD的密码子优化序列
154： Hh.araD突变体的序列
155： 位置434处半胱氨酸突变为丝氨酸的Hh.araD突变体162,172,174,176,178,
180,182： Ec.Mdh突变体的序列
163： 具有突变I12V R81A M85Q D86S G179D的Ec.Mdh突变体168,169,170,171,184：质粒序列
173：具有突变I12V R81A M85Q D86S G179D D34G的Ec.Mdh突变体
175：具有突变I12V R81A M85Q D86S G179D I35S的Ec.Mdh突变体
177：具有突变I12V R81AM85Q D86S G179D D34G I35K的Ec.Mdh突变体
179：具有突变I12V R81AM85Q D86S G179D D34G I35R的Ec.Mdh突变体
181：具有突变I12V R81A M85Q D86S G179D D34G I35S的Ec.Mdh突变体
183：具有突变I12V R81A M85Q D86S G179D D34G I35T的Ec.Mdh突变体
使用的参考数字和缩写列表
I 木糖异构酶
II 木酮糖-1-激酶
III 木酮糖-1P-醛缩酶
IV 乙二醇脱氢酶(膜结合)
V 乙二醇脱氢酶(胞质)
VI 甲醇脱氢酶
VII 乙醇醛合酶
VIII D-苏糖醛缩酶
IX D-苏糖脱氢酶
X D-苏糖酸-1,4-内酯酶
XI D-苏糖酸脱水酶
XII L-高丝氨酸转氨酶
XIII L-高丝氨酸激酶
XIV L-苏氨酸合酶
XV 2-酮基-4-羟基丁酸(OHB)还原酶
ATCC 美国模式培养物保藏所
DH 脱氢酶
DHB 2,4-二羟基丁酸(2,4-dihydroxybutyrate，2,4-dihydroxybutyric acid)EG乙二醇
HEPES 2-(4-(2-羟乙基)-1-哌嗪基)-乙磺酸
HMTB D/L-2-羟基-4-(甲硫基)丁酸
IPTG异丙基-β-D-硫代吡喃半乳糖苷
LB溶原肉汤(LB)培养基，用于培养细菌的复合营养培养基NAD烟酰胺腺嘌呤二核苷酸
NADH质子化或还原形式的烟酰胺腺嘌呤二核苷酸
NADP烟酰胺腺嘌呤二核苷酸磷酸
NADPH质子化或还原形式的烟酰胺腺嘌呤二核苷酸磷酸
OD光密度
OD600 600nm波长下的光密度
OHB 2-酮基-4-羟基丁酸盐或酸2-酮基-4-羟基丁酸形式的2-酮基-4-羟基丁酸PCR 聚合酶链式反应
HiFi 高保真聚合酶
RBS 核糖体结合序列
RE 限制酶
UniProt.所有生物体和病毒蛋白质的生物信息学数据库(英语：universalproteindatabase)
序列表
<110> 德累斯顿工业大学（Technische Universität Dresden）
<120> 使用微生物代谢途径生产
2,4-二羟基丁酸或L-苏氨酸的方法
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<223> 质粒构建的引物序列
<400> 29
taagcaattt aaatgtttaa ctttaagaag gagatatacc atgaatcggc gcacagg 57
<210> 30
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 30
tgcttatcta gactaaacgg gctcgtgtgt 30
<210> 31
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 31
ggttctgcct atggcagcgc accggcaccg tcg 33
<210> 32
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 32
cgacggtgcc ggtgcgctgc cataggcaga acc 33
<210> 33
<211> 75
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 33
taagcaggat ccgtttaact ttaagaagga gatataccat gaaaaatgtt ataaagataa 60
atgaaaaaga taatg 75
<210> 34
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 34
tgcttatcta gattatagtg ttacaccgtt tttaaatata gatatttc 48
<210> 35
<211> 59
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 35
taagcaggat ccgtttaact ttaagaagga gatataccat gaagacctca acagcagac 59
<210> 36
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 36
tgcttatcta gatcaggtga tcgcgccgat c 31
<210> 37
<211> 70
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 37
tgtctttgct gatctgctac gtaccctctc atggaagtta ggagtctgac gtgtaggctg 60
gagctgcttc 70
<210> 38
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 38
gatagggacg acgtggtgtt agctgtgcat atgaatatcc tccttag 47
<210> 39
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 39
cacagctaac accacgtcgt 20
<210> 40
<211> 86
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 40
aacccgacgc tcatattggc actggaagcc ggggcataaa ctttaaccat ataatacctc 60
ctaaagttaa acaaaattat ttgtag 86
<210> 41
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 41
attgccgaag tggatggtaa 20
<210> 42
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 42
gtgactacat ctccgagcaa 20
<210> 43
<211> 70
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 43
tgcgacagta gcgtattgtg gcacaaaaat agacacaccg ggagttcatc gtgtaggctg 60
gagctgcttc 70
<210> 44
<211> 86
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 44
cttaaaatga tcgatgtcag caggtaggca aaccaccatt ctaaggtcat ataatacctc 60
ctaaagttaa acaaaattat ttgtag 86
<210> 45
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 45
catggtaaaa gcagcaaacg cgt 23
<210> 46
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 46
tatgaatcgc cagtccggtc tga 23
<210> 47
<211> 58
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 47
taagcagagc tcgtttaact ttaagaagga gatataccat gtttgagaac attaccgc 58
<210> 48
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 质粒构建的引物序列
<400> 48
tgcttaggat ccttacagca ctgccacaat c 31
<210> 49
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 49
agatatcata tggaactgta tctggatact tcagac 36
<210> 50
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 50
agatatgaat tcttaaatcg acgttctgcc aaacgc 36
<210> 51
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 51
agatatcata tgaaagtcgc agtcctcggc 30
<210> 52
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 52
agatatgaat tcttacttat taacgaactc ttcgcccag 39
<210> 53
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 53
ctggtgccgc gcggcagcca tatgcgcaaa ctgcttggc 39
<210> 54
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 54
gtcgacggag ctcgaattcg gatccttaga accccagtcc tttgg 45
<210> 55
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 55
actttaagaa ggagatatac catgcgcaaa ctgcttgg 38
<210> 56
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 56
ggtgctcgag tgcggccgca agcttgaacc ccagtccttt ggttttc 47
<210> 57
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 57
actttaagaa ggagatatac catggaaccg agtcagaatc c 41
<210> 58
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 58
actttaagaa ggagatatac catggaacgc gcagatc 37
<210> 59
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 59
actttaagaa ggagatatac catgtggagc gaaggtcc 38
<210> 60
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 60
cagccatatg tcttcttcag tagcctcaac cgaaaac 37
<210> 61
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 61
gaattcggat ccttaatact ttaaattgtc caagtttgg 39
<210> 62
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 62
cagccatatg gttaatgaaa aagtgaatcc attcgac 37
<210> 63
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 63
agctcgaatt cttatatcat ttctggatga ggaatacc 38
<210> 64
<211> 64
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 64
catcacagca gcggcctggt gccgcgcggc agccatatgt ctaccgatag tttacaacag 60
tttc 64
<210> 65
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 65
tgcggccgca agcttgtcga cggagctcga attcttatgc cggaaccggt gcac 54
<210> 66
<211> 58
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 66
catcacagca gcggcctggt gccgcgcggc agccatatga gtaatgccga aaaagcac 58
<210> 67
<211> 52
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 67
tgcggccgca agcttgtcga cggagctcga attcttaaaa attcaccggc tg 52
<210> 68
<211> 56
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 68
catcacagca gcggcctggt gccgcgcggc agccatatgt cttctactga acctgc 56
<210> 69
<211> 53
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 69
tgcggccgca agcttgtcga cggagctcga attcttacgg ggtcggaatt aag 53
<210> 70
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 70
catcacagca gcggcctggt gccgcgcggc agccatatgg agaacgtgaa tatggtg 57
<210> 71
<211> 52
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 71
tgcggccgca agcttgtcga cggagctcga attcttacgg ggtgataact tg 52
<210> 72
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 72
gcaggagcca tatggatctg aatctgcagg acaaggtcgt 40
<210> 73
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 73
gcaggagcaa gctttcagac gagcgcacga tcgagatgcg tat 43
<210> 74
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 74
agatatgcta gcatgcctaa gtaccgttcc gcc 33
<210> 75
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 75
agatatgaat tcttaacccc ccagtttcga tttatcg 37
<210> 76
<211> 53
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 76
catcacagca gcggcctggt gccgcgcggc agccatatgc cggcaaaatt aaa 53
<210> 77
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 77
tgcggccgca agcttgtcga cggagctcga attcttaagc gggacgg 47
<210> 78
<211> 53
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 78
catcacagca gcggcctggt gccgcgcggc agccatatgc gtaccattat cgc 53
<210> 79
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 79
tgcggccgca agcttgtcga cggagctcga attcttaggc tttggctt 48
<210> 80
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 80
ctggtgccgc gcggcagcca tatgaacagt gcccccgac 39
<210> 81
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 81
gtcgacggag ctcgaattcg gatcctcagg aacggttgat gtcg 44
<210> 82
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 82
ctggtgccgc gcggcagcca tatgtccgtc cgcatcgtc 39
<210> 83
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 83
gtcgacggag ctcgaattcg gatccttact ccgccagcgc ctt 43
<210> 84
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 84
ctggtgccgc gcggcagcca tatgtcgact gatgcactgg c 41
<210> 85
<211> 38
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 85
cggagctcga attcggatcc tcacatcacc gcgccgag 38
<210> 86
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 86
ctggtgccgc gcggcagcca tatgaaagcc aactctcccg 40
<210> 87
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 87
cggagctcga attcggatcc tcaggtgtag acgccgatg 39
<210> 88
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 88
ctggtgccgc gcggcagcca tatgcaatac atcaagatcc atgc 44
<210> 89
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 用于将靶基因克隆到
pET28表达载体中的引物序列
<400> 89
gtcgacggag ctcgaattcg gatccttata gcgttacgcc gcttttg 47
<210> 90
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 90
tcagaactct ctctgtatgg catcgctcca gtgactcccg g 41
<210> 91
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 91
ccgggagtca ctggagcgat gccatacaga gagagttctg a 41
<210> 92
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 92
gaactctctc tgtatgattc tgctccagtg actcccggtg 40
<210> 93
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 93
caccgggagt cactggagca gaatcataca gagagagttc 40
<210> 94
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 94
gaactctctc tgtatggctc tgctccagtg actcccggtg 40
<210> 95
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 95
caccgggagt cactggagca gagccataca gagagagttc 40
<210> 96
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 96
gaactctctc tgtatggcaa agctccagtg actcccggtg 40
<210> 97
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 97
caccgggagt cactggagct ttgccataca gagagagttc 40
<210> 98
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 98
gaactctctc tgtatggccg tgctccagtg actcccggtg 40
<210> 99
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 99
caccgggagt cactggagca cggccataca gagagagttc 40
<210> 100
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 100
gaactctctc tgtatggcac cgctccagtg actcccggtg 40
<210> 101
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 用于将突变引入Ec.Mdh5Q酶的引物序列
<400> 101
caccgggagt cactggagcg gtgccataca gagagagttc 40
<210> 102
<211> 1149
<212> DNA
<213> 大肠杆菌
<400> 102
atggctaaca gaatgattct gaacgaaacg gcatggtttg gtcggggtgc tgttggggct 60
ttaaccgatg aggtgaaacg ccgtggttat cagaaggcgc tgatcgtcac cgataaaacg 120
ctggtgcaat gcggcgtggt ggcgaaagtg accgataaga tggatgctgc agggctggca 180
tgggcgattt acgacggcgt agtgcccaac ccaacaatta ctgtcgtcaa agaagggctc 240
ggtgtattcc agaatagcgg cgcggattac ctgatcgcta ttggtggtgg ttctccacag 300
gatacttgta aagcgattgg cattatcagc aacaacccgg agtttgccga tgtgcgtagc 360
ctggaagggc tttccccgac caataaaccc agtgtaccga ttctggcaat tcctaccaca 420
gcaggtactg cggcagaagt gaccattaac tacgtgatca ctgacgaaga gaaacggcgc 480
aagtttgttt gcgttgatcc gcatgatatc ccgcaggtgg cgtttattga cgctgacatg 540
atggatggta tgcctccagc gctgaaagct gcgacgggtg tcgatgcgct cactcatgct 600
attgaggggt atattacccg tggcgcgtgg gcgctaaccg atgcactgca cattaaagcg 660
attgaaatca ttgctggggc gctgcgagga tcggttgctg gtgataagga tgccggagaa 720
gaaatggcgc tcgggcagta tgttgcgggt atgggcttct cgaatgttgg gttagggttg 780
gtgcatggta tggcgcatcc actgggcgcg ttttataaca ctccacacgg tgttgcgaac 840
gccatcctgt taccgcatgt catgcgttat aacgctgact ttaccggtga gaagtaccgc 900
gatatcgcgc gcgttatggg cgtgaaagtg gaaggtatga gcctggaaga ggcgcgtaat 960
gccgctgttg aagcggtgtt tgctctcaac cgtgatgtcg gtattccgcc acatttgcgt 1020
gatgttggtg tacgcaagga agacattccg gcactggcgc aggcggcact ggatgatgtt 1080
tgtaccggtg gcaacccgcg tgaagcaacg cttgaggata ttgtagagct ttaccatacc 1140
gcctggtaa 1149
<210> 103
<211> 382
<212> PRT
<213> 大肠杆菌
<400> 103
Met Ala Asn Arg Met Ile Leu Asn Glu Thr Ala Trp Phe Gly Arg Gly
1 5 10 15
Ala Val Gly Ala Leu Thr Asp Glu Val Lys Arg Arg Gly Tyr Gln Lys
20 25 30
Ala Leu Ile Val Thr Asp Lys Thr Leu Val Gln Cys Gly Val Val Ala
35 40 45
Lys Val Thr Asp Lys Met Asp Ala Ala Gly Leu Ala Trp Ala Ile Tyr
50 55 60
Asp Gly Val Val Pro Asn Pro Thr Ile Thr Val Val Lys Glu Gly Leu
65 70 75 80
Gly Val Phe Gln Asn Ser Gly Ala Asp Tyr Leu Ile Ala Ile Gly Gly
85 90 95
Gly Ser Pro Gln Asp Thr Cys Lys Ala Ile Gly Ile Ile Ser Asn Asn
100 105 110
Pro Glu Phe Ala Asp Val Arg Ser Leu Glu Gly Leu Ser Pro Thr Asn
115 120 125
Lys Pro Ser Val Pro Ile Leu Ala Ile Pro Thr Thr Ala Gly Thr Ala
130 135 140
Ala Glu Val Thr Ile Asn Tyr Val Ile Thr Asp Glu Glu Lys Arg Arg
145 150 155 160
Lys Phe Val Cys Val Asp Pro His Asp Ile Pro Gln Val Ala Phe Ile
165 170 175
Asp Ala Asp Met Met Asp Gly Met Pro Pro Ala Leu Lys Ala Ala Thr
180 185 190
Gly Val Asp Ala Leu Thr His Ala Ile Glu Gly Tyr Ile Thr Arg Gly
195 200 205
Ala Trp Ala Leu Thr Asp Ala Leu His Ile Lys Ala Ile Glu Ile Ile
210 215 220
Ala Gly Ala Leu Arg Gly Ser Val Ala Gly Asp Lys Asp Ala Gly Glu
225 230 235 240
Glu Met Ala Leu Gly Gln Tyr Val Ala Gly Met Gly Phe Ser Asn Val
245 250 255
Gly Leu Gly Leu Val His Gly Met Ala His Pro Leu Gly Ala Phe Tyr
260 265 270
Asn Thr Pro His Gly Val Ala Asn Ala Ile Leu Leu Pro His Val Met
275 280 285
Arg Tyr Asn Ala Asp Phe Thr Gly Glu Lys Tyr Arg Asp Ile Ala Arg
290 295 300
Val Met Gly Val Lys Val Glu Gly Met Ser Leu Glu Glu Ala Arg Asn
305 310 315 320
Ala Ala Val Glu Ala Val Phe Ala Leu Asn Arg Asp Val Gly Ile Pro
325 330 335
Pro His Leu Arg Asp Val Gly Val Arg Lys Glu Asp Ile Pro Ala Leu
340 345 350
Ala Gln Ala Ala Leu Asp Asp Val Cys Thr Gly Gly Asn Pro Arg Glu
355 360 365
Ala Thr Leu Glu Asp Ile Val Glu Leu Tyr His Thr Ala Trp
370 375 380
<210> 104
<211> 1149
<212> DNA
<213> 人工序列
<220>
<223> Ec.FucO突变体的序列
<400> 104
atggctaaca gaatgctggt gaacgaaacg gcatggtttg gtcggggtgc tgttggggct 60
ttaaccgatg aggtgaaacg ccgtggttat cagaaggcgc tgatcgtcac cgataaaacg 120
ctggtgcaat gcggcgtggt ggcgaaagtg accgataaga tggatgctgc agggctggca 180
tgggcgattt acgacggcgt agtgcccaac ccaacaatta ctgtcgtcaa agaagggctc 240
ggtgtattcc agaatagcgg cgcggattac ctgatcgcta ttggtggtgg ttctccacag 300
gatacttgta aagcgattgg cattatcagc aacaacccgg agtttgccga tgtgcgtagc 360
ctggaagggc tttccccgac caataaaccc agtgtaccga ttctggcaat tcctaccaca 420
gcaggtactg cggcagaagt gaccattaac tacgtgatca ctgacgaaga gaaacggcgc 480
aagtttgttt gcgttgatcc gcatgatatc ccgcaggtgg cgtttattga cgctgacatg 540
atggatggta tgcctccagc gctgaaagct gcgacgggtg tcgatgcgct cactcatgct 600
attgaggggt atattacccg tggcgcgtgg gcgctaaccg atgcactgca cattaaagcg 660
attgaaatca ttgctggggc gctgcgagga tcggttgctg gtgataagga tgccggagaa 720
gaaatggcgc tcgggcagta tgttgcgggt atgggcttct cgaatgttgg gttagggttg 780
gtgcatggta tggcgcatcc actgggcgcg ttttataaca ctccacacgg tgttgcgaac 840
gccatcctgt taccgcatgt catgcgttat aacgctgact ttaccggtga gaagtaccgc 900
gatatcgcgc gcgttatggg cgtgaaagtg gaaggtatga gcctggaaga ggcgcgtaat 960
gccgctgttg aagcggtgtt tgctctcaac cgtgatgtcg gtattccgcc acatttgcgt 1020
gatgttggtg tacgcaagga agacattccg gcactggcgc aggcggcact ggatgatgtt 1080
tgtaccggtg gcaacccgcg tgaagcaacg cttgaggata ttgtagagct ttaccatacc 1140
gcctggtaa 1149
<210> 105
<211> 382
<212> PRT
<213> 人工序列
<220>
<223> Ec.FucO突变体
<400> 105
Met Ala Asn Arg Met Leu Val Asn Glu Thr Ala Trp Phe Gly Arg Gly
1 5 10 15
Ala Val Gly Ala Leu Thr Asp Glu Val Lys Arg Arg Gly Tyr Gln Lys
20 25 30
Ala Leu Ile Val Thr Asp Lys Thr Leu Val Gln Cys Gly Val Val Ala
35 40 45
Lys Val Thr Asp Lys Met Asp Ala Ala Gly Leu Ala Trp Ala Ile Tyr
50 55 60
Asp Gly Val Val Pro Asn Pro Thr Ile Thr Val Val Lys Glu Gly Leu
65 70 75 80
Gly Val Phe Gln Asn Ser Gly Ala Asp Tyr Leu Ile Ala Ile Gly Gly
85 90 95
Gly Ser Pro Gln Asp Thr Cys Lys Ala Ile Gly Ile Ile Ser Asn Asn
100 105 110
Pro Glu Phe Ala Asp Val Arg Ser Leu Glu Gly Leu Ser Pro Thr Asn
115 120 125
Lys Pro Ser Val Pro Ile Leu Ala Ile Pro Thr Thr Ala Gly Thr Ala
130 135 140
Ala Glu Val Thr Ile Asn Tyr Val Ile Thr Asp Glu Glu Lys Arg Arg
145 150 155 160
Lys Phe Val Cys Val Asp Pro His Asp Ile Pro Gln Val Ala Phe Ile
165 170 175
Asp Ala Asp Met Met Asp Gly Met Pro Pro Ala Leu Lys Ala Ala Thr
180 185 190
Gly Val Asp Ala Leu Thr His Ala Ile Glu Gly Tyr Ile Thr Arg Gly
195 200 205
Ala Trp Ala Leu Thr Asp Ala Leu His Ile Lys Ala Ile Glu Ile Ile
210 215 220
Ala Gly Ala Leu Arg Gly Ser Val Ala Gly Asp Lys Asp Ala Gly Glu
225 230 235 240
Glu Met Ala Leu Gly Gln Tyr Val Ala Gly Met Gly Phe Ser Asn Val
245 250 255
Gly Leu Gly Leu Val His Gly Met Ala His Pro Leu Gly Ala Phe Tyr
260 265 270
Asn Thr Pro His Gly Val Ala Asn Ala Ile Leu Leu Pro His Val Met
275 280 285
Arg Tyr Asn Ala Asp Phe Thr Gly Glu Lys Tyr Arg Asp Ile Ala Arg
290 295 300
Val Met Gly Val Lys Val Glu Gly Met Ser Leu Glu Glu Ala Arg Asn
305 310 315 320
Ala Ala Val Glu Ala Val Phe Ala Leu Asn Arg Asp Val Gly Ile Pro
325 330 335
Pro His Leu Arg Asp Val Gly Val Arg Lys Glu Asp Ile Pro Ala Leu
340 345 350
Ala Gln Ala Ala Leu Asp Asp Val Cys Thr Gly Gly Asn Pro Arg Glu
355 360 365
Ala Thr Leu Glu Asp Ile Val Glu Leu Tyr His Thr Ala Trp
370 375 380
<210> 106
<211> 1032
<212> DNA
<213> 人工序列
<220>
<223> Go.Adh的密码子优化序列
<400> 106
atggctgata caatgctcgc cgccgtcgtc cgtgaattcg gcaagccgct ctccatcgag 60
cggctaccca tcccggacat caagccccac cagatcctcg tgaaggtcga tacctgtggc 120
gtctgccaca ctgacctgca cgccgcgcgc ggggactggc cgtccaagcc caacccgccg 180
ttcattcccg ggcatgaagg cgtcggacac atcgtcgccg tcggcagtca ggtcggcgat 240
ttcgtcaaga ccggcgatgt cgtgggcgtg ccctggctct actccgcctg cggtcactgc 300
gaacactgtc tgggcggctg ggaaacactc tgcgaaaagc aggacgacac cggctacacc 360
gtcaatggct gcttcgccga atatgtcgtg gcagacccga actacgtcgc acacctgccc 420
tcgaccatcg acccgcttca ggcctcgccg gtcctgtgcg cggggctgac ggtctataag 480
ggcctgaaaa tgacggaggc ccgccccggc cagtgggtcg cagtctcggg cgtcggcggt 540
ctcggccaga tggccgtgca gtacgccgtc gccatgggca tgaatgtcgt cgcggtggac 600
atcgatgacg aaaaactcgc cacagccaaa aagctcggcg catccctgac cgtcaacgcc 660
aaggacacgg acccggccag gttcatccag cagcagatcg gcggcgcaca tggcgctctc 720
gtcaccgctg tcggacggac ggcgttttcg caggccatgg gctatgcccg ccgcggcggc 780
accatcgtcc tgaacggact gccgcccggc gatttcccgg tctcgatctt cgacatggtc 840
atgaacggca ccaccatccg tggctccatc gtcggaacac ggctggacat gatcgaggcc 900
atggatttct tcgcccgcgg caaggtcaaa tccgtcgtca cccccggaaa acttgaaaac 960
atcaatacga tcttcgacga tctgcagaat ggtcgcctcg aaggccggac agtgctcgac 1020
ttccggtcct aa 1032
<210> 107
<211> 343
<212> PRT
<213> 氧化葡糖杆菌
<400> 107
Met Ala Asp Thr Met Leu Ala Ala Val Val Arg Glu Phe Gly Lys Pro
1 5 10 15
Leu Ser Ile Glu Arg Leu Pro Ile Pro Asp Ile Lys Pro His Gln Ile
20 25 30
Leu Val Lys Val Asp Thr Cys Gly Val Cys His Thr Asp Leu His Ala
35 40 45
Ala Arg Gly Asp Trp Pro Ser Lys Pro Asn Pro Pro Phe Ile Pro Gly
50 55 60
His Glu Gly Val Gly His Ile Val Ala Val Gly Ser Gln Val Gly Asp
65 70 75 80
Phe Val Lys Thr Gly Asp Val Val Gly Val Pro Trp Leu Tyr Ser Ala
85 90 95
Cys Gly His Cys Glu His Cys Leu Gly Gly Trp Glu Thr Leu Cys Glu
100 105 110
Lys Gln Asp Asp Thr Gly Tyr Thr Val Asn Gly Cys Phe Ala Glu Tyr
115 120 125
Val Val Ala Asp Pro Asn Tyr Val Ala His Leu Pro Ser Thr Ile Asp
130 135 140
Pro Leu Gln Ala Ser Pro Val Leu Cys Ala Gly Leu Thr Val Tyr Lys
145 150 155 160
Gly Leu Lys Met Thr Glu Ala Arg Pro Gly Gln Trp Val Ala Val Ser
165 170 175
Gly Val Gly Gly Leu Gly Gln Met Ala Val Gln Tyr Ala Val Ala Met
180 185 190
Gly Met Asn Val Val Ala Val Asp Ile Asp Asp Glu Lys Leu Ala Thr
195 200 205
Ala Lys Lys Leu Gly Ala Ser Leu Thr Val Asn Ala Lys Asp Thr Asp
210 215 220
Pro Ala Arg Phe Ile Gln Gln Gln Ile Gly Gly Ala His Gly Ala Leu
225 230 235 240
Val Thr Ala Val Gly Arg Thr Ala Phe Ser Gln Ala Met Gly Tyr Ala
245 250 255
Arg Arg Gly Gly Thr Ile Val Leu Asn Gly Leu Pro Pro Gly Asp Phe
260 265 270
Pro Val Ser Ile Phe Asp Met Val Met Asn Gly Thr Thr Ile Arg Gly
275 280 285
Ser Ile Val Gly Thr Arg Leu Asp Met Ile Glu Ala Met Asp Phe Phe
290 295 300
Ala Arg Gly Lys Val Lys Ser Val Val Thr Pro Gly Lys Leu Glu Asn
305 310 315 320
Ile Asn Thr Ile Phe Asp Asp Leu Gln Asn Gly Arg Leu Glu Gly Arg
325 330 335
Thr Val Leu Asp Phe Arg Ser
340
<210> 108
<211> 663
<212> DNA
<213> 大肠杆菌
<400> 108
atggaactgt atctggatac ttcagacgtt gttgcggtga aggcgctgtc acgtattttt 60
ccgctggcgg gtgtgaccac taacccaagc attatcgccg cgggtaaaaa accgctggat 120
gttgtgcttc cgcaacttca tgaagcgatg ggcggtcagg ggcgtctgtt tgcccaggta 180
atggctacca ctgccgaagg gatggttaat gacgcgctta agctgcgttc tattattgcg 240
gatatcgtgg tgaaagttcc ggtgaccgcc gaggggctgg cagctattaa gatgttaaaa 300
gcggaaggga ttccgacgct gggaaccgcg gtatatggcg cagcacaagg gctgctgtcg 360
gcgctggcag gtgcggaata tgttgcgcct tacgttaatc gtattgatgc tcagggcggt 420
agcggcattc agactgtgac cgacttacac cagttattga aaatgcatgc gccgcaggcg 480
aaagtgctgg cagcgagttt caaaaccccg cgtcaggcgc tggactgctt actggcagga 540
tgtgaatcaa ttactctgcc actggatgtg gcacaacaga tgattagcta tccggcggtt 600
gatgccgctg tggcgaagtt tgagcaggac tggcagggag cgtttggcag aacgtcgatt 660
taa 663
<210> 109
<211> 220
<212> PRT
<213> 大肠杆菌
<400> 109
Met Glu Leu Tyr Leu Asp Thr Ser Asp Val Val Ala Val Lys Ala Leu
1 5 10 15
Ser Arg Ile Phe Pro Leu Ala Gly Val Thr Thr Asn Pro Ser Ile Ile
20 25 30
Ala Ala Gly Lys Lys Pro Leu Asp Val Val Leu Pro Gln Leu His Glu
35 40 45
Ala Met Gly Gly Gln Gly Arg Leu Phe Ala Gln Val Met Ala Thr Thr
50 55 60
Ala Glu Gly Met Val Asn Asp Ala Leu Lys Leu Arg Ser Ile Ile Ala
65 70 75 80
Asp Ile Val Val Lys Val Pro Val Thr Ala Glu Gly Leu Ala Ala Ile
85 90 95
Lys Met Leu Lys Ala Glu Gly Ile Pro Thr Leu Gly Thr Ala Val Tyr
100 105 110
Gly Ala Ala Gln Gly Leu Leu Ser Ala Leu Ala Gly Ala Glu Tyr Val
115 120 125
Ala Pro Tyr Val Asn Arg Ile Asp Ala Gln Gly Gly Ser Gly Ile Gln
130 135 140
Thr Val Thr Asp Leu His Gln Leu Leu Lys Met His Ala Pro Gln Ala
145 150 155 160
Lys Val Leu Ala Ala Ser Phe Lys Thr Pro Arg Gln Ala Leu Asp Cys
165 170 175
Leu Leu Ala Gly Cys Glu Ser Ile Thr Leu Pro Leu Asp Val Ala Gln
180 185 190
Gln Met Ile Ser Tyr Pro Ala Val Asp Ala Ala Val Ala Lys Phe Glu
195 200 205
Gln Asp Trp Gln Gly Ala Phe Gly Arg Thr Ser Ile
210 215 220
<210> 110
<211> 663
<212> DNA
<213> 人工序列
<220>
<223> Ec.FsaA突变体的序列
<400> 110
atggaactgt atctggatac ttcagacgtt gttgcggtga aggcgctgtc acgtattttt 60
ccgctggcgg gtgtgaccac taacccaagc attatcgccg cgggtaaaaa accgctggat 120
gttgtgcttc cgcaacttca tgaagcgatg ggcggtcagg ggcgtctgtt tgcccaggta 180
atggctacca ctgccgaagg gatggttaat gacgcgctta agctgcgttc tattattgcg 240
gatatcgtgg tgaaagttcc ggtgaccgcc gaggggctgg cagctattaa gatgttaaaa 300
gcggaaggga ttccgacgta tggaaccgcg gtatatggcg cagcacaagg gctgctgtcg 360
gcgctggcag gtgcggaata tgttggccct tacgttaatc gtattgatgc tcagggcggt 420
agcggcattc agactgtgac cgacttacac cagttattga aaatgcatgc gccgcaggcg 480
aaagtgctgg cagcgagttt caaaaccccg cgtcaggcgc tggactgctt actggcagga 540
tgtgaatcaa ttactctgcc actggatgtg gcacaacaga tgattagcta tccggcggtt 600
gatgccgctg tggcgaagtt tgagcaggac tggcagggag cgtttggcag aacgtcgatt 660
taa 663
<210> 111
<211> 220
<212> PRT
<213> 人工序列
<220>
<223> Ec.FsaA突变体
<400> 111
Met Glu Leu Tyr Leu Asp Thr Ser Asp Val Val Ala Val Lys Ala Leu
1 5 10 15
Ser Arg Ile Phe Pro Leu Ala Gly Val Thr Thr Asn Pro Ser Ile Ile
20 25 30
Ala Ala Gly Lys Lys Pro Leu Asp Val Val Leu Pro Gln Leu His Glu
35 40 45
Ala Met Gly Gly Gln Gly Arg Leu Phe Ala Gln Val Met Ala Thr Thr
50 55 60
Ala Glu Gly Met Val Asn Asp Ala Leu Lys Leu Arg Ser Ile Ile Ala
65 70 75 80
Asp Ile Val Val Lys Val Pro Val Thr Ala Glu Gly Leu Ala Ala Ile
85 90 95
Lys Met Leu Lys Ala Glu Gly Ile Pro Thr Tyr Gly Thr Ala Val Tyr
100 105 110
Gly Ala Ala Gln Gly Leu Leu Ser Ala Leu Ala Gly Ala Glu Tyr Val
115 120 125
Gly Pro Tyr Val Asn Arg Ile Asp Ala Gln Gly Gly Ser Gly Ile Gln
130 135 140
Thr Val Thr Asp Leu His Gln Leu Leu Lys Met His Ala Pro Gln Ala
145 150 155 160
Lys Val Leu Ala Ala Ser Phe Lys Thr Pro Arg Gln Ala Leu Asp Cys
165 170 175
Leu Leu Ala Gly Cys Glu Ser Ile Thr Leu Pro Leu Asp Val Ala Gln
180 185 190
Gln Met Ile Ser Tyr Pro Ala Val Asp Ala Ala Val Ala Lys Phe Glu
195 200 205
Gln Asp Trp Gln Gly Ala Phe Gly Arg Thr Ser Ile
210 215 220
<210> 112
<211> 1035
<212> DNA
<213> 酿酒酵母
<400> 112
atgtcttctt cagtagcctc aaccgaaaac atagtcgaaa atatgttgca tccaaagact 60
acagaaatat acttttcact caacaatggt gttcgtatcc cagcactggg tttggggaca 120
gcaaatcctc acgaaaagtt agctgaaaca aaacaagccg taaaagctgc aatcaaagct 180
ggatacaggc acattgatac tgcttgggcc tacgagacag agccattcgt aggtgaagcc 240
atcaaggagt tattagaaga tggatctatc aaaagggagg atcttttcat aaccacaaaa 300
gtgtggccgg ttctatggga cgaagtggac agatcattga atgaatcttt gaaagcttta 360
ggcttggaat acgtcgactt gctcttgcaa cattggccgc tatgttttga aaagattaag 420
gaccctaagg ggatcagcgg actggtgaag actccggttg atgattctgg aaaaacaatg 480
tatgctgccg acggtgacta tttagaaact tacaagcaat tggaaaaaat ttaccttgat 540
cctaacgatc atcgtgtgag agccattggt gtctcaaatt tttccattga gtatttggaa 600
cgtctcatta aggaatgcag agttaagcca acggtgaacc aagtggaaac tcaccctcac 660
ttaccacaaa tggaactaag aaagttctgc tttatgcacg acattctgtt aacagcatac 720
tcaccattag gttcccatgg cgcaccaaac ttgaaaatcc cactagtgaa aaagcttgcc 780
gaaaagtaca atgtcacagg aaatgacttg ctaatttctt accatattag acaaggcact 840
atcgtaattc cgagatcctt gaatccagtt aggatttcct cgagtattga attcgcatct 900
ttgacaaagg atgaattaca agagttgaac gacttcggtg aaaaataccc agtgagattc 960
atcgatgagc catttgcagc catccttcca gagtttactg gtaacggacc aaacttggac 1020
aatttaaagt attaa 1035
<210> 113
<211> 344
<212> PRT
<213> 酿酒酵母
<400> 113
Met Ser Ser Ser Val Ala Ser Thr Glu Asn Ile Val Glu Asn Met Leu
1 5 10 15
His Pro Lys Thr Thr Glu Ile Tyr Phe Ser Leu Asn Asn Gly Val Arg
20 25 30
Ile Pro Ala Leu Gly Leu Gly Thr Ala Asn Pro His Glu Lys Leu Ala
35 40 45
Glu Thr Lys Gln Ala Val Lys Ala Ala Ile Lys Ala Gly Tyr Arg His
50 55 60
Ile Asp Thr Ala Trp Ala Tyr Glu Thr Glu Pro Phe Val Gly Glu Ala
65 70 75 80
Ile Lys Glu Leu Leu Glu Asp Gly Ser Ile Lys Arg Glu Asp Leu Phe
85 90 95
Ile Thr Thr Lys Val Trp Pro Val Leu Trp Asp Glu Val Asp Arg Ser
100 105 110
Leu Asn Glu Ser Leu Lys Ala Leu Gly Leu Glu Tyr Val Asp Leu Leu
115 120 125
Leu Gln His Trp Pro Leu Cys Phe Glu Lys Ile Lys Asp Pro Lys Gly
130 135 140
Ile Ser Gly Leu Val Lys Thr Pro Val Asp Asp Ser Gly Lys Thr Met
145 150 155 160
Tyr Ala Ala Asp Gly Asp Tyr Leu Glu Thr Tyr Lys Gln Leu Glu Lys
165 170 175
Ile Tyr Leu Asp Pro Asn Asp His Arg Val Arg Ala Ile Gly Val Ser
180 185 190
Asn Phe Ser Ile Glu Tyr Leu Glu Arg Leu Ile Lys Glu Cys Arg Val
195 200 205
Lys Pro Thr Val Asn Gln Val Glu Thr His Pro His Leu Pro Gln Met
210 215 220
Glu Leu Arg Lys Phe Cys Phe Met His Asp Ile Leu Leu Thr Ala Tyr
225 230 235 240
Ser Pro Leu Gly Ser His Gly Ala Pro Asn Leu Lys Ile Pro Leu Val
245 250 255
Lys Lys Leu Ala Glu Lys Tyr Asn Val Thr Gly Asn Asp Leu Leu Ile
260 265 270
Ser Tyr His Ile Arg Gln Gly Thr Ile Val Ile Pro Arg Ser Leu Asn
275 280 285
Pro Val Arg Ile Ser Ser Ser Ile Glu Phe Ala Ser Leu Thr Lys Asp
290 295 300
Glu Leu Gln Glu Leu Asn Asp Phe Gly Glu Lys Tyr Pro Val Arg Phe
305 310 315 320
Ile Asp Glu Pro Phe Ala Ala Ile Leu Pro Glu Phe Thr Gly Asn Gly
325 330 335
Pro Asn Leu Asp Asn Leu Lys Tyr
340
<210> 114
<211> 1008
<212> DNA
<213> 酿酒酵母
<400> 114
atggttaatg aaaaagtgaa tccattcgac ttagcttcgg tttctccttt agtcttagga 60
ggtgcaatat tgaaccagca atatacagat gagccagaat ccattccact ggaagacata 120
atcaaatacg cattttcgca tggtatcaat gcaattgaca cttctcccta ctatggccct 180
agtgaggttc tttatggtag ggcactgtcc aatttaagga atgaatttcc cagagacact 240
tattttatat gcacaaaggt tgggcgtatt ggtgcggaag agtttaacta ttctagagat 300
ttcgtgagat ttagtgttca cagatcgtgc gaaagattac acactacata tctcgatttg 360
gtgtatttgc acgatgtcga gtttgtcaaa tttccagaca tcttagaggc attaaaggaa 420
ttgaggacct tgaagaacaa aggagttatc aaaaattttg gcatatcagg ctacccaatc 480
gactttatca cctggcttgc tgagtactgc tctaccgagg aaagtgatat tgggtcattg 540
gatgcagtat tgtcctattg taacttgaac ttacaaaaca ataagctatt gaactttcga 600
gaaagactgc tacgcaatgc taaactgaaa atggtttgca acgcatcaat cttgagcatg 660
tcgctactaa ggtcacagga gacaagacag tttcacccgt gttctcatga attgagagaa 720
tgcgcatctc aggctgccaa atactgtcaa gaacaaaatg tcgacttagc cgatttggcc 780
acaagatacg cgatttcgga atgggtagga aagggacccg ttgttcttgg agtcagtagc 840
atggaagaat tgaaacttgc tctggataat tacgaaatag tgaaatcgaa tggcaacaga 900
ttatcttcta aggacggaca actagtagaa tatatccaaa agaatatttt taaagaacat 960
tttaatgaag agtggtcttc aggtattcct catccagaaa tgatataa 1008
<210> 115
<211> 335
<212> PRT
<213> 酿酒酵母
<400> 115
Met Val Asn Glu Lys Val Asn Pro Phe Asp Leu Ala Ser Val Ser Pro
1 5 10 15
Leu Val Leu Gly Gly Ala Ile Leu Asn Gln Gln Tyr Thr Asp Glu Pro
20 25 30
Glu Ser Ile Pro Leu Glu Asp Ile Ile Lys Tyr Ala Phe Ser His Gly
35 40 45
Ile Asn Ala Ile Asp Thr Ser Pro Tyr Tyr Gly Pro Ser Glu Val Leu
50 55 60
Tyr Gly Arg Ala Leu Ser Asn Leu Arg Asn Glu Phe Pro Arg Asp Thr
65 70 75 80
Tyr Phe Ile Cys Thr Lys Val Gly Arg Ile Gly Ala Glu Glu Phe Asn
85 90 95
Tyr Ser Arg Asp Phe Val Arg Phe Ser Val His Arg Ser Cys Glu Arg
100 105 110
Leu His Thr Thr Tyr Leu Asp Leu Val Tyr Leu His Asp Val Glu Phe
115 120 125
Val Lys Phe Pro Asp Ile Leu Glu Ala Leu Lys Glu Leu Arg Thr Leu
130 135 140
Lys Asn Lys Gly Val Ile Lys Asn Phe Gly Ile Ser Gly Tyr Pro Ile
145 150 155 160
Asp Phe Ile Thr Trp Leu Ala Glu Tyr Cys Ser Thr Glu Glu Ser Asp
165 170 175
Ile Gly Ser Leu Asp Ala Val Leu Ser Tyr Cys Asn Leu Asn Leu Gln
180 185 190
Asn Asn Lys Leu Leu Asn Phe Arg Glu Arg Leu Leu Arg Asn Ala Lys
195 200 205
Leu Lys Met Val Cys Asn Ala Ser Ile Leu Ser Met Ser Leu Leu Arg
210 215 220
Ser Gln Glu Thr Arg Gln Phe His Pro Cys Ser His Glu Leu Arg Glu
225 230 235 240
Cys Ala Ser Gln Ala Ala Lys Tyr Cys Gln Glu Gln Asn Val Asp Leu
245 250 255
Ala Asp Leu Ala Thr Arg Tyr Ala Ile Ser Glu Trp Val Gly Lys Gly
260 265 270
Pro Val Val Leu Gly Val Ser Ser Met Glu Glu Leu Lys Leu Ala Leu
275 280 285
Asp Asn Tyr Glu Ile Val Lys Ser Asn Gly Asn Arg Leu Ser Ser Lys
290 295 300
Asp Gly Gln Leu Val Glu Tyr Ile Gln Lys Asn Ile Phe Lys Glu His
305 310 315 320
Phe Asn Glu Glu Trp Ser Ser Gly Ile Pro His Pro Glu Met Ile
325 330 335
<210> 116
<211> 1005
<212> DNA
<213> 人工序列
<220>
<223> Pc.TadH的密码子优化序列
<400> 116
atgtctaccg atagtttaca acagtttcgt cgccgtgtgg caattccggg tccggctggt 60
tttggtgcag caccgctggg caatctgttt agccgtgtta ccgatgaggc cgcacaagaa 120
actttaagtg ccgcatgggg tgctggtatc cgctactttg ataccgcacc gttctatggc 180
atgggtctga gtgaacgccg tctgggtcgc gcactggcca gctatccgcg tgatgccttc 240
gtgattagca ccaaagtggg tcgtttactg atcgccgacg acagtgtgcc ggaaacccag 300
cacggttatg ttggtggttt accgtaccgc gttgaatacg actatactgc cgatggcgcc 360
cgccgtagca ttgaaagctc tttagaacgt ttaggtttag atcgtattga cattgtgtat 420
attcacgacg tggccgagga tacccatggt ccgctgtggc gtcagcatta tcgtaccgcc 480
gctggtggtg caatgaaagc tttaagccgt ttacgcgatg aaggtgtgat tggtggctgg 540
ggtttaggcg tgaatcgtgt tgaaccgtgt ctgatggctt tagccgatgc cgatccggac 600
gtctttttaa tcgccggccg ctatacttta ctggatacca cagctttaga tgctttaatt 660
ccggcatgcg aagcacgcgg tgcacgttta gttgttggtg gtccgtacaa tagtggttta 720
ctggctggtg gcgatacctt tgaatatgcc cgcgcagatg ccgcaatgtt agcccgtcgc 780
gcacgtttat taagccactg cgaacgcttt ggtgttgatt taaaggccgc cgctttacag 840
ttttgtaaag ccccgggcgt tgttgcatgc gttattgccg gtgcccgcaa tgcagatgag 900
gttcgtcaga actgcgcagc aatgacagca ccggtgccgc gcgaattttg gcaagcttta 960
aaggaagagg gcttagttcc gcaaggtgca ccggttccgg cataa 1005
<210> 117
<211> 334
<212> PRT
<213> 石竹副伯克霍尔德菌
<400> 117
Met Ser Thr Asp Ser Leu Gln Gln Phe Arg Arg Arg Val Ala Ile Pro
1 5 10 15
Gly Pro Ala Gly Phe Gly Ala Ala Pro Leu Gly Asn Leu Phe Ser Arg
20 25 30
Val Thr Asp Glu Ala Ala Gln Glu Thr Leu Ser Ala Ala Trp Gly Ala
35 40 45
Gly Ile Arg Tyr Phe Asp Thr Ala Pro Phe Tyr Gly Met Gly Leu Ser
50 55 60
Glu Arg Arg Leu Gly Arg Ala Leu Ala Ser Tyr Pro Arg Asp Ala Phe
65 70 75 80
Val Ile Ser Thr Lys Val Gly Arg Leu Leu Ile Ala Asp Asp Ser Val
85 90 95
Pro Glu Thr Gln His Gly Tyr Val Gly Gly Leu Pro Tyr Arg Val Glu
100 105 110
Tyr Asp Tyr Thr Ala Asp Gly Ala Arg Arg Ser Ile Glu Ser Ser Leu
115 120 125
Glu Arg Leu Gly Leu Asp Arg Ile Asp Ile Val Tyr Ile His Asp Val
130 135 140
Ala Glu Asp Thr His Gly Pro Leu Trp Arg Gln His Tyr Arg Thr Ala
145 150 155 160
Ala Gly Gly Ala Met Lys Ala Leu Ser Arg Leu Arg Asp Glu Gly Val
165 170 175
Ile Gly Gly Trp Gly Leu Gly Val Asn Arg Val Glu Pro Cys Leu Met
180 185 190
Ala Leu Ala Asp Ala Asp Pro Asp Val Phe Leu Ile Ala Gly Arg Tyr
195 200 205
Thr Leu Leu Asp Thr Thr Ala Leu Asp Ala Leu Ile Pro Ala Cys Glu
210 215 220
Ala Arg Gly Ala Arg Leu Val Val Gly Gly Pro Tyr Asn Ser Gly Leu
225 230 235 240
Leu Ala Gly Gly Asp Thr Phe Glu Tyr Ala Arg Ala Asp Ala Ala Met
245 250 255
Leu Ala Arg Arg Ala Arg Leu Leu Ser His Cys Glu Arg Phe Gly Val
260 265 270
Asp Leu Lys Ala Ala Ala Leu Gln Phe Cys Lys Ala Pro Gly Val Val
275 280 285
Ala Cys Val Ile Ala Gly Ala Arg Asn Ala Asp Glu Val Arg Gln Asn
290 295 300
Cys Ala Ala Met Thr Ala Pro Val Pro Arg Glu Phe Trp Gln Ala Leu
305 310 315 320
Lys Glu Glu Gly Leu Val Pro Gln Gly Ala Pro Val Pro Ala
325 330
<210> 118
<211> 1119
<212> DNA
<213> 人工序列
<220>
<223> Pl.LgdA的密码子优化序列
<400> 118
atgagtaatg ccgaaaaagc actgggcgtg gctttaatcg gtaccggctt tatgggcaaa 60
tgccatgcca tggcatggcg caatgtggcc accgcatttg gtggtttacc gccgcgttta 120
gaggtgctgg cagatatgcc ggcagataag gcacattctt tagcaagcag ctttggcttt 180
gcccgcggta ccgcagattg gcgtgaagcc gttaatgatc cggcagtgga cgtggtgagc 240
attaccaccc cgaacggctt acatcgcgaa atggccgaag cagcactggc agctggtaaa 300
catgtttggc tggagaagcc gatggcttta agtgtggaag atgcccaagc tatggaagca 360
gccgcccgtg caagcgatcg tcgcaccatc atcggctaca actatactcg cagcccggcc 420
tttcgcgccg ccgtggatct gattgcagaa ggcgcaattg gccgtccgat tcactttcgc 480
ggcatgtatg acgaggacta catggccgat ccggatctgc cgtggagctg gcgtctgaca 540
cgtaaagatg gtggtttagg tgcactgggt gatctgggtt gccatctggt tagcgtgatg 600
gtgagcttaa tgggtccggt tgcacgcgtg tatgcacaag ctgataccgt gattaccgat 660
cgccctcatc aaggtggcac agcacgcgtt gaaaatgagg atcaagctca agctttaatc 720
cgcttcgcaa gcggtaccag cggtgaattt agctgcagcc gtgtggcccg cggttatcgt 780
tgccgtctgg catgggaagt gcaaggtacc gaaggtactt tacgctttga tcaagaacgc 840
atgaacgaac tgtggctgta tcagccgggt cgcccggaaa tcgatggctt tcgccgcatc 900
ttaactggtc cggcccagcc gggctttgca gcattttgcc cgggcggtgg ccataacttt 960
ggctttaatg agcagaaagt ggttgaagcc gaaatgctgc gccaagctat tgctggtcgc 1020
ggtaaagcat ggccggactt caccgatggt ctgaccattg aacgcgtgat tcacggtatg 1080
gcaaccagtg cccaaactgg tcagccggtg aatttttaa 1119
<210> 119
<211> 372
<212> PRT
<213> Paracoccus laeviglucosivorans
<400> 119
Met Ser Asn Ala Glu Lys Ala Leu Gly Val Ala Leu Ile Gly Thr Gly
1 5 10 15
Phe Met Gly Lys Cys His Ala Met Ala Trp Arg Asn Val Ala Thr Ala
20 25 30
Phe Gly Gly Leu Pro Pro Arg Leu Glu Val Leu Ala Asp Met Pro Ala
35 40 45
Asp Lys Ala His Ser Leu Ala Ser Ser Phe Gly Phe Ala Arg Gly Thr
50 55 60
Ala Asp Trp Arg Glu Ala Val Asn Asp Pro Ala Val Asp Val Val Ser
65 70 75 80
Ile Thr Thr Pro Asn Gly Leu His Arg Glu Met Ala Glu Ala Ala Leu
85 90 95
Ala Ala Gly Lys His Val Trp Leu Glu Lys Pro Met Ala Leu Ser Val
100 105 110
Glu Asp Ala Gln Ala Met Glu Ala Ala Ala Arg Ala Ser Asp Arg Arg
115 120 125
Thr Ile Ile Gly Tyr Asn Tyr Thr Arg Ser Pro Ala Phe Arg Ala Ala
130 135 140
Val Asp Leu Ile Ala Glu Gly Ala Ile Gly Arg Pro Ile His Phe Arg
145 150 155 160
Gly Met Tyr Asp Glu Asp Tyr Met Ala Asp Pro Asp Leu Pro Trp Ser
165 170 175
Trp Arg Leu Thr Arg Lys Asp Gly Gly Leu Gly Ala Leu Gly Asp Leu
180 185 190
Gly Cys His Leu Val Ser Val Met Val Ser Leu Met Gly Pro Val Ala
195 200 205
Arg Val Tyr Ala Gln Ala Asp Thr Val Ile Thr Asp Arg Pro His Gln
210 215 220
Gly Gly Thr Ala Arg Val Glu Asn Glu Asp Gln Ala Gln Ala Leu Ile
225 230 235 240
Arg Phe Ala Ser Gly Thr Ser Gly Glu Phe Ser Cys Ser Arg Val Ala
245 250 255
Arg Gly Tyr Arg Cys Arg Leu Ala Trp Glu Val Gln Gly Thr Glu Gly
260 265 270
Thr Leu Arg Phe Asp Gln Glu Arg Met Asn Glu Leu Trp Leu Tyr Gln
275 280 285
Pro Gly Arg Pro Glu Ile Asp Gly Phe Arg Arg Ile Leu Thr Gly Pro
290 295 300
Ala Gln Pro Gly Phe Ala Ala Phe Cys Pro Gly Gly Gly His Asn Phe
305 310 315 320
Gly Phe Asn Glu Gln Lys Val Val Glu Ala Glu Met Leu Arg Gln Ala
325 330 335
Ile Ala Gly Arg Gly Lys Ala Trp Pro Asp Phe Thr Asp Gly Leu Thr
340 345 350
Ile Glu Arg Val Ile His Gly Met Ala Thr Ser Ala Gln Thr Gly Gln
355 360 365
Pro Val Asn Phe
370
<210> 120
<211> 990
<212> DNA
<213> 人工序列
<220>
<223> Ps.Fdh的密码子优化序列
<400> 120
atgtcttcta ctgaacctgc tgcagcagct gctggtctgg ccattccggc actgggttat 60
ggcgccgcaa acgtgggcaa tttatttcgt gcactgagcg acgatgaagc atgggccgtt 120
ctggaagcag cttgggatgc cggcattcgt tattatgaca ccgccccgca ttatggttta 180
ggtctgagcg aaaagcgtct gggtgcattt ttacagacca agccgcgcga cgaatttgtg 240
gttagcacca aagctggtcg tttattacgt cctaatccgg aacgccgtcc gagcggtctg 300
gataccgata acgactttca cgtgccggat gatctgcgcc gcgaatggga tttcaccgaa 360
caaggtattc gcgccagtat tgccgagagt caagaacgtc tgggtctgga tcgcattgat 420
ctgctgtatt tacatgatcc ggaacgtcat gatctggatc tggcactggc aagtgccttt 480
ccggctttag aaaaagtgcg cgccgaaggc gtggtgaaag ccattggcat tggcagcatg 540
gtgagtgatg cactgacccg cgccgttcgt gaagcagatt tagatttaat catggtggcc 600
ggtcgctata ctttattaga acagccggca gccaccgaag ttctgccggc ttgtgcagaa 660
aatgccaccg gcattgtggc cgccagtgtg tttaatagcg gtttactggc acagagtgag 720
ccgaaacgtg atggccgcta tgagtatggc cagctgccgg atgaactgtg ggatcgtctg 780
gtgcgcattg ccgccatttg tcgcaaccat gatgttccgc tgccggccgc agccatccag 840
tttccgctgc agagtgcttt agtgcgtagc gtggttgtgg gtggtagtcg cccggcacag 900
ctgacccaga atgcagagta tgccgcttta gaaattccgg ctggtttatg ggcagaactg 960
gcagaagccc gcttaattcc gaccccgtaa 990
<210> 121
<211> 329
<212> PRT
<213> 假单胞菌属1143
<400> 121
Met Ser Ser Thr Glu Pro Ala Ala Ala Ala Ala Gly Leu Ala Ile Pro
1 5 10 15
Ala Leu Gly Tyr Gly Ala Ala Asn Val Gly Asn Leu Phe Arg Ala Leu
20 25 30
Ser Asp Asp Glu Ala Trp Ala Val Leu Glu Ala Ala Trp Asp Ala Gly
35 40 45
Ile Arg Tyr Tyr Asp Thr Ala Pro His Tyr Gly Leu Gly Leu Ser Glu
50 55 60
Lys Arg Leu Gly Ala Phe Leu Gln Thr Lys Pro Arg Asp Glu Phe Val
65 70 75 80
Val Ser Thr Lys Ala Gly Arg Leu Leu Arg Pro Asn Pro Glu Arg Arg
85 90 95
Pro Ser Gly Leu Asp Thr Asp Asn Asp Phe His Val Pro Asp Asp Leu
100 105 110
Arg Arg Glu Trp Asp Phe Thr Glu Gln Gly Ile Arg Ala Ser Ile Ala
115 120 125
Glu Ser Gln Glu Arg Leu Gly Leu Asp Arg Ile Asp Leu Leu Tyr Leu
130 135 140
His Asp Pro Glu Arg His Asp Leu Asp Leu Ala Leu Ala Ser Ala Phe
145 150 155 160
Pro Ala Leu Glu Lys Val Arg Ala Glu Gly Val Val Lys Ala Ile Gly
165 170 175
Ile Gly Ser Met Val Ser Asp Ala Leu Thr Arg Ala Val Arg Glu Ala
180 185 190
Asp Leu Asp Leu Ile Met Val Ala Gly Arg Tyr Thr Leu Leu Glu Gln
195 200 205
Pro Ala Ala Thr Glu Val Leu Pro Ala Cys Ala Glu Asn Ala Thr Gly
210 215 220
Ile Val Ala Ala Ser Val Phe Asn Ser Gly Leu Leu Ala Gln Ser Glu
225 230 235 240
Pro Lys Arg Asp Gly Arg Tyr Glu Tyr Gly Gln Leu Pro Asp Glu Leu
245 250 255
Trp Asp Arg Leu Val Arg Ile Ala Ala Ile Cys Arg Asn His Asp Val
260 265 270
Pro Leu Pro Ala Ala Ala Ile Gln Phe Pro Leu Gln Ser Ala Leu Val
275 280 285
Arg Ser Val Val Val Gly Gly Ser Arg Pro Ala Gln Leu Thr Gln Asn
290 295 300
Ala Glu Tyr Ala Ala Leu Glu Ile Pro Ala Gly Leu Trp Ala Glu Leu
305 310 315 320
Ala Glu Ala Arg Leu Ile Pro Thr Pro
325
<210> 122
<211> 1149
<212> DNA
<213> 油菜黄单胞菌
<400> 122
atgaatacac gtcgccaatt cctgtctgct gccgcggccg gcaccacggc gctggccgca 60
tcgccattgc tggcacagtc gtccgcaccc ggaagcgtga tgcccacgcg cggccgcacg 120
gcggcctctg ccgtgcaaac tagcgcgccg gccaagggcg cacgttatcg gccgaccacg 180
cggttgggct ttggcggtgt tgcaatcggc aatgggtttg cgccagccac cgatgcgcaa 240
agcgagcaga cgctggcggc ggtgtgggcc tcgggcgtgc gctatttcga tacctcgccg 300
tggtacgggc tgggcctgtc cgaacgccgc accggccatc atctgcataa ccatgccgcc 360
gatagctacg tgctctcgac caaggtgggg cgcttgctga ccgcgaccga caagccgccg 420
aagaccatgt ggcagcaacc gtccccgttc gattaccgct acgactacag cgccgccggc 480
gtgcgccgct cgatcgaaga cagcctgcaa cggctgggcg tgtcgcagat cgacatcgtg 540
tacatccacg acctctcgcc ggagaacgaa aaagacctcg gcatgccctg ggaacagcgc 600
tttgccgagg ccgccaaggg cgcgatgccg gagctgacca agatgcgcaa ggagggcctg 660
atcaaggcct ggggctttgg cgtcaatcgc ccggaaccgg ccttgcgtgc gattgaagaa 720
gccgacccgg atatcttctt gctggcctgc cagtactcgt tgctcgacca cgcgcaggcc 780
ttgcatgaca ccttcccgaa gatcgccaga cacggcgcct cggtggtggt gggcgcgccg 840
ctgctggccg gttatctggc cgggcgcgac cgttatctgt acgagggcac ggtgccggag 900
tgggcgccgg gcaaacgcca gaaggccctg gcaatctgcg acaaacacgg cgtggatctg 960
cgtacggtgt cgctgcaatt tgctgccgca ccggaggtgg tgtcggcggt gattcccggc 1020
gcacgcacgg ccgaacaggc gcaggccaat gcggcctcga tgcgggtggc gattcctgcc 1080
gcgctctggg acgacctcaa gcgcgcgcag gtgatcgaag cggatgcgcc ggtgccggcg 1140
gctggatga 1149
<210> 123
<211> 382
<212> PRT
<213> 油菜黄单胞菌
<400> 123
Met Asn Thr Arg Arg Gln Phe Leu Ser Ala Ala Ala Ala Gly Thr Thr
1 5 10 15
Ala Leu Ala Ala Ser Pro Leu Leu Ala Gln Ser Ser Ala Pro Gly Ser
20 25 30
Val Met Pro Thr Arg Gly Arg Thr Ala Ala Ser Ala Val Gln Thr Ser
35 40 45
Ala Pro Ala Lys Gly Ala Arg Tyr Arg Pro Thr Thr Arg Leu Gly Phe
50 55 60
Gly Gly Val Ala Ile Gly Asn Gly Phe Ala Pro Ala Thr Asp Ala Gln
65 70 75 80
Ser Glu Gln Thr Leu Ala Ala Val Trp Ala Ser Gly Val Arg Tyr Phe
85 90 95
Asp Thr Ser Pro Trp Tyr Gly Leu Gly Leu Ser Glu Arg Arg Thr Gly
100 105 110
His His Leu His Asn His Ala Ala Asp Ser Tyr Val Leu Ser Thr Lys
115 120 125
Val Gly Arg Leu Leu Thr Ala Thr Asp Lys Pro Pro Lys Thr Met Trp
130 135 140
Gln Gln Pro Ser Pro Phe Asp Tyr Arg Tyr Asp Tyr Ser Ala Ala Gly
145 150 155 160
Val Arg Arg Ser Ile Glu Asp Ser Leu Gln Arg Leu Gly Val Ser Gln
165 170 175
Ile Asp Ile Val Tyr Ile His Asp Leu Ser Pro Glu Asn Glu Lys Asp
180 185 190
Leu Gly Met Pro Trp Glu Gln Arg Phe Ala Glu Ala Ala Lys Gly Ala
195 200 205
Met Pro Glu Leu Thr Lys Met Arg Lys Glu Gly Leu Ile Lys Ala Trp
210 215 220
Gly Phe Gly Val Asn Arg Pro Glu Pro Ala Leu Arg Ala Ile Glu Glu
225 230 235 240
Ala Asp Pro Asp Ile Phe Leu Leu Ala Cys Gln Tyr Ser Leu Leu Asp
245 250 255
His Ala Gln Ala Leu His Asp Thr Phe Pro Lys Ile Ala Arg His Gly
260 265 270
Ala Ser Val Val Val Gly Ala Pro Leu Leu Ala Gly Tyr Leu Ala Gly
275 280 285
Arg Asp Arg Tyr Leu Tyr Glu Gly Thr Val Pro Glu Trp Ala Pro Gly
290 295 300
Lys Arg Gln Lys Ala Leu Ala Ile Cys Asp Lys His Gly Val Asp Leu
305 310 315 320
Arg Thr Val Ser Leu Gln Phe Ala Ala Ala Pro Glu Val Val Ser Ala
325 330 335
Val Ile Pro Gly Ala Arg Thr Ala Glu Gln Ala Gln Ala Asn Ala Ala
340 345 350
Ser Met Arg Val Ala Ile Pro Ala Ala Leu Trp Asp Asp Leu Lys Arg
355 360 365
Ala Gln Val Ile Glu Ala Asp Ala Pro Val Pro Ala Ala Gly
370 375 380
<210> 124
<211> 1014
<212> DNA
<213> 燕麦食酸菌
<400> 124
atgaaggtca cagaaacacg ccgcatcggc gaaggccccg cacggctcac gcgcttcggc 60
gtgggttgcg gcacgctggc cgggctgtac aagcccacgt ccgaggcaga cgcccgggcc 120
acgctgcagg cggcatggga tgccggcctg cgctacttcg acaccgcccc tttctacggg 180
cacacgctga gcgagcaccg cgtgggcgcc ttcctgcgcg agcagccccg cgatgccttc 240
gtcatcagca ccaaggtcgg ccggctgatg cgccccgatt cctccgtgca gccgatggac 300
aacggctggg ccgccccgct gccgttccgc ccggactacg actacagcta cggcggcgtc 360
atgcgctcgt tcgaggacag cctgcagcgg ctcggcctgc agcgcatcga cctgctgttc 420
gtgcacgaca tcggcaccta cacccatggc gaccggaacg ccgggcactg gaaggccctc 480
acggcacaag gcggcttccg cgcgctggaa gagctccggg ccgatggccg catcgccggc 540
ttcggcctgg gcgtcaacga agcccaagtc atcctcgaca gcctgcagga gacgcggctg 600
gactgctccc tgctcgccgg gcgctacacg ctgctggagc agcatagcct gccgctcatg 660
aacacatgcc agcggctcgg cagtgccatc gtcatcggcg gtccgttcaa ctccgggctg 720
ctggtgggca acggcaagtt cgactatgcg gatgcgccgg cggacatgct ggcgcgtgcc 780
caggcgctgg acgaggcgtg cgccgcgcac ggcgtggccc tgccagcggc cgcgctgcaa 840
tttccgatgg cccatggcgc cgtggcatcg tgcgtctccg gcgcgcgcaa cccggcccag 900
ctggaccaga ccgtgggctg gttcgagcag gacattcccg cagcgctctg ggccgacctg 960
cagcagcaag gcctgatcgc ggaaggctgc cccgttcccg gagccgcgcc atga 1014
<210> 125
<211> 337
<212> PRT
<213> 燕麦食酸菌
<400> 125
Met Lys Val Thr Glu Thr Arg Arg Ile Gly Glu Gly Pro Ala Arg Leu
1 5 10 15
Thr Arg Phe Gly Val Gly Cys Gly Thr Leu Ala Gly Leu Tyr Lys Pro
20 25 30
Thr Ser Glu Ala Asp Ala Arg Ala Thr Leu Gln Ala Ala Trp Asp Ala
35 40 45
Gly Leu Arg Tyr Phe Asp Thr Ala Pro Phe Tyr Gly His Thr Leu Ser
50 55 60
Glu His Arg Val Gly Ala Phe Leu Arg Glu Gln Pro Arg Asp Ala Phe
65 70 75 80
Val Ile Ser Thr Lys Val Gly Arg Leu Met Arg Pro Asp Ser Ser Val
85 90 95
Gln Pro Met Asp Asn Gly Trp Ala Ala Pro Leu Pro Phe Arg Pro Asp
100 105 110
Tyr Asp Tyr Ser Tyr Gly Gly Val Met Arg Ser Phe Glu Asp Ser Leu
115 120 125
Gln Arg Leu Gly Leu Gln Arg Ile Asp Leu Leu Phe Val His Asp Ile
130 135 140
Gly Thr Tyr Thr His Gly Asp Arg Asn Ala Gly His Trp Lys Ala Leu
145 150 155 160
Thr Ala Gln Gly Gly Phe Arg Ala Leu Glu Glu Leu Arg Ala Asp Gly
165 170 175
Arg Ile Ala Gly Phe Gly Leu Gly Val Asn Glu Ala Gln Val Ile Leu
180 185 190
Asp Ser Leu Gln Glu Thr Arg Leu Asp Cys Ser Leu Leu Ala Gly Arg
195 200 205
Tyr Thr Leu Leu Glu Gln His Ser Leu Pro Leu Met Asn Thr Cys Gln
210 215 220
Arg Leu Gly Ser Ala Ile Val Ile Gly Gly Pro Phe Asn Ser Gly Leu
225 230 235 240
Leu Val Gly Asn Gly Lys Phe Asp Tyr Ala Asp Ala Pro Ala Asp Met
245 250 255
Leu Ala Arg Ala Gln Ala Leu Asp Glu Ala Cys Ala Ala His Gly Val
260 265 270
Ala Leu Pro Ala Ala Ala Leu Gln Phe Pro Met Ala His Gly Ala Val
275 280 285
Ala Ser Cys Val Ser Gly Ala Arg Asn Pro Ala Gln Leu Asp Gln Thr
290 295 300
Val Gly Trp Phe Glu Gln Asp Ile Pro Ala Ala Leu Trp Ala Asp Leu
305 310 315 320
Gln Gln Gln Gly Leu Ile Ala Glu Gly Cys Pro Val Pro Gly Ala Ala
325 330 335
Pro
<210> 126
<211> 1086
<212> DNA
<213> 人工序列
<220>
<223> Ppi.TadH的密码子优化序列
<400> 126
atgaatcggc gcacaggcgc ggcgagcgag acctatatca acgttgcgag gcaatccgtg 60
aacaacaggc aagccctgat tccgccggga ccgttgggtt tcggcgcggc ccccttgggc 120
aacctctttg cgcgcgtcag cgaagaagcg gctgaagaga cattgagcgc ggcatgggaa 180
gccggcatgc gctatttcga taccgcgccg ttctacggca tgggtctgtc cgaacagcgc 240
ctcggccgcg cgctcgcgca ccgtgcgcgc gacgaattcg tcgtctcgac gaaagtgggc 300
cgcctgctga ttcccgatgc gtccgttccc gaagtccagc acggctatgt cggcgcgctg 360
ccgtttcgcg tcgaattcga ttattcggcg caaggcgtgc tccgctccat cgacgagagt 420
ctcaagcgtc tttcactcga tcgcatcgat atcgtctata tccacgacgt cgccgaagat 480
gcgcatcgtg aggactggct gcggcaatac cgtatcgccg tcgaaggcgc gatgcgggcg 540
ctgagcgatc tgcgcgatca gggcgtgatc ggcgcatggg ggctcggcgt caaccgggtc 600
gagccgtgca tgatgacgtt gaacgacgcg gaccctgacg tgtttctgat cgccggaaga 660
tacacgctgc tcgacacgtc ggcgctcgat gcgctgatcc cggcttgcga tgctcgcggc 720
gtgcagttgg tcgcgggcgg tccgttcaac tcggggctgc tcgtgggcgg cacgacgttc 780
gaatatgccg aggcgccggc ggaaatgctc gagaagcggc gcagacttct cgcgcactgc 840
gagcgcttcg gcgtggacct gaaggcggcc gcgctgcagt tcacggatgc gccgcgagcg 900
gtcgcgtgcg tgatcgcggg cgcgagaaac gcgagcgaga tcagacagaa ctgcgcggca 960
atgaccgagc cgattccggc ggcattctgg gcggcattga aggccgatgg cctgattccc 1020
gccgatgcgc cggagcccct tgcttcaggc ggcgcgctcc acgcgacgac acacgagccc 1080
gtttag 1086
<210> 127
<211> 361
<212> PRT
<213> Paraburkholderia piptadeniae
<400> 127
Met Asn Arg Arg Thr Gly Ala Ala Ser Glu Thr Tyr Ile Asn Val Ala
1 5 10 15
Arg Gln Ser Val Asn Asn Arg Gln Ala Leu Ile Pro Pro Gly Pro Leu
20 25 30
Gly Phe Gly Ala Ala Pro Leu Gly Asn Leu Phe Ala Arg Val Ser Glu
35 40 45
Glu Ala Ala Glu Glu Thr Leu Ser Ala Ala Trp Glu Ala Gly Met Arg
50 55 60
Tyr Phe Asp Thr Ala Pro Phe Tyr Gly Met Gly Leu Ser Glu Gln Arg
65 70 75 80
Leu Gly Arg Ala Leu Ala His Arg Ala Arg Asp Glu Phe Val Val Ser
85 90 95
Thr Lys Val Gly Arg Leu Leu Ile Pro Asp Ala Ser Val Pro Glu Val
100 105 110
Gln His Gly Tyr Val Gly Ala Leu Pro Phe Arg Val Glu Phe Asp Tyr
115 120 125
Ser Ala Gln Gly Val Leu Arg Ser Ile Asp Glu Ser Leu Lys Arg Leu
130 135 140
Ser Leu Asp Arg Ile Asp Ile Val Tyr Ile His Asp Val Ala Glu Asp
145 150 155 160
Ala His Arg Glu Asp Trp Leu Arg Gln Tyr Arg Ile Ala Val Glu Gly
165 170 175
Ala Met Arg Ala Leu Ser Asp Leu Arg Asp Gln Gly Val Ile Gly Ala
180 185 190
Trp Gly Leu Gly Val Asn Arg Val Glu Pro Cys Met Met Thr Leu Asn
195 200 205
Asp Ala Asp Pro Asp Val Phe Leu Ile Ala Gly Arg Tyr Thr Leu Leu
210 215 220
Asp Thr Ser Ala Leu Asp Ala Leu Ile Pro Ala Cys Asp Ala Arg Gly
225 230 235 240
Val Gln Leu Val Ala Gly Gly Pro Phe Asn Ser Gly Leu Leu Val Gly
245 250 255
Gly Thr Thr Phe Glu Tyr Ala Glu Ala Pro Ala Glu Met Leu Glu Lys
260 265 270
Arg Arg Arg Leu Leu Ala His Cys Glu Arg Phe Gly Val Asp Leu Lys
275 280 285
Ala Ala Ala Leu Gln Phe Thr Asp Ala Pro Arg Ala Val Ala Cys Val
290 295 300
Ile Ala Gly Ala Arg Asn Ala Ser Glu Ile Arg Gln Asn Cys Ala Ala
305 310 315 320
Met Thr Glu Pro Ile Pro Ala Ala Phe Trp Ala Ala Leu Lys Ala Asp
325 330 335
Gly Leu Ile Pro Ala Asp Ala Pro Glu Pro Leu Ala Ser Gly Gly Ala
340 345 350
Leu His Ala Thr Thr His Glu Pro Val
355 360
<210> 128
<211> 1050
<212> DNA
<213> 人工序列
<220>
<223> Ss.Adh4的密码子优化序列
<400> 128
atggagaacg tgaatatggt gaaaagcaag gccgctttac tgaagaaatt cagcgaaccg 60
ctgagcatcg aggatgtgaa cattcccgaa ccgcaaggtg aagaagtgct gatccgcatt 120
ggtggtgctg gtgtgtgccg caccgatctg cgtgtgtgga agggcgtgga agccaaacaa 180
ggtttccgtt taccgatcat tctgggccat gaaaacgccg gcacaatcgt tgaagtgggc 240
gagctggcaa aagtgaagaa gggcgataac gtggtggtgt acgcaacttg gggcgattta 300
acttgtcgct attgccgcga gggtaaattt aatatttgta aaaaccaaat tatcccgggc 360
cagaccacca acggtggctt tagcgagtac atgctggtga aaagtagccg ctggctggtg 420
aaactgaaca gtctgagccc cgttgaagcc gcacctctgg ccgatgccgg tacaaccagc 480
atgggcgcca tccgtcaagc actgccgttc attagcaagt tcgccgaacc ggttgtgatt 540
gtgaatggta tcggcggttt agccgtgtac accattcaga ttctgaaggc tttaatgaaa 600
aacattacca tcgtgggtat cagccgcagc aaaaagcatc gcgacttcgc tttagaactg 660
ggcgccgatt acgtgagcga aatgaaggat gccgaatctt taatcaataa gctgaccgac 720
ggtttaggtg caagcatcgc aatcgatctg gtgggtaccg aagagaccac ctacaattta 780
ggcaagctgc tggcccaaga aggtgccatt attttagtgg gtatggaagg caaacgcgtg 840
agtctggaag ccttcgacac agcagtgtgg aacaagaaac tgctgggcag caactacggc 900
tctttaaatg atttagaaga tgtggtgcgt ctgagcgaaa gcggcaaaat taagccgtac 960
atcatcaagg tgccgctgga tgacatcaac aaagccttca ccaatctgga tgagggccgt 1020
gtggatggtc gccaagttat caccccgtaa 1050
<210> 129
<211> 349
<212> PRT
<213> 硫化叶菌
<400> 129
Met Glu Asn Val Asn Met Val Lys Ser Lys Ala Ala Leu Leu Lys Lys
1 5 10 15
Phe Ser Glu Pro Leu Ser Ile Glu Asp Val Asn Ile Pro Glu Pro Gln
20 25 30
Gly Glu Glu Val Leu Ile Arg Ile Gly Gly Ala Gly Val Cys Arg Thr
35 40 45
Asp Leu Arg Val Trp Lys Gly Val Glu Ala Lys Gln Gly Phe Arg Leu
50 55 60
Pro Ile Ile Leu Gly His Glu Asn Ala Gly Thr Ile Val Glu Val Gly
65 70 75 80
Glu Leu Ala Lys Val Lys Lys Gly Asp Asn Val Val Val Tyr Ala Thr
85 90 95
Trp Gly Asp Leu Thr Cys Arg Tyr Cys Arg Glu Gly Lys Phe Asn Ile
100 105 110
Cys Lys Asn Gln Ile Ile Pro Gly Gln Thr Thr Asn Gly Gly Phe Ser
115 120 125
Glu Tyr Met Leu Val Lys Ser Ser Arg Trp Leu Val Lys Leu Asn Ser
130 135 140
Leu Ser Pro Val Glu Ala Ala Pro Leu Ala Asp Ala Gly Thr Thr Ser
145 150 155 160
Met Gly Ala Ile Arg Gln Ala Leu Pro Phe Ile Ser Lys Phe Ala Glu
165 170 175
Pro Val Val Ile Val Asn Gly Ile Gly Gly Leu Ala Val Tyr Thr Ile
180 185 190
Gln Ile Leu Lys Ala Leu Met Lys Asn Ile Thr Ile Val Gly Ile Ser
195 200 205
Arg Ser Lys Lys His Arg Asp Phe Ala Leu Glu Leu Gly Ala Asp Tyr
210 215 220
Val Ser Glu Met Lys Asp Ala Glu Ser Leu Ile Asn Lys Leu Thr Asp
225 230 235 240
Gly Leu Gly Ala Ser Ile Ala Ile Asp Leu Val Gly Thr Glu Glu Thr
245 250 255
Thr Tyr Asn Leu Gly Lys Leu Leu Ala Gln Glu Gly Ala Ile Ile Leu
260 265 270
Val Gly Met Glu Gly Lys Arg Val Ser Leu Glu Ala Phe Asp Thr Ala
275 280 285
Val Trp Asn Lys Lys Leu Leu Gly Ser Asn Tyr Gly Ser Leu Asn Asp
290 295 300
Leu Glu Asp Val Val Arg Leu Ser Glu Ser Gly Lys Ile Lys Pro Tyr
305 310 315 320
Ile Ile Lys Val Pro Leu Asp Asp Ile Asn Lys Ala Phe Thr Asn Leu
325 330 335
Asp Glu Gly Arg Val Asp Gly Arg Gln Val Ile Thr Pro
340 345
<210> 130
<211> 777
<212> DNA
<213> 多食伯克霍尔德菌
<400> 130
atggatctga atctgcagga caaggtcgtg atcgtgacgg gcggcgcgtc gggcatcggc 60
ggcgcgatct cgatgcggct cgccgaagaa cgcgcgattc cggtcgtgtt cgcgcgccac 120
gcgcccgacg gcgcgttcct cgacgcgctc gcgcaacggc agccgcgcgc gacgtatctg 180
ccggtcgagc tgcaggacga tgcgcaatgc cgcgacgcgg tcgctcagac gatcgcgacg 240
ttcggacgcc tcgacgggct cgtgaacaac gcgggcgtca acgacggcat cgggctcgac 300
gcagggcgcg acgcgttcgt cgcatcgctc gagcgcaacc tgatccacta ctacgcgatg 360
gcgcattact gcgtgccgca tctgaaggcg acgcgcggcg cgatcgtcaa catctcgtcg 420
aagacggccg tgaccgggca gggcaatacg agcggctatt gcgcgtcgaa gggcgcgcag 480
ctcgcgctga cgcgcgaatg ggcggtcgcg ctgcgcgagc acggcgtgcg cgtgaacgcc 540
gtgattccgg ccgaggtcat gacgccgctc taccggaact ggatcgcgac gttcgaggat 600
cccgaggcga agctcgccga gatcgcggcg aaggtgccgc tcggcaagcg cttcacgaca 660
cccgacgaga tcgccgatac ggccgtgttc ctgctgtcgc cgcgcgcgtc gcacacgacg 720
ggcgaatggc tgttcgtcga cggcggctat acgcatctcg atcgtgcgct cgtctga 777
<210> 131
<211> 258
<212> PRT
<213> 多食伯克霍尔德菌
<400> 131
Met Asp Leu Asn Leu Gln Asp Lys Val Val Ile Val Thr Gly Gly Ala
1 5 10 15
Ser Gly Ile Gly Gly Ala Ile Ser Met Arg Leu Ala Glu Glu Arg Ala
20 25 30
Ile Pro Val Val Phe Ala Arg His Ala Pro Asp Gly Ala Phe Leu Asp
35 40 45
Ala Leu Ala Gln Arg Gln Pro Arg Ala Thr Tyr Leu Pro Val Glu Leu
50 55 60
Gln Asp Asp Ala Gln Cys Arg Asp Ala Val Ala Gln Thr Ile Ala Thr
65 70 75 80
Phe Gly Arg Leu Asp Gly Leu Val Asn Asn Ala Gly Val Asn Asp Gly
85 90 95
Ile Gly Leu Asp Ala Gly Arg Asp Ala Phe Val Ala Ser Leu Glu Arg
100 105 110
Asn Leu Ile His Tyr Tyr Ala Met Ala His Tyr Cys Val Pro His Leu
115 120 125
Lys Ala Thr Arg Gly Ala Ile Val Asn Ile Ser Ser Lys Thr Ala Val
130 135 140
Thr Gly Gln Gly Asn Thr Ser Gly Tyr Cys Ala Ser Lys Gly Ala Gln
145 150 155 160
Leu Ala Leu Thr Arg Glu Trp Ala Val Ala Leu Arg Glu His Gly Val
165 170 175
Arg Val Asn Ala Val Ile Pro Ala Glu Val Met Thr Pro Leu Tyr Arg
180 185 190
Asn Trp Ile Ala Thr Phe Glu Asp Pro Glu Ala Lys Leu Ala Glu Ile
195 200 205
Ala Ala Lys Val Pro Leu Gly Lys Arg Phe Thr Thr Pro Asp Glu Ile
210 215 220
Ala Asp Thr Ala Val Phe Leu Leu Ser Pro Arg Ala Ser His Thr Thr
225 230 235 240
Gly Glu Trp Leu Phe Val Asp Gly Gly Tyr Thr His Leu Asp Arg Ala
245 250 255
Leu Val
<210> 132
<211> 1068
<212> DNA
<213> 人工序列
<220>
<223> Tt.lac11的密码子优化序列
<400> 132
atgcgcaaac tgcttggcag ctctgccatc cacgtgctgt cccgtcgcac cggttatggt 60
atgtacttcc tgattctggc cctgggtgtc gctacacagg ttgtctttgg agcagaaccg 120
agtcagaatc cgagcgaaaa ggtggcatgg attgaacgcg cagatcccgg cctcgacgat 180
ctgattgctc cagatgcgca agtggaagtg ctggcagaag gttttgagtg gagcgaaggt 240
ccggtatgga ttcccgaagg cggctacttg ctgtttagcg atgttccgaa gaacaccatt 300
taccgctgga aagaaggtca ggggattgac atcttcctga aaccgtcggg atataccggc 360
tttcgtgaac gtggtggtga atcgggctca aacggcttag ccctggatcg ccaaggccgc 420
cttctgttgt gtcagcatgg tgatcgtcgt gtagctcgtt gggagaaagg ttgctttatc 480
acactggcag accagtatga gggcaaaccg ctgaattccc cgaatgactt ggtagtcaag 540
tcgaacggcg atatctactt cacggatcca ccatatggca tgactcctga ggcccaacgc 600
gatcccaatg cgctcggctt ttgcggcgtt tatcgcatta gtgcggatgg gaagctgacg 660
ctgttagttc gtgacatgac tcgccctaac gggatcgcct tcagcccaga cgagaaaacg 720
ctctatgttg cccagtcaga tccgcaacgc ccgttatgga tggcgttccc ggttcgcgaa 780
gatgggacct taggggaagg caaagtgttc ttcgatgcga aaccgtggca gcaaagtggc 840
ttgccgggac tcccagatgg catgaaagtg gaccagaaag gcaatctgtt tgcgactggt 900
ccgggtggtg tcaacatctt tcgccctgat ggtacctttc tgggccggat tcgggtgaaa 960
gtccctaccg ctaactgtgc gtttggtgat gacggatcta cgctttacat taccgcggac 1020
atgtatctgc ttcgtgtgaa aacgaaaacc aaaggactgg ggttctaa 1068
<210> 133
<211> 355
<212> PRT
<213> Thermogutta terrifontis
<400> 133
Met Arg Lys Leu Leu Gly Ser Ser Ala Ile His Val Leu Ser Arg Arg
1 5 10 15
Thr Gly Tyr Gly Met Tyr Phe Leu Ile Leu Ala Leu Gly Val Ala Thr
20 25 30
Gln Val Val Phe Gly Ala Glu Pro Ser Gln Asn Pro Ser Glu Lys Val
35 40 45
Ala Trp Ile Glu Arg Ala Asp Pro Gly Leu Asp Asp Leu Ile Ala Pro
50 55 60
Asp Ala Gln Val Glu Val Leu Ala Glu Gly Phe Glu Trp Ser Glu Gly
65 70 75 80
Pro Val Trp Ile Pro Glu Gly Gly Tyr Leu Leu Phe Ser Asp Val Pro
85 90 95
Lys Asn Thr Ile Tyr Arg Trp Lys Glu Gly Gln Gly Ile Asp Ile Phe
100 105 110
Leu Lys Pro Ser Gly Tyr Thr Gly Phe Arg Glu Arg Gly Gly Glu Ser
115 120 125
Gly Ser Asn Gly Leu Ala Leu Asp Arg Gln Gly Arg Leu Leu Leu Cys
130 135 140
Gln His Gly Asp Arg Arg Val Ala Arg Trp Glu Lys Gly Cys Phe Ile
145 150 155 160
Thr Leu Ala Asp Gln Tyr Glu Gly Lys Pro Leu Asn Ser Pro Asn Asp
165 170 175
Leu Val Val Lys Ser Asn Gly Asp Ile Tyr Phe Thr Asp Pro Pro Tyr
180 185 190
Gly Met Thr Pro Glu Ala Gln Arg Asp Pro Asn Ala Leu Gly Phe Cys
195 200 205
Gly Val Tyr Arg Ile Ser Ala Asp Gly Lys Leu Thr Leu Leu Val Arg
210 215 220
Asp Met Thr Arg Pro Asn Gly Ile Ala Phe Ser Pro Asp Glu Lys Thr
225 230 235 240
Leu Tyr Val Ala Gln Ser Asp Pro Gln Arg Pro Leu Trp Met Ala Phe
245 250 255
Pro Val Arg Glu Asp Gly Thr Leu Gly Glu Gly Lys Val Phe Phe Asp
260 265 270
Ala Lys Pro Trp Gln Gln Ser Gly Leu Pro Gly Leu Pro Asp Gly Met
275 280 285
Lys Val Asp Gln Lys Gly Asn Leu Phe Ala Thr Gly Pro Gly Gly Val
290 295 300
Asn Ile Phe Arg Pro Asp Gly Thr Phe Leu Gly Arg Ile Arg Val Lys
305 310 315 320
Val Pro Thr Ala Asn Cys Ala Phe Gly Asp Asp Gly Ser Thr Leu Tyr
325 330 335
Ile Thr Ala Asp Met Tyr Leu Leu Arg Val Lys Thr Lys Thr Lys Gly
340 345 350
Leu Gly Phe
355
<210> 134
<211> 957
<212> DNA
<213> 人工序列
<220>
<223> Tt.lac11截短变体的密码子优化序列
<400> 134
atggaaccga gtcagaatcc gagcgaaaag gtggcatgga ttgaacgcgc agatcccggc 60
ctcgacgatc tgattgctcc agatgcgcaa gtggaagtgc tggcagaagg ttttgagtgg 120
agcgaaggtc cggtatggat tcccgaaggc ggctacttgc tgtttagcga tgttccgaag 180
aacaccattt accgctggaa agaaggtcag gggattgaca tcttcctgaa accgtcggga 240
tataccggct ttcgtgaacg tggtggtgaa tcgggctcaa acggcttagc cctggatcgc 300
caaggccgcc ttctgttgtg tcagcatggt gatcgtcgtg tagctcgttg ggagaaaggt 360
tgctttatca cactggcaga ccagtatgag ggcaaaccgc tgaattcccc gaatgacttg 420
gtagtcaagt cgaacggcga tatctacttc acggatccac catatggcat gactcctgag 480
gcccaacgcg atcccaatgc gctcggcttt tgcggcgttt atcgcattag tgcggatggg 540
aagctgacgc tgttagttcg tgacatgact cgccctaacg ggatcgcctt cagcccagac 600
gagaaaacgc tctatgttgc ccagtcagat ccgcaacgcc cgttatggat ggcgttcccg 660
gttcgcgaag atgggacctt aggggaaggc aaagtgttct tcgatgcgaa accgtggcag 720
caaagtggct tgccgggact cccagatggc atgaaagtgg accagaaagg caatctgttt 780
gcgactggtc cgggtggtgt caacatcttt cgccctgatg gtacctttct gggccggatt 840
cgggtgaaag tccctaccgc taactgtgcg tttggtgatg acggatctac gctttacatt 900
accgcggaca tgtatctgct tcgtgtgaaa acgaaaacca aaggactggg gttctaa 957
<210> 135
<211> 318
<212> PRT
<213> 人工序列
<220>
<223> 截短了38个氨基酸的Tt.Lac11变体
<400> 135
Met Glu Pro Ser Gln Asn Pro Ser Glu Lys Val Ala Trp Ile Glu Arg
1 5 10 15
Ala Asp Pro Gly Leu Asp Asp Leu Ile Ala Pro Asp Ala Gln Val Glu
20 25 30
Val Leu Ala Glu Gly Phe Glu Trp Ser Glu Gly Pro Val Trp Ile Pro
35 40 45
Glu Gly Gly Tyr Leu Leu Phe Ser Asp Val Pro Lys Asn Thr Ile Tyr
50 55 60
Arg Trp Lys Glu Gly Gln Gly Ile Asp Ile Phe Leu Lys Pro Ser Gly
65 70 75 80
Tyr Thr Gly Phe Arg Glu Arg Gly Gly Glu Ser Gly Ser Asn Gly Leu
85 90 95
Ala Leu Asp Arg Gln Gly Arg Leu Leu Leu Cys Gln His Gly Asp Arg
100 105 110
Arg Val Ala Arg Trp Glu Lys Gly Cys Phe Ile Thr Leu Ala Asp Gln
115 120 125
Tyr Glu Gly Lys Pro Leu Asn Ser Pro Asn Asp Leu Val Val Lys Ser
130 135 140
Asn Gly Asp Ile Tyr Phe Thr Asp Pro Pro Tyr Gly Met Thr Pro Glu
145 150 155 160
Ala Gln Arg Asp Pro Asn Ala Leu Gly Phe Cys Gly Val Tyr Arg Ile
165 170 175
Ser Ala Asp Gly Lys Leu Thr Leu Leu Val Arg Asp Met Thr Arg Pro
180 185 190
Asn Gly Ile Ala Phe Ser Pro Asp Glu Lys Thr Leu Tyr Val Ala Gln
195 200 205
Ser Asp Pro Gln Arg Pro Leu Trp Met Ala Phe Pro Val Arg Glu Asp
210 215 220
Gly Thr Leu Gly Glu Gly Lys Val Phe Phe Asp Ala Lys Pro Trp Gln
225 230 235 240
Gln Ser Gly Leu Pro Gly Leu Pro Asp Gly Met Lys Val Asp Gln Lys
245 250 255
Gly Asn Leu Phe Ala Thr Gly Pro Gly Gly Val Asn Ile Phe Arg Pro
260 265 270
Asp Gly Thr Phe Leu Gly Arg Ile Arg Val Lys Val Pro Thr Ala Asn
275 280 285
Cys Ala Phe Gly Asp Asp Gly Ser Thr Leu Tyr Ile Thr Ala Asp Met
290 295 300
Tyr Leu Leu Arg Val Lys Thr Lys Thr Lys Gly Leu Gly Phe
305 310 315
<210> 136
<211> 918
<212> DNA
<213> 人工序列
<220>
<223> Tt.lac111截短变体的密码子优化序列
<400> 136
atggaacgcg cagatcccgg cctcgacgat ctgattgctc cagatgcgca agtggaagtg 60
ctggcagaag gttttgagtg gagcgaaggt ccggtatgga ttcccgaagg cggctacttg 120
ctgtttagcg atgttccgaa gaacaccatt taccgctgga aagaaggtca ggggattgac 180
atcttcctga aaccgtcggg atataccggc tttcgtgaac gtggtggtga atcgggctca 240
aacggcttag ccctggatcg ccaaggccgc cttctgttgt gtcagcatgg tgatcgtcgt 300
gtagctcgtt gggagaaagg ttgctttatc acactggcag accagtatga gggcaaaccg 360
ctgaattccc cgaatgactt ggtagtcaag tcgaacggcg atatctactt cacggatcca 420
ccatatggca tgactcctga ggcccaacgc gatcccaatg cgctcggctt ttgcggcgtt 480
tatcgcatta gtgcggatgg gaagctgacg ctgttagttc gtgacatgac tcgccctaac 540
gggatcgcct tcagcccaga cgagaaaacg ctctatgttg cccagtcaga tccgcaacgc 600
ccgttatgga tggcgttccc ggttcgcgaa gatgggacct taggggaagg caaagtgttc 660
ttcgatgcga aaccgtggca gcaaagtggc ttgccgggac tcccagatgg catgaaagtg 720
gaccagaaag gcaatctgtt tgcgactggt ccgggtggtg tcaacatctt tcgccctgat 780
ggtacctttc tgggccggat tcgggtgaaa gtccctaccg ctaactgtgc gtttggtgat 840
gacggatcta cgctttacat taccgcggac atgtatctgc ttcgtgtgaa aacgaaaacc 900
aaaggactgg ggttctaa 918
<210> 137
<211> 305
<212> PRT
<213> 人工序列
<220>
<223> 截短了51个氨基酸的Tt.Lac11变体
<400> 137
Met Glu Arg Ala Asp Pro Gly Leu Asp Asp Leu Ile Ala Pro Asp Ala
1 5 10 15
Gln Val Glu Val Leu Ala Glu Gly Phe Glu Trp Ser Glu Gly Pro Val
20 25 30
Trp Ile Pro Glu Gly Gly Tyr Leu Leu Phe Ser Asp Val Pro Lys Asn
35 40 45
Thr Ile Tyr Arg Trp Lys Glu Gly Gln Gly Ile Asp Ile Phe Leu Lys
50 55 60
Pro Ser Gly Tyr Thr Gly Phe Arg Glu Arg Gly Gly Glu Ser Gly Ser
65 70 75 80
Asn Gly Leu Ala Leu Asp Arg Gln Gly Arg Leu Leu Leu Cys Gln His
85 90 95
Gly Asp Arg Arg Val Ala Arg Trp Glu Lys Gly Cys Phe Ile Thr Leu
100 105 110
Ala Asp Gln Tyr Glu Gly Lys Pro Leu Asn Ser Pro Asn Asp Leu Val
115 120 125
Val Lys Ser Asn Gly Asp Ile Tyr Phe Thr Asp Pro Pro Tyr Gly Met
130 135 140
Thr Pro Glu Ala Gln Arg Asp Pro Asn Ala Leu Gly Phe Cys Gly Val
145 150 155 160
Tyr Arg Ile Ser Ala Asp Gly Lys Leu Thr Leu Leu Val Arg Asp Met
165 170 175
Thr Arg Pro Asn Gly Ile Ala Phe Ser Pro Asp Glu Lys Thr Leu Tyr
180 185 190
Val Ala Gln Ser Asp Pro Gln Arg Pro Leu Trp Met Ala Phe Pro Val
195 200 205
Arg Glu Asp Gly Thr Leu Gly Glu Gly Lys Val Phe Phe Asp Ala Lys
210 215 220
Pro Trp Gln Gln Ser Gly Leu Pro Gly Leu Pro Asp Gly Met Lys Val
225 230 235 240
Asp Gln Lys Gly Asn Leu Phe Ala Thr Gly Pro Gly Gly Val Asn Ile
245 250 255
Phe Arg Pro Asp Gly Thr Phe Leu Gly Arg Ile Arg Val Lys Val Pro
260 265 270
Thr Ala Asn Cys Ala Phe Gly Asp Asp Gly Ser Thr Leu Tyr Ile Thr
275 280 285
Ala Asp Met Tyr Leu Leu Arg Val Lys Thr Lys Thr Lys Gly Leu Gly
290 295 300
Phe
305
<210> 138
<211> 843
<212> DNA
<213> 人工序列
<220>
<223> Tt.lac11截短变体的密码子优化序列
<400> 138
atgtggagcg aaggtccggt atggattccc gaaggcggct acttgctgtt tagcgatgtt 60
ccgaagaaca ccatttaccg ctggaaagaa ggtcagggga ttgacatctt cctgaaaccg 120
tcgggatata ccggctttcg tgaacgtggt ggtgaatcgg gctcaaacgg cttagccctg 180
gatcgccaag gccgccttct gttgtgtcag catggtgatc gtcgtgtagc tcgttgggag 240
aaaggttgct ttatcacact ggcagaccag tatgagggca aaccgctgaa ttccccgaat 300
gacttggtag tcaagtcgaa cggcgatatc tacttcacgg atccaccata tggcatgact 360
cctgaggccc aacgcgatcc caatgcgctc ggcttttgcg gcgtttatcg cattagtgcg 420
gatgggaagc tgacgctgtt agttcgtgac atgactcgcc ctaacgggat cgccttcagc 480
ccagacgaga aaacgctcta tgttgcccag tcagatccgc aacgcccgtt atggatggcg 540
ttcccggttc gcgaagatgg gaccttaggg gaaggcaaag tgttcttcga tgcgaaaccg 600
tggcagcaaa gtggcttgcc gggactccca gatggcatga aagtggacca gaaaggcaat 660
ctgtttgcga ctggtccggg tggtgtcaac atctttcgcc ctgatggtac ctttctgggc 720
cggattcggg tgaaagtccc taccgctaac tgtgcgtttg gtgatgacgg atctacgctt 780
tacattaccg cggacatgta tctgcttcgt gtgaaaacga aaaccaaagg actggggttc 840
taa 843
<210> 139
<211> 280
<212> PRT
<213> 人工序列
<220>
<223> 截短了76个氨基酸的Tt.Lac11变体
<400> 139
Met Trp Ser Glu Gly Pro Val Trp Ile Pro Glu Gly Gly Tyr Leu Leu
1 5 10 15
Phe Ser Asp Val Pro Lys Asn Thr Ile Tyr Arg Trp Lys Glu Gly Gln
20 25 30
Gly Ile Asp Ile Phe Leu Lys Pro Ser Gly Tyr Thr Gly Phe Arg Glu
35 40 45
Arg Gly Gly Glu Ser Gly Ser Asn Gly Leu Ala Leu Asp Arg Gln Gly
50 55 60
Arg Leu Leu Leu Cys Gln His Gly Asp Arg Arg Val Ala Arg Trp Glu
65 70 75 80
Lys Gly Cys Phe Ile Thr Leu Ala Asp Gln Tyr Glu Gly Lys Pro Leu
85 90 95
Asn Ser Pro Asn Asp Leu Val Val Lys Ser Asn Gly Asp Ile Tyr Phe
100 105 110
Thr Asp Pro Pro Tyr Gly Met Thr Pro Glu Ala Gln Arg Asp Pro Asn
115 120 125
Ala Leu Gly Phe Cys Gly Val Tyr Arg Ile Ser Ala Asp Gly Lys Leu
130 135 140
Thr Leu Leu Val Arg Asp Met Thr Arg Pro Asn Gly Ile Ala Phe Ser
145 150 155 160
Pro Asp Glu Lys Thr Leu Tyr Val Ala Gln Ser Asp Pro Gln Arg Pro
165 170 175
Leu Trp Met Ala Phe Pro Val Arg Glu Asp Gly Thr Leu Gly Glu Gly
180 185 190
Lys Val Phe Phe Asp Ala Lys Pro Trp Gln Gln Ser Gly Leu Pro Gly
195 200 205
Leu Pro Asp Gly Met Lys Val Asp Gln Lys Gly Asn Leu Phe Ala Thr
210 215 220
Gly Pro Gly Gly Val Asn Ile Phe Arg Pro Asp Gly Thr Phe Leu Gly
225 230 235 240
Arg Ile Arg Val Lys Val Pro Thr Ala Asn Cys Ala Phe Gly Asp Asp
245 250 255
Gly Ser Thr Leu Tyr Ile Thr Ala Asp Met Tyr Leu Leu Arg Val Lys
260 265 270
Thr Lys Thr Lys Gly Leu Gly Phe
275 280
<210> 140
<211> 1851
<212> DNA
<213> 大肠杆菌
<400> 140
atgcctaagt accgttccgc caccaccact catggtcgta atatggcggg tgctcgtgcg 60
ctgtggcgcg ccaccggaat gaccgacgcc gatttcggta agccgattat cgcggttgtg 120
aactcgttca cccaatttgt accgggtcac gtccatctgc gcgatctcgg taaactggtc 180
gccgaacaaa ttgaagcggc tggcggcgtt gccaaagagt tcaacaccat tgcggtggat 240
gatgggattg ccatgggcca cggggggatg ctttattcac tgccatctcg cgaactgatc 300
gctgattccg ttgagtatat ggtcaacgcc cactgcgccg acgccatggt ctgcatctct 360
aactgcgaca aaatcacccc ggggatgctg atggcttccc tgcgcctgaa tattccggtg 420
atctttgttt ccggcggccc gatggaggcc gggaaaacca aactttccga tcagatcatc 480
aagctcgatc tggttgatgc gatgatccag ggcgcagacc cgaaagtatc tgactcccag 540
agcgatcagg ttgaacgttc cgcgtgtccg acctgcggtt cctgctccgg gatgtttacc 600
gctaactcaa tgaactgcct gaccgaagcg ctgggcctgt cgcagccggg caacggctcg 660
ctgctggcaa cccacgccga ccgtaagcag ctgttcctta atgctggtaa acgcattgtt 720
gaattgacca aacgttatta cgagcaaaac gacgaaagtg cactgccgcg taatatcgcc 780
agtaaggcgg cgtttgaaaa cgccatgacg ctggatatcg cgatgggtgg atcgactaac 840
accgtacttc acctgctggc ggcggcgcag gaagcggaaa tcgacttcac catgagtgat 900
atcgataagc tttcccgcaa ggttccacag ctgtgtaaag ttgcgccgag cacccagaaa 960
taccatatgg aagatgttca ccgtgctggt ggtgttatcg gtattctcgg cgaactggat 1020
cgcgcggggt tactgaaccg tgatgtgaaa aacgtacttg gcctgacgtt gccgcaaacg 1080
ctggaacaat acgacgttat gctgacccag gatgacgcgg taaaaaatat gttccgcgca 1140
ggtcctgcag gcattcgtac cacacaggca ttctcgcaag attgccgttg ggatacgctg 1200
gacgacgatc gcgccaatgg ctgtatccgc tcgctggaac acgcctacag caaagacggc 1260
ggcctggcgg tgctctacgg taactttgcg gaaaacggct gcatcgtgaa aacggcaggc 1320
gtcgatgaca gcatcctcaa attcaccggc ccggcgaaag tgtacgaaag ccaggacgat 1380
gcggtagaag cgattctcgg cggtaaagtt gtcgccggag atgtggtagt aattcgctat 1440
gaaggcccga aaggcggtcc ggggatgcag gaaatgctct acccaaccag cttcctgaaa 1500
tcaatgggtc tcggcaaagc ctgtgcgctg atcaccgacg gtcgtttctc tggtggcacc 1560
tctggtcttt ccatcggcca cgtctcaccg gaagcggcaa gcggcggcag cattggcctg 1620
attgaagatg gtgacctgat cgctatcgac atcccgaacc gtggcattca gttacaggta 1680
agcgatgccg aactggcggc gcgtcgtgaa gcgcaggacg ctcgaggtga caaagcctgg 1740
acgccgaaaa atcgtgaacg tcaggtctcc tttgccctgc gtgcttatgc cagcctggca 1800
accagcgccg acaaaggcgc ggtgcgcgat aaatcgaaac tggggggtta a 1851
<210> 141
<211> 616
<212> PRT
<213> 大肠杆菌
<400> 141
Met Pro Lys Tyr Arg Ser Ala Thr Thr Thr His Gly Arg Asn Met Ala
1 5 10 15
Gly Ala Arg Ala Leu Trp Arg Ala Thr Gly Met Thr Asp Ala Asp Phe
20 25 30
Gly Lys Pro Ile Ile Ala Val Val Asn Ser Phe Thr Gln Phe Val Pro
35 40 45
Gly His Val His Leu Arg Asp Leu Gly Lys Leu Val Ala Glu Gln Ile
50 55 60
Glu Ala Ala Gly Gly Val Ala Lys Glu Phe Asn Thr Ile Ala Val Asp
65 70 75 80
Asp Gly Ile Ala Met Gly His Gly Gly Met Leu Tyr Ser Leu Pro Ser
85 90 95
Arg Glu Leu Ile Ala Asp Ser Val Glu Tyr Met Val Asn Ala His Cys
100 105 110
Ala Asp Ala Met Val Cys Ile Ser Asn Cys Asp Lys Ile Thr Pro Gly
115 120 125
Met Leu Met Ala Ser Leu Arg Leu Asn Ile Pro Val Ile Phe Val Ser
130 135 140
Gly Gly Pro Met Glu Ala Gly Lys Thr Lys Leu Ser Asp Gln Ile Ile
145 150 155 160
Lys Leu Asp Leu Val Asp Ala Met Ile Gln Gly Ala Asp Pro Lys Val
165 170 175
Ser Asp Ser Gln Ser Asp Gln Val Glu Arg Ser Ala Cys Pro Thr Cys
180 185 190
Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Thr
195 200 205
Glu Ala Leu Gly Leu Ser Gln Pro Gly Asn Gly Ser Leu Leu Ala Thr
210 215 220
His Ala Asp Arg Lys Gln Leu Phe Leu Asn Ala Gly Lys Arg Ile Val
225 230 235 240
Glu Leu Thr Lys Arg Tyr Tyr Glu Gln Asn Asp Glu Ser Ala Leu Pro
245 250 255
Arg Asn Ile Ala Ser Lys Ala Ala Phe Glu Asn Ala Met Thr Leu Asp
260 265 270
Ile Ala Met Gly Gly Ser Thr Asn Thr Val Leu His Leu Leu Ala Ala
275 280 285
Ala Gln Glu Ala Glu Ile Asp Phe Thr Met Ser Asp Ile Asp Lys Leu
290 295 300
Ser Arg Lys Val Pro Gln Leu Cys Lys Val Ala Pro Ser Thr Gln Lys
305 310 315 320
Tyr His Met Glu Asp Val His Arg Ala Gly Gly Val Ile Gly Ile Leu
325 330 335
Gly Glu Leu Asp Arg Ala Gly Leu Leu Asn Arg Asp Val Lys Asn Val
340 345 350
Leu Gly Leu Thr Leu Pro Gln Thr Leu Glu Gln Tyr Asp Val Met Leu
355 360 365
Thr Gln Asp Asp Ala Val Lys Asn Met Phe Arg Ala Gly Pro Ala Gly
370 375 380
Ile Arg Thr Thr Gln Ala Phe Ser Gln Asp Cys Arg Trp Asp Thr Leu
385 390 395 400
Asp Asp Asp Arg Ala Asn Gly Cys Ile Arg Ser Leu Glu His Ala Tyr
405 410 415
Ser Lys Asp Gly Gly Leu Ala Val Leu Tyr Gly Asn Phe Ala Glu Asn
420 425 430
Gly Cys Ile Val Lys Thr Ala Gly Val Asp Asp Ser Ile Leu Lys Phe
435 440 445
Thr Gly Pro Ala Lys Val Tyr Glu Ser Gln Asp Asp Ala Val Glu Ala
450 455 460
Ile Leu Gly Gly Lys Val Val Ala Gly Asp Val Val Val Ile Arg Tyr
465 470 475 480
Glu Gly Pro Lys Gly Gly Pro Gly Met Gln Glu Met Leu Tyr Pro Thr
485 490 495
Ser Phe Leu Lys Ser Met Gly Leu Gly Lys Ala Cys Ala Leu Ile Thr
500 505 510
Asp Gly Arg Phe Ser Gly Gly Thr Ser Gly Leu Ser Ile Gly His Val
515 520 525
Ser Pro Glu Ala Ala Ser Gly Gly Ser Ile Gly Leu Ile Glu Asp Gly
530 535 540
Asp Leu Ile Ala Ile Asp Ile Pro Asn Arg Gly Ile Gln Leu Gln Val
545 550 555 560
Ser Asp Ala Glu Leu Ala Ala Arg Arg Glu Ala Gln Asp Ala Arg Gly
565 570 575
Asp Lys Ala Trp Thr Pro Lys Asn Arg Glu Arg Gln Val Ser Phe Ala
580 585 590
Leu Arg Ala Tyr Ala Ser Leu Ala Thr Ser Ala Asp Lys Gly Ala Val
595 600 605
Arg Asp Lys Ser Lys Leu Gly Gly
610 615
<210> 142
<211> 1677
<212> DNA
<213> 人工序列
<220>
<223> Ss.IlvD的密码子优化序列
<400> 142
atgccggcaa aattaaattc accgagccgt tatcatggta tttacaatgc accgcaccgc 60
gccttcttac gtagcgtggg tttaacagat gaggagatcg gcaaaccgct ggtggcaatc 120
gccaccgcat ggagcgaagc tggtccgtgt aatttccaca ctttagcttt agcccgtgtt 180
gccaaagagg gtaccaagga agctggtctg agccctctgg cattcccgac catggtggtg 240
aacgacaata tcggcatggg cagcgaaggc atgcgttatt ctctggtgag tcgcgatctg 300
atcgccgata tggtggaggc ccagtttaac gcccacgcat tcgatggctt agtgggcatc 360
ggcggctgcg ataagaccac cccgggcatt ttaatggcca tggcccgttt aaatgtgccg 420
agcatttata tttacggcgg tagcgccgaa ccgggctatt ttatgggcaa gcgtttaaca 480
atcgaggacg ttcatgaagc catcggcgca tatttagcca agcgcattac agaaaacgaa 540
ctgtatgaaa ttgaaaaacg cgcccaccct acactgggta cttgtagcgg tctgttcacc 600
gccaacacca tgggtagtat gagcgaagca ctgggcatgg ctttaccggg tagtgcaagt 660
ccgacagcaa caagcagccg ccgtgttatg tatgtgaaag agaccggcaa agcactgggt 720
tctttaattg aaaatggcat caagagccgc gagattttaa cctttgaagc attcgagaac 780
gccatcacca ctttaatggc tatgggtggt agcacaaatg cagtgctgca tctgctggcc 840
attgcatacg aagccggcgt gaaactgact ttagatgact tcaaccgcat cagcaagcgt 900
acaccgtata tcgccagcat gaaacccggt ggtgattatg tgatggccga tttagatgaa 960
gttggcggcg tgccggtggt gctgaaaaaa ctgctggacg ccggtttact gcatggcgat 1020
gttctgaccg tgaccggtaa gaccatgaag cagaatttag agcagtacaa gtaccctaac 1080
gtgccgcata gccatatcgt gcgtgatgtg aagaacccga tcaaaccgcg cggcggtatc 1140
gttattttaa aaggctcttt agcccccgaa ggcgcagtta ttaaagtggc cgccaccaac 1200
gtggttaagt tcgaaggcaa ggccaaggtg tacaatagcg aggacgatgc cttcaaaggc 1260
gtgcaaagtg gcgaggtgag tgagggtgaa gttgtgatca tccgctatga gggtccgaaa 1320
ggcgcaccgg gtatgcccga aatgctgcgt gttaccgccg ccatcatggg tgccggttta 1380
aacaatgtgg cactggttac cgacggtcgt tttagcggcg caacacgcgg tccgatggtt 1440
ggtcatgttg caccggaggc aatggttggc ggcccgatcg caattgtgga agacggtgac 1500
accattgtga tcgatgtgga gagtgagcgt ttagatctga aactgagcga agaggagatc 1560
aaaaatcgtc tgaaacgttg gagcccgccg agccctcgct acaaaagtgg tttactggca 1620
aaatacgcca gtctggttag tcaagctagc atgggcgcag tgacccgtcc cgcttaa 1677
<210> 143
<211> 558
<212> PRT
<213> 硫化叶菌
<400> 143
Met Pro Ala Lys Leu Asn Ser Pro Ser Arg Tyr His Gly Ile Tyr Asn
1 5 10 15
Ala Pro His Arg Ala Phe Leu Arg Ser Val Gly Leu Thr Asp Glu Glu
20 25 30
Ile Gly Lys Pro Leu Val Ala Ile Ala Thr Ala Trp Ser Glu Ala Gly
35 40 45
Pro Cys Asn Phe His Thr Leu Ala Leu Ala Arg Val Ala Lys Glu Gly
50 55 60
Thr Lys Glu Ala Gly Leu Ser Pro Leu Ala Phe Pro Thr Met Val Val
65 70 75 80
Asn Asp Asn Ile Gly Met Gly Ser Glu Gly Met Arg Tyr Ser Leu Val
85 90 95
Ser Arg Asp Leu Ile Ala Asp Met Val Glu Ala Gln Phe Asn Ala His
100 105 110
Ala Phe Asp Gly Leu Val Gly Ile Gly Gly Cys Asp Lys Thr Thr Pro
115 120 125
Gly Ile Leu Met Ala Met Ala Arg Leu Asn Val Pro Ser Ile Tyr Ile
130 135 140
Tyr Gly Gly Ser Ala Glu Pro Gly Tyr Phe Met Gly Lys Arg Leu Thr
145 150 155 160
Ile Glu Asp Val His Glu Ala Ile Gly Ala Tyr Leu Ala Lys Arg Ile
165 170 175
Thr Glu Asn Glu Leu Tyr Glu Ile Glu Lys Arg Ala His Pro Thr Leu
180 185 190
Gly Thr Cys Ser Gly Leu Phe Thr Ala Asn Thr Met Gly Ser Met Ser
195 200 205
Glu Ala Leu Gly Met Ala Leu Pro Gly Ser Ala Ser Pro Thr Ala Thr
210 215 220
Ser Ser Arg Arg Val Met Tyr Val Lys Glu Thr Gly Lys Ala Leu Gly
225 230 235 240
Ser Leu Ile Glu Asn Gly Ile Lys Ser Arg Glu Ile Leu Thr Phe Glu
245 250 255
Ala Phe Glu Asn Ala Ile Thr Thr Leu Met Ala Met Gly Gly Ser Thr
260 265 270
Asn Ala Val Leu His Leu Leu Ala Ile Ala Tyr Glu Ala Gly Val Lys
275 280 285
Leu Thr Leu Asp Asp Phe Asn Arg Ile Ser Lys Arg Thr Pro Tyr Ile
290 295 300
Ala Ser Met Lys Pro Gly Gly Asp Tyr Val Met Ala Asp Leu Asp Glu
305 310 315 320
Val Gly Gly Val Pro Val Val Leu Lys Lys Leu Leu Asp Ala Gly Leu
325 330 335
Leu His Gly Asp Val Leu Thr Val Thr Gly Lys Thr Met Lys Gln Asn
340 345 350
Leu Glu Gln Tyr Lys Tyr Pro Asn Val Pro His Ser His Ile Val Arg
355 360 365
Asp Val Lys Asn Pro Ile Lys Pro Arg Gly Gly Ile Val Ile Leu Lys
370 375 380
Gly Ser Leu Ala Pro Glu Gly Ala Val Ile Lys Val Ala Ala Thr Asn
385 390 395 400
Val Val Lys Phe Glu Gly Lys Ala Lys Val Tyr Asn Ser Glu Asp Asp
405 410 415
Ala Phe Lys Gly Val Gln Ser Gly Glu Val Ser Glu Gly Glu Val Val
420 425 430
Ile Ile Arg Tyr Glu Gly Pro Lys Gly Ala Pro Gly Met Pro Glu Met
435 440 445
Leu Arg Val Thr Ala Ala Ile Met Gly Ala Gly Leu Asn Asn Val Ala
450 455 460
Leu Val Thr Asp Gly Arg Phe Ser Gly Ala Thr Arg Gly Pro Met Val
465 470 475 480
Gly His Val Ala Pro Glu Ala Met Val Gly Gly Pro Ile Ala Ile Val
485 490 495
Glu Asp Gly Asp Thr Ile Val Ile Asp Val Glu Ser Glu Arg Leu Asp
500 505 510
Leu Lys Leu Ser Glu Glu Glu Ile Lys Asn Arg Leu Lys Arg Trp Ser
515 520 525
Pro Pro Ser Pro Arg Tyr Lys Ser Gly Leu Leu Ala Lys Tyr Ala Ser
530 535 540
Leu Val Ser Gln Ala Ser Met Gly Ala Val Thr Arg Pro Ala
545 550 555
<210> 144
<211> 1326
<212> DNA
<213> 人工序列
<220>
<223> Xc.FucD的密码子优化序列
<400> 144
atgcgtacca ttatcgcttt agaaacccat gatgttcgct ttccgacaag ccgtgagctg 60
gatggcagcg acgcaatgaa cccggacccg gactatagcg ccgcctacgt tgttctgcgt 120
acagatggcg cagaagatct ggccggctac ggtctggtgt ttaccattgg tcgcggcaac 180
gatgttcaga cagcagccgt ggcagcttta gcagaacacg tggtgggttt aagcgttgac 240
aaggtgattg ccgatctggg tgcatttgca cgccgtttaa ccaatgatag ccaactgcgt 300
tggctgggtc cggaaaaagg cgtgatgcac atggccatcg gcgccgttat taatgccgca 360
tgggatctgg cagcacgcgc cgcaaataaa ccgctgtggc gctttatcgc cgaactgacc 420
ccggaacagc tggtggacac catcgacttt cgctatctga gcgatgcact gacccgtgat 480
gaagcactgg caattctgcg tgatgcacag ccgcagcgtg cagcacgtac cgccacactg 540
atcgagcaag gttatccggc atataccacc agcccgggct ggctgggcta cagtgacgaa 600
aaattagtgc gtttagccaa ggaagccgtt gccgatggct tccgcaccat taaactgaaa 660
gtgggcgcca acgtgcaaga tgatattcgc cgttgccgtc tggcacgtgc cgcaattggt 720
ccggacatcg ccatggcagt ggatgccaac cagcgctggg atgttggtcc ggccattgat 780
tggatgcgcc agctggccga gtttgatatc gcttggattg aggaaccgac aagtccggat 840
gatgtgctgg gccatgcagc aatccgtcaa ggtatcaccc cggttccggt gagtaccggc 900
gaacacaccc agaaccgcgt tgttttcaaa cagctgctgc aagctggtgc cgtggatctg 960
attcaaatcg atgccgcccg tgttggcggc gttaatgaga atttagccat tttactgctg 1020
gccgccaaat tcggcgtgcg tgtgtttccg catgctggtg gcgtgggtct gtgcgaactg 1080
gttcagcatc tggcaatggc cgatttcgtt gcaattaccg gcaagatgga ggatcgtgcc 1140
atcgagtttg tggatcatct gcaccagcac tttttagacc cggtgcgcat tcagcacggt 1200
cgctatctgg ccccggaagt tccgggtttc agcgccgaaa tgcacccggc aagcattgcc 1260
gagtttagct acccggacgg ccgtttctgg gttgaagatt tagccgccag caaagccaaa 1320
gcctaa 1326
<210> 145
<211> 441
<212> PRT
<213> 油菜黄单胞菌
<400> 145
Met Arg Thr Ile Ile Ala Leu Glu Thr His Asp Val Arg Phe Pro Thr
1 5 10 15
Ser Arg Glu Leu Asp Gly Ser Asp Ala Met Asn Pro Asp Pro Asp Tyr
20 25 30
Ser Ala Ala Tyr Val Val Leu Arg Thr Asp Gly Ala Glu Asp Leu Ala
35 40 45
Gly Tyr Gly Leu Val Phe Thr Ile Gly Arg Gly Asn Asp Val Gln Thr
50 55 60
Ala Ala Val Ala Ala Leu Ala Glu His Val Val Gly Leu Ser Val Asp
65 70 75 80
Lys Val Ile Ala Asp Leu Gly Ala Phe Ala Arg Arg Leu Thr Asn Asp
85 90 95
Ser Gln Leu Arg Trp Leu Gly Pro Glu Lys Gly Val Met His Met Ala
100 105 110
Ile Gly Ala Val Ile Asn Ala Ala Trp Asp Leu Ala Ala Arg Ala Ala
115 120 125
Asn Lys Pro Leu Trp Arg Phe Ile Ala Glu Leu Thr Pro Glu Gln Leu
130 135 140
Val Asp Thr Ile Asp Phe Arg Tyr Leu Ser Asp Ala Leu Thr Arg Asp
145 150 155 160
Glu Ala Leu Ala Ile Leu Arg Asp Ala Gln Pro Gln Arg Ala Ala Arg
165 170 175
Thr Ala Thr Leu Ile Glu Gln Gly Tyr Pro Ala Tyr Thr Thr Ser Pro
180 185 190
Gly Trp Leu Gly Tyr Ser Asp Glu Lys Leu Val Arg Leu Ala Lys Glu
195 200 205
Ala Val Ala Asp Gly Phe Arg Thr Ile Lys Leu Lys Val Gly Ala Asn
210 215 220
Val Gln Asp Asp Ile Arg Arg Cys Arg Leu Ala Arg Ala Ala Ile Gly
225 230 235 240
Pro Asp Ile Ala Met Ala Val Asp Ala Asn Gln Arg Trp Asp Val Gly
245 250 255
Pro Ala Ile Asp Trp Met Arg Gln Leu Ala Glu Phe Asp Ile Ala Trp
260 265 270
Ile Glu Glu Pro Thr Ser Pro Asp Asp Val Leu Gly His Ala Ala Ile
275 280 285
Arg Gln Gly Ile Thr Pro Val Pro Val Ser Thr Gly Glu His Thr Gln
290 295 300
Asn Arg Val Val Phe Lys Gln Leu Leu Gln Ala Gly Ala Val Asp Leu
305 310 315 320
Ile Gln Ile Asp Ala Ala Arg Val Gly Gly Val Asn Glu Asn Leu Ala
325 330 335
Ile Leu Leu Leu Ala Ala Lys Phe Gly Val Arg Val Phe Pro His Ala
340 345 350
Gly Gly Val Gly Leu Cys Glu Leu Val Gln His Leu Ala Met Ala Asp
355 360 365
Phe Val Ala Ile Thr Gly Lys Met Glu Asp Arg Ala Ile Glu Phe Val
370 375 380
Asp His Leu His Gln His Phe Leu Asp Pro Val Arg Ile Gln His Gly
385 390 395 400
Arg Tyr Leu Ala Pro Glu Val Pro Gly Phe Ser Ala Glu Met His Pro
405 410 415
Ala Ser Ile Ala Glu Phe Ser Tyr Pro Asp Gly Arg Phe Trp Val Glu
420 425 430
Asp Leu Ala Ala Ser Lys Ala Lys Ala
435 440
<210> 146
<211> 1236
<212> DNA
<213> 恶臭假单胞菌
<400> 146
atgaacagtg cccccgacta ttcggcggcc tacgtggtgc tgcacaccga cgctgccgcc 60
ctcgaaggcc acggcctgac cttcaccatc ggccgtggca acgaaatatg cgcagcagcg 120
gtgcagtcgc tggccccctt gatcgtcggc cttacgctcg aagaaatcag cgccgacatg 180
ggcgctttct ggcatcgctt taccgtcagc gacagccagt tgcgctggct gggcccggaa 240
aagggcgtga tccacctggc caccgccgcc atcatcaacg cagtgtggga cctgtgggcc 300
aagcacgaag gcaagcccgt gtggaaactg ctggccgaca tgacgccgga gcagctggtg 360
cgctgcctgg acttcagcta cgtcaccgac gtgctcaccc ccgaagaggc catcgccctg 420
ctgcgccgcc aggcgccggg caaagccgag cgcgaggcac acatgctgcg tgaaggctac 480
cccgggtaca ccactgcccc gggctggctg ggctacagcg aagagaaaat gcgcaaactg 540
gcccgcgagg ccgtggcaga cggctggacc cacatcaaac agaagatcgg ggccgacctg 600
gaagaagaca tccgccgcgc cagcatcctg cgcgacgaaa tcggctggga acgcaccctg 660
atgatggatg ccaaccaagt gtggggcgtc gaagagtcgg tggccaacat gcgccgcctg 720
gccgcattcg aaccgctttg gatcgaagaa ccgaccagcc ccgatgacat tcttggccac 780
gccaccattc gccagcgcat tgcgccaatc ggtgtcgcca ccggtgagca ttgccacaac 840
cgggtgatgt tcaagcagat gttccaggcc ggtgcactgg acttctgcca gctggacgca 900
gcccgcctgg gtggcctgaa cgaggtcctg atcgtgctgc tgatggccgc caagtacgac 960
gtgccggtct gcccgcacgg cggtggcgtc gggctgtgcg aatatgtgca gaacatcgcg 1020
cttttcgact acatcgcggt ttctgcctcg ctgcacaacc gggtgctgga gtacgtcgac 1080
cacctgcacg agcacttcat cgacccggtg gtgatccatc gcgggcgcta catgccgcca 1140
caacgccctg gctacagcat cgagatgcac gctgagaccc tggaacgtta ccagtacccc 1200
aacggcgccg tgtggctcga catcaaccgt tcctga 1236
<210> 147
<211> 411
<212> PRT
<213> 恶臭假单胞菌
<400> 147
Met Asn Ser Ala Pro Asp Tyr Ser Ala Ala Tyr Val Val Leu His Thr
1 5 10 15
Asp Ala Ala Ala Leu Glu Gly His Gly Leu Thr Phe Thr Ile Gly Arg
20 25 30
Gly Asn Glu Ile Cys Ala Ala Ala Val Gln Ser Leu Ala Pro Leu Ile
35 40 45
Val Gly Leu Thr Leu Glu Glu Ile Ser Ala Asp Met Gly Ala Phe Trp
50 55 60
His Arg Phe Thr Val Ser Asp Ser Gln Leu Arg Trp Leu Gly Pro Glu
65 70 75 80
Lys Gly Val Ile His Leu Ala Thr Ala Ala Ile Ile Asn Ala Val Trp
85 90 95
Asp Leu Trp Ala Lys His Glu Gly Lys Pro Val Trp Lys Leu Leu Ala
100 105 110
Asp Met Thr Pro Glu Gln Leu Val Arg Cys Leu Asp Phe Ser Tyr Val
115 120 125
Thr Asp Val Leu Thr Pro Glu Glu Ala Ile Ala Leu Leu Arg Arg Gln
130 135 140
Ala Pro Gly Lys Ala Glu Arg Glu Ala His Met Leu Arg Glu Gly Tyr
145 150 155 160
Pro Gly Tyr Thr Thr Ala Pro Gly Trp Leu Gly Tyr Ser Glu Glu Lys
165 170 175
Met Arg Lys Leu Ala Arg Glu Ala Val Ala Asp Gly Trp Thr His Ile
180 185 190
Lys Gln Lys Ile Gly Ala Asp Leu Glu Glu Asp Ile Arg Arg Ala Ser
195 200 205
Ile Leu Arg Asp Glu Ile Gly Trp Glu Arg Thr Leu Met Met Asp Ala
210 215 220
Asn Gln Val Trp Gly Val Glu Glu Ser Val Ala Asn Met Arg Arg Leu
225 230 235 240
Ala Ala Phe Glu Pro Leu Trp Ile Glu Glu Pro Thr Ser Pro Asp Asp
245 250 255
Ile Leu Gly His Ala Thr Ile Arg Gln Arg Ile Ala Pro Ile Gly Val
260 265 270
Ala Thr Gly Glu His Cys His Asn Arg Val Met Phe Lys Gln Met Phe
275 280 285
Gln Ala Gly Ala Leu Asp Phe Cys Gln Leu Asp Ala Ala Arg Leu Gly
290 295 300
Gly Leu Asn Glu Val Leu Ile Val Leu Leu Met Ala Ala Lys Tyr Asp
305 310 315 320
Val Pro Val Cys Pro His Gly Gly Gly Val Gly Leu Cys Glu Tyr Val
325 330 335
Gln Asn Ile Ala Leu Phe Asp Tyr Ile Ala Val Ser Ala Ser Leu His
340 345 350
Asn Arg Val Leu Glu Tyr Val Asp His Leu His Glu His Phe Ile Asp
355 360 365
Pro Val Val Ile His Arg Gly Arg Tyr Met Pro Pro Gln Arg Pro Gly
370 375 380
Tyr Ser Ile Glu Met His Ala Glu Thr Leu Glu Arg Tyr Gln Tyr Pro
385 390 395 400
Asn Gly Ala Val Trp Leu Asp Ile Asn Arg Ser
405 410
<210> 148
<211> 1170
<212> DNA
<213> 大豆慢生根瘤菌
<400> 148
atgtccgtcc gcatcgtcga cgtccgcgag atcaccaagc cgatctcatc gccgatccgc 60
aacgcctata tcgatttcac caagatgacg acgagcctcg tcgccgtcgt cactgacgtt 120
gtgcgcgaag gcaagcgcgt cgtcggctac ggcttcaatt ccaacggccg ctacgggcag 180
ggcggcctga tccgcgaacg cttcgcctcg cgcatcctgg aggccgatcc gaagaagctt 240
ctgaacgagg ccggcgacaa tctcgatccc gacaaggtct gggccgcgat gatgatcaac 300
gagaagccgg gcggccatgg cgagcgctcg gtcgcggtcg gcaccatcga catggcggtg 360
tgggacgcgg tggcgaagat cgcgggcaag ccgctgtttc gcctgctcgc cgaacgtcac 420
ggcgtcaaag ccaacccgcg cgtgttcgtc tatgccgccg gcggctacta ctatcccggc 480
aaggggctat cgatgctgcg cggcgagatg cgtggctatc tcgaccgcgg ctacaacgtc 540
gtgaagatga agatcggcgg cgcgccgatc gaggaggatc gcatgcgcat cgaggcggtg 600
ctggaggaga tcggcaagga cgcgcagctc gccgtcgacg ccaacggccg cttcaacctc 660
gagaccggca tcgcctatgc caagatgctg cgcgattatc cgctgttctg gtacgaggag 720
gtcggcgatc ccctcgacta cgcgctgcag gccgcgctcg ccgaattcta tcccggcccg 780
atggcgacag gcgaaaacct gttcagccac caggacgcgc gcaacctgct ccgctatggc 840
ggcatgcgcc cggaccgcga ctggctgcaa ttcgactgcg cgctgtccta tggcctctgc 900
gaataccagc gcacgctcga agtgctgaag acccatggct ggtcgccaag ccgctgcatc 960
ccgcatggcg gccaccagat gtcgctcaac atcgccgctg gcctcggcct cggcggcaac 1020
gaaagctacc cggacctgtt ccagccctac ggcggcttcc ccgacggcgt gcgcgtcgag 1080
aacggccaca tcaccatgcc ggaccttccg ggcatcggct tcgaaggcaa gtcggatctc 1140
tacaaggaga tgaaggcgct ggcggagtaa 1170
<210> 149
<211> 389
<212> PRT
<213> 大豆慢生根瘤菌
<400> 149
Met Ser Val Arg Ile Val Asp Val Arg Glu Ile Thr Lys Pro Ile Ser
1 5 10 15
Ser Pro Ile Arg Asn Ala Tyr Ile Asp Phe Thr Lys Met Thr Thr Ser
20 25 30
Leu Val Ala Val Val Thr Asp Val Val Arg Glu Gly Lys Arg Val Val
35 40 45
Gly Tyr Gly Phe Asn Ser Asn Gly Arg Tyr Gly Gln Gly Gly Leu Ile
50 55 60
Arg Glu Arg Phe Ala Ser Arg Ile Leu Glu Ala Asp Pro Lys Lys Leu
65 70 75 80
Leu Asn Glu Ala Gly Asp Asn Leu Asp Pro Asp Lys Val Trp Ala Ala
85 90 95
Met Met Ile Asn Glu Lys Pro Gly Gly His Gly Glu Arg Ser Val Ala
100 105 110
Val Gly Thr Ile Asp Met Ala Val Trp Asp Ala Val Ala Lys Ile Ala
115 120 125
Gly Lys Pro Leu Phe Arg Leu Leu Ala Glu Arg His Gly Val Lys Ala
130 135 140
Asn Pro Arg Val Phe Val Tyr Ala Ala Gly Gly Tyr Tyr Tyr Pro Gly
145 150 155 160
Lys Gly Leu Ser Met Leu Arg Gly Glu Met Arg Gly Tyr Leu Asp Arg
165 170 175
Gly Tyr Asn Val Val Lys Met Lys Ile Gly Gly Ala Pro Ile Glu Glu
180 185 190
Asp Arg Met Arg Ile Glu Ala Val Leu Glu Glu Ile Gly Lys Asp Ala
195 200 205
Gln Leu Ala Val Asp Ala Asn Gly Arg Phe Asn Leu Glu Thr Gly Ile
210 215 220
Ala Tyr Ala Lys Met Leu Arg Asp Tyr Pro Leu Phe Trp Tyr Glu Glu
225 230 235 240
Val Gly Asp Pro Leu Asp Tyr Ala Leu Gln Ala Ala Leu Ala Glu Phe
245 250 255
Tyr Pro Gly Pro Met Ala Thr Gly Glu Asn Leu Phe Ser His Gln Asp
260 265 270
Ala Arg Asn Leu Leu Arg Tyr Gly Gly Met Arg Pro Asp Arg Asp Trp
275 280 285
Leu Gln Phe Asp Cys Ala Leu Ser Tyr Gly Leu Cys Glu Tyr Gln Arg
290 295 300
Thr Leu Glu Val Leu Lys Thr His Gly Trp Ser Pro Ser Arg Cys Ile
305 310 315 320
Pro His Gly Gly His Gln Met Ser Leu Asn Ile Ala Ala Gly Leu Gly
325 330 335
Leu Gly Gly Asn Glu Ser Tyr Pro Asp Leu Phe Gln Pro Tyr Gly Gly
340 345 350
Phe Pro Asp Gly Val Arg Val Glu Asn Gly His Ile Thr Met Pro Asp
355 360 365
Leu Pro Gly Ile Gly Phe Glu Gly Lys Ser Asp Leu Tyr Lys Glu Met
370 375 380
Lys Ala Leu Ala Glu
385
<210> 150
<211> 1539
<212> DNA
<213> 燕麦食酸菌
<400> 150
atgtcgactg atgcactggc gacgcagtcg ctgctcatcc tgaacgaccg cgacaacgtg 60
gctgtcgcgc gcaccgaact cggcgcgggc atggagcttc ggctggccgg cggcgaagta 120
ctggaggtga tggccactgt gcccgccggc cacaagatcg cactcaggcc catcgcgaag 180
ggcgatgccg tgctgaagta cggccaggtc atcggcgctg ccagcacgcc gatcggggcc 240
ggcgagcacg tgcatgtgca caacgtcgag atgggcgact ccagccccac ccacctggtg 300
ggccgcgcct acgtcccgac cgatcccgtg gagcagcctg cgaccttcat gggctacgtc 360
agggccgatg ggcgcgtcgg cacgcgcaac tacatcggcg tcatttcttc ggtgaactgc 420
tccgccacgg tctgcaaggc catcgcgcag gccttcacgg gcccggcgct cggtgcctat 480
ccgggtgtgg acggtgtcgt cgccatcacg cacggcaccg gctgcggcat gagcggtgaa 540
ggcgaaggca tcgaactcct gcgccgcacg ctcagcggct acatcgccca ccccaatttc 600
gccgcggtgc tggtgatcgg actgggctgc gaggtcaacc aggtcgaggg cctgaccggc 660
cgcctgccgt tgccggaagg ccgggtgcgc accatgacca tccaggacgc cggtggcacc 720
cgcgaggcca tcgcccaggg catcgatatc gtgcgcgaca tggcagcggc ggcatccggc 780
gcccggcgca ctcaggtgcc ggcatcgcac ctcgtggtgg ggctgcagtg cggcggatcg 840
gacggctact cgggcatcag cgccaacccg gtactgggcg cggcggtcga catgctggtg 900
cgccacggcg gcacggccat cctgtcggaa acacccgaga tctatggcgc cgagcatttg 960
ctgacggcgc gcgccgcatc ggccgaagtg gcagaacgcc tgatggcgcg cctggcctgg 1020
tgggaggcct acacccagcg caacggcgcg aacatgaaca acaacccctc gcccggcaac 1080
aaggccggcg gcatcaccac catcctcgag aaatcgctgg gcgcggtggc caagggcggt 1140
ggcagcgggc tgatggatgt ggtcgaatac gcgcagcccg tgcacagccg gggcctggtg 1200
ttcatggaca cccccggcta cgatccggtg tcggccaccg gccaggtcgc gggcggtgcc 1260
aacctcatct gtttcactac cgggcgggga tcgacctacg ggtgcaagcc cacgccgtcg 1320
ctcaagctcg ccaccaacac cgcgctgttc cgcaacatgg gcatggacat ggacttcaac 1380
gccggcgacg tggtggacgg aacgctgggc atcgcgcagg cgggccgcca tcttttcgaa 1440
ctgatgctcg ccacggcgtc cggcgaccgc acgctgagcg agacgcacgg tctgggcgac 1500
aacgaattcg tgccctggca gctcggcgcg gtgatgtga 1539
<210> 151
<211> 512
<212> PRT
<213> 燕麦食酸菌
<400> 151
Met Ser Thr Asp Ala Leu Ala Thr Gln Ser Leu Leu Ile Leu Asn Asp
1 5 10 15
Arg Asp Asn Val Ala Val Ala Arg Thr Glu Leu Gly Ala Gly Met Glu
20 25 30
Leu Arg Leu Ala Gly Gly Glu Val Leu Glu Val Met Ala Thr Val Pro
35 40 45
Ala Gly His Lys Ile Ala Leu Arg Pro Ile Ala Lys Gly Asp Ala Val
50 55 60
Leu Lys Tyr Gly Gln Val Ile Gly Ala Ala Ser Thr Pro Ile Gly Ala
65 70 75 80
Gly Glu His Val His Val His Asn Val Glu Met Gly Asp Ser Ser Pro
85 90 95
Thr His Leu Val Gly Arg Ala Tyr Val Pro Thr Asp Pro Val Glu Gln
100 105 110
Pro Ala Thr Phe Met Gly Tyr Val Arg Ala Asp Gly Arg Val Gly Thr
115 120 125
Arg Asn Tyr Ile Gly Val Ile Ser Ser Val Asn Cys Ser Ala Thr Val
130 135 140
Cys Lys Ala Ile Ala Gln Ala Phe Thr Gly Pro Ala Leu Gly Ala Tyr
145 150 155 160
Pro Gly Val Asp Gly Val Val Ala Ile Thr His Gly Thr Gly Cys Gly
165 170 175
Met Ser Gly Glu Gly Glu Gly Ile Glu Leu Leu Arg Arg Thr Leu Ser
180 185 190
Gly Tyr Ile Ala His Pro Asn Phe Ala Ala Val Leu Val Ile Gly Leu
195 200 205
Gly Cys Glu Val Asn Gln Val Glu Gly Leu Thr Gly Arg Leu Pro Leu
210 215 220
Pro Glu Gly Arg Val Arg Thr Met Thr Ile Gln Asp Ala Gly Gly Thr
225 230 235 240
Arg Glu Ala Ile Ala Gln Gly Ile Asp Ile Val Arg Asp Met Ala Ala
245 250 255
Ala Ala Ser Gly Ala Arg Arg Thr Gln Val Pro Ala Ser His Leu Val
260 265 270
Val Gly Leu Gln Cys Gly Gly Ser Asp Gly Tyr Ser Gly Ile Ser Ala
275 280 285
Asn Pro Val Leu Gly Ala Ala Val Asp Met Leu Val Arg His Gly Gly
290 295 300
Thr Ala Ile Leu Ser Glu Thr Pro Glu Ile Tyr Gly Ala Glu His Leu
305 310 315 320
Leu Thr Ala Arg Ala Ala Ser Ala Glu Val Ala Glu Arg Leu Met Ala
325 330 335
Arg Leu Ala Trp Trp Glu Ala Tyr Thr Gln Arg Asn Gly Ala Asn Met
340 345 350
Asn Asn Asn Pro Ser Pro Gly Asn Lys Ala Gly Gly Ile Thr Thr Ile
355 360 365
Leu Glu Lys Ser Leu Gly Ala Val Ala Lys Gly Gly Gly Ser Gly Leu
370 375 380
Met Asp Val Val Glu Tyr Ala Gln Pro Val His Ser Arg Gly Leu Val
385 390 395 400
Phe Met Asp Thr Pro Gly Tyr Asp Pro Val Ser Ala Thr Gly Gln Val
405 410 415
Ala Gly Gly Ala Asn Leu Ile Cys Phe Thr Thr Gly Arg Gly Ser Thr
420 425 430
Tyr Gly Cys Lys Pro Thr Pro Ser Leu Lys Leu Ala Thr Asn Thr Ala
435 440 445
Leu Phe Arg Asn Met Gly Met Asp Met Asp Phe Asn Ala Gly Asp Val
450 455 460
Val Asp Gly Thr Leu Gly Ile Ala Gln Ala Gly Arg His Leu Phe Glu
465 470 475 480
Leu Met Leu Ala Thr Ala Ser Gly Asp Arg Thr Leu Ser Glu Thr His
485 490 495
Gly Leu Gly Asp Asn Glu Phe Val Pro Trp Gln Leu Gly Ala Val Met
500 505 510
<210> 152
<211> 1536
<212> DNA
<213> 赫氏草螺菌
<400> 152
atgaaagcca actctcccgt catccgcctg aacccggtcg atgatgtcgt catcgcccgc 60
cagcagctca tctccggcac cgtgctgcag gacgaaggcg gcctcaaagt ccagggcctc 120
attcccgccg gccacaagat ggcgacccgc gccatctctg cgggcgagcc ggtcaagcgc 180
tatggccaga tcatcggcac ggccagccag gacatcgcgc ccggacagca cgtgcacacg 240
cacaacctgg ccatggccga gttctcgcgt gaacaccatt tcggcgctga cgtgaagccg 300
gtcgatttcg tcgccgagcc cgcaaccttc atgggcatcg tgcgtcccga tggccgcgtg 360
gccacgcgca actacatcgg cgtactgacc tcggtgaact gctcggccac cgcggcacgc 420
gccatcgccg attacttccg ccgcgacatc catcccgaag tgctggccga ctatcccaac 480
atcgatggcg tggtggccct gacccatggc cagggttgcg ccacggattc gcagggcgag 540
ccgctgcaga tcctgcgccg cacgctggcg ggttacgcca cgcatcccaa cttcgctgcg 600
gtgctggtgg tgggcctggg ctgcgagacc aaccagatct cgggcctgat ggaaagccac 660
aacctgaagg aaggcgaata cttccacacc ttcaccatcc agggcaccgg cggcaccgcc 720
aagaccgtgg cgctggggat cgagaagatc aagaagatgc tgcccaaggc caatgacatc 780
aagcgcgagc cggtgtcggc caagcatctg acgctgggcc tgcagtgcgg tggttcggat 840
ggctattcgg gcatcacggc caatccggcg ctgggtgcgg cagtggactt gctggtgcgg 900
catggcggca cggccatcct ttccgaaacg ccggaaatct atggggccga acacctgctc 960
acccgccgcg cggtctcgcc ggaagtcggt gagaagctgc tggcccgcat cgcctggtgg 1020
gaggagtatt gcgccaagaa cgatgccgag atgaacaaca atccctcagc gggcaacaag 1080
gccggtggct tgaccaccat cctcgagaag tcgctgggcg cggttgccaa gggtggcact 1140
accaatctgg tggatgtcta caagtatgcg gagacggtca ccgcacgcgg cttcgtcttc 1200
atggatacgc cgggctacga cccgatctcc gccaccggcc aggtcgccgg aggggccaac 1260
atgatctgct tcaccacggg ccggggttct gcctatggct gcgcaccggc accgtcgctc 1320
aagctggcca ccaatactgc gctgtggcaa cgtcaggaag aagacatgga tatcaactgc 1380
ggcgagattg ccgacagcaa tgtcaccccg caggaggtcg gcgaacgctt cttccagatg 1440
atcctcgata cggcatcggg caagaagacc aagagcgagc tgcatggcta cgggcaagac 1500
gaattcgtgc cctggcacat cggcgtctac acctga 1536
<210> 153
<211> 511
<212> PRT
<213> 赫氏草螺菌
<400> 153
Met Lys Ala Asn Ser Pro Val Ile Arg Leu Asn Pro Val Asp Asp Val
1 5 10 15
Val Ile Ala Arg Gln Gln Leu Ile Ser Gly Thr Val Leu Gln Asp Glu
20 25 30
Gly Gly Leu Lys Val Gln Gly Leu Ile Pro Ala Gly His Lys Met Ala
35 40 45
Thr Arg Ala Ile Ser Ala Gly Glu Pro Val Lys Arg Tyr Gly Gln Ile
50 55 60
Ile Gly Thr Ala Ser Gln Asp Ile Ala Pro Gly Gln His Val His Thr
65 70 75 80
His Asn Leu Ala Met Ala Glu Phe Ser Arg Glu His His Phe Gly Ala
85 90 95
Asp Val Lys Pro Val Asp Phe Val Ala Glu Pro Ala Thr Phe Met Gly
100 105 110
Ile Val Arg Pro Asp Gly Arg Val Ala Thr Arg Asn Tyr Ile Gly Val
115 120 125
Leu Thr Ser Val Asn Cys Ser Ala Thr Ala Ala Arg Ala Ile Ala Asp
130 135 140
Tyr Phe Arg Arg Asp Ile His Pro Glu Val Leu Ala Asp Tyr Pro Asn
145 150 155 160
Ile Asp Gly Val Val Ala Leu Thr His Gly Gln Gly Cys Ala Thr Asp
165 170 175
Ser Gln Gly Glu Pro Leu Gln Ile Leu Arg Arg Thr Leu Ala Gly Tyr
180 185 190
Ala Thr His Pro Asn Phe Ala Ala Val Leu Val Val Gly Leu Gly Cys
195 200 205
Glu Thr Asn Gln Ile Ser Gly Leu Met Glu Ser His Asn Leu Lys Glu
210 215 220
Gly Glu Tyr Phe His Thr Phe Thr Ile Gln Gly Thr Gly Gly Thr Ala
225 230 235 240
Lys Thr Val Ala Leu Gly Ile Glu Lys Ile Lys Lys Met Leu Pro Lys
245 250 255
Ala Asn Asp Ile Lys Arg Glu Pro Val Ser Ala Lys His Leu Thr Leu
260 265 270
Gly Leu Gln Cys Gly Gly Ser Asp Gly Tyr Ser Gly Ile Thr Ala Asn
275 280 285
Pro Ala Leu Gly Ala Ala Val Asp Leu Leu Val Arg His Gly Gly Thr
290 295 300
Ala Ile Leu Ser Glu Thr Pro Glu Ile Tyr Gly Ala Glu His Leu Leu
305 310 315 320
Thr Arg Arg Ala Val Ser Pro Glu Val Gly Glu Lys Leu Leu Ala Arg
325 330 335
Ile Ala Trp Trp Glu Glu Tyr Cys Ala Lys Asn Asp Ala Glu Met Asn
340 345 350
Asn Asn Pro Ser Ala Gly Asn Lys Ala Gly Gly Leu Thr Thr Ile Leu
355 360 365
Glu Lys Ser Leu Gly Ala Val Ala Lys Gly Gly Thr Thr Asn Leu Val
370 375 380
Asp Val Tyr Lys Tyr Ala Glu Thr Val Thr Ala Arg Gly Phe Val Phe
385 390 395 400
Met Asp Thr Pro Gly Tyr Asp Pro Ile Ser Ala Thr Gly Gln Val Ala
405 410 415
Gly Gly Ala Asn Met Ile Cys Phe Thr Thr Gly Arg Gly Ser Ala Tyr
420 425 430
Gly Cys Ala Pro Ala Pro Ser Leu Lys Leu Ala Thr Asn Thr Ala Leu
435 440 445
Trp Gln Arg Gln Glu Glu Asp Met Asp Ile Asn Cys Gly Glu Ile Ala
450 455 460
Asp Ser Asn Val Thr Pro Gln Glu Val Gly Glu Arg Phe Phe Gln Met
465 470 475 480
Ile Leu Asp Thr Ala Ser Gly Lys Lys Thr Lys Ser Glu Leu His Gly
485 490 495
Tyr Gly Gln Asp Glu Phe Val Pro Trp His Ile Gly Val Tyr Thr
500 505 510
<210> 154
<211> 1536
<212> DNA
<213> 人工序列
<220>
<223> Hh.araD突变体的序列
<400> 154
atgaaagcca actctcccgt catccgcctg aacccggtcg atgatgtcgt catcgcccgc 60
cagcagctca tctccggcac cgtgctgcag gacgaaggcg gcctcaaagt ccagggcctc 120
attcccgccg gccacaagat ggcgacccgc gccatctctg cgggcgagcc ggtcaagcgc 180
tatggccaga tcatcggcac ggccagccag gacatcgcgc ccggacagca cgtgcacacg 240
cacaacctgg ccatggccga gttctcgcgt gaacaccatt tcggcgctga cgtgaagccg 300
gtcgatttcg tcgccgagcc cgcaaccttc atgggcatcg tgcgtcccga tggccgcgtg 360
gccacgcgca actacatcgg cgtactgacc tcggtgaact gctcggccac cgcggcacgc 420
gccatcgccg attacttccg ccgcgacatc catcccgaag tgctggccga ctatcccaac 480
atcgatggcg tggtggccct gacccatggc cagggttgcg ccacggattc gcagggcgag 540
ccgctgcaga tcctgcgccg cacgctggcg ggttacgcca cgcatcccaa cttcgctgcg 600
gtgctggtgg tgggcctggg ctgcgagacc aaccagatct cgggcctgat ggaaagccac 660
aacctgaagg aaggcgaata cttccacacc ttcaccatcc agggcaccgg cggcaccgcc 720
aagaccgtgg cgctggggat cgagaagatc aagaagatgc tgcccaaggc caatgacatc 780
aagcgcgagc cggtgtcggc caagcatctg acgctgggcc tgcagtgcgg tggttcggat 840
ggctattcgg gcatcacggc caatccggcg ctgggtgcgg cagtggactt gctggtgcgg 900
catggcggca cggccatcct ttccgaaacg ccggaaatct atggggccga acacctgctc 960
acccgccgcg cggtctcgcc ggaagtcggt gagaagctgc tggcccgcat cgcctggtgg 1020
gaggagtatt gcgccaagaa cgatgccgag atgaacaaca atccctcagc gggcaacaag 1080
gccggtggct tgaccaccat cctcgagaag tcgctgggcg cggttgccaa gggtggcact 1140
accaatctgg tggatgtcta caagtatgcg gagacggtca ccgcacgcgg cttcgtcttc 1200
atggatacgc cgggctacga cccgatctcc gccaccggcc aggtcgccgg aggggccaac 1260
atgatctgct tcaccacggg ccggggttct gcctatggca gcgcaccggc accgtcgctc 1320
aagctggcca ccaatactgc gctgtggcaa cgtcaggaag aagacatgga tatcaactgc 1380
ggcgagattg ccgacagcaa tgtcaccccg caggaggtcg gcgaacgctt cttccagatg 1440
atcctcgata cggcatcggg caagaagacc aagagcgagc tgcatggcta cgggcaagac 1500
gaattcgtgc cctggcacat cggcgtctac acctga 1536
<210> 155
<211> 511
<212> PRT
<213> 人工序列
<220>
<223> 位置434处半胱氨酸突变为丝氨酸的Hh.araD
突变体
<400> 155
Met Lys Ala Asn Ser Pro Val Ile Arg Leu Asn Pro Val Asp Asp Val
1 5 10 15
Val Ile Ala Arg Gln Gln Leu Ile Ser Gly Thr Val Leu Gln Asp Glu
20 25 30
Gly Gly Leu Lys Val Gln Gly Leu Ile Pro Ala Gly His Lys Met Ala
35 40 45
Thr Arg Ala Ile Ser Ala Gly Glu Pro Val Lys Arg Tyr Gly Gln Ile
50 55 60
Ile Gly Thr Ala Ser Gln Asp Ile Ala Pro Gly Gln His Val His Thr
65 70 75 80
His Asn Leu Ala Met Ala Glu Phe Ser Arg Glu His His Phe Gly Ala
85 90 95
Asp Val Lys Pro Val Asp Phe Val Ala Glu Pro Ala Thr Phe Met Gly
100 105 110
Ile Val Arg Pro Asp Gly Arg Val Ala Thr Arg Asn Tyr Ile Gly Val
115 120 125
Leu Thr Ser Val Asn Cys Ser Ala Thr Ala Ala Arg Ala Ile Ala Asp
130 135 140
Tyr Phe Arg Arg Asp Ile His Pro Glu Val Leu Ala Asp Tyr Pro Asn
145 150 155 160
Ile Asp Gly Val Val Ala Leu Thr His Gly Gln Gly Cys Ala Thr Asp
165 170 175
Ser Gln Gly Glu Pro Leu Gln Ile Leu Arg Arg Thr Leu Ala Gly Tyr
180 185 190
Ala Thr His Pro Asn Phe Ala Ala Val Leu Val Val Gly Leu Gly Cys
195 200 205
Glu Thr Asn Gln Ile Ser Gly Leu Met Glu Ser His Asn Leu Lys Glu
210 215 220
Gly Glu Tyr Phe His Thr Phe Thr Ile Gln Gly Thr Gly Gly Thr Ala
225 230 235 240
Lys Thr Val Ala Leu Gly Ile Glu Lys Ile Lys Lys Met Leu Pro Lys
245 250 255
Ala Asn Asp Ile Lys Arg Glu Pro Val Ser Ala Lys His Leu Thr Leu
260 265 270
Gly Leu Gln Cys Gly Gly Ser Asp Gly Tyr Ser Gly Ile Thr Ala Asn
275 280 285
Pro Ala Leu Gly Ala Ala Val Asp Leu Leu Val Arg His Gly Gly Thr
290 295 300
Ala Ile Leu Ser Glu Thr Pro Glu Ile Tyr Gly Ala Glu His Leu Leu
305 310 315 320
Thr Arg Arg Ala Val Ser Pro Glu Val Gly Glu Lys Leu Leu Ala Arg
325 330 335
Ile Ala Trp Trp Glu Glu Tyr Cys Ala Lys Asn Asp Ala Glu Met Asn
340 345 350
Asn Asn Pro Ser Ala Gly Asn Lys Ala Gly Gly Leu Thr Thr Ile Leu
355 360 365
Glu Lys Ser Leu Gly Ala Val Ala Lys Gly Gly Thr Thr Asn Leu Val
370 375 380
Asp Val Tyr Lys Tyr Ala Glu Thr Val Thr Ala Arg Gly Phe Val Phe
385 390 395 400
Met Asp Thr Pro Gly Tyr Asp Pro Ile Ser Ala Thr Gly Gln Val Ala
405 410 415
Gly Gly Ala Asn Met Ile Cys Phe Thr Thr Gly Arg Gly Ser Ala Tyr
420 425 430
Gly Ser Ala Pro Ala Pro Ser Leu Lys Leu Ala Thr Asn Thr Ala Leu
435 440 445
Trp Gln Arg Gln Glu Glu Asp Met Asp Ile Asn Cys Gly Glu Ile Ala
450 455 460
Asp Ser Asn Val Thr Pro Gln Glu Val Gly Glu Arg Phe Phe Gln Met
465 470 475 480
Ile Leu Asp Thr Ala Ser Gly Lys Lys Thr Lys Ser Glu Leu His Gly
485 490 495
Tyr Gly Gln Asp Glu Phe Val Pro Trp His Ile Gly Val Tyr Thr
500 505 510
<210> 156
<211> 1479
<212> DNA
<213> 丙酮丁醇梭菌
<400> 156
atgaaaaatg ttataaagat aaatgaaaaa gataatgttg tagttgcttt aaatgattta 60
aacaaaggcg atgtaattga aatagatgga aaagtaatta cagcagaaga gcctgtaaaa 120
aaaggtcata aaattgcaat tacagatata caaaagaact ctaatatata taagtatgga 180
tttcctattg gtcatgcgtt agaagaaata aaaaaaggtc aatgggtaca tactcataat 240
ataaaaacca atttggatgg aataaaggat tatgagtaca ataaacaaac ttttgagaat 300
ccatttaaaa atgaaaattt aacttttaag ggttatagaa gagaagacgg aacagtagga 360
ataagaaatg aattatggat agttccaact gttggttgtg tgaatggtac agctgattta 420
atagctgaaa gatttaaaag tgaaactgag tttaaagatg tacatgtatt taagcataat 480
tttggatgtt cacagcttgg tgatgatcac aataacacaa gaactatatt aggtaacatt 540
gtaaagcatc ctaatgcagg aggagtatta gttcttggac ttggttgcga aaataatact 600
atggagagtt tcaaggaatc acttcatagc tacaataaag aaagagtaag atttttaata 660
gctcaagatg tagaagatga aatatcttcc ggatgtgagt tgttaaaaga attatatgaa 720
aagatacaaa aggatgaaag agaagaggta tctatttcag aacttaaaat aggacttaaa 780
tgtggagcat cagatggttt ttctggaata actgcaaatc cacttttagg aaagttatca 840
gactttttaa tagcacaggg tggaacaact atacttacag aagttcctga aatgtttgga 900
gcagaaacaa tactcatgaa tagggcaaaa gatgagaaag tatttgcaaa aacggtaaac 960
ttgattaatg attttaaaaa atatttcatg agctataatc aacctgtata tgaaaatcct 1020
tctccaggaa acaaagcagg tggaataact acattagaag ataaatcatt aggatgtaca 1080
caaaaatcag gaagctctga agtagttgga gtacttaagt acggagaaac tttagaaaat 1140
aaagggttaa atcttttaag tgcgccaggt aatgatttag tggcatctac tgctcttgca 1200
tcggcaggat gtcatatggt gttgtttaca actggtagag gtacaccatt tggaactttt 1260
gtgccaactg ttaagatttc aactaactca gatatatata ataagaagaa aaattggata 1320
gattttaatg cgggtgcact tttagaaaat caatctatgg atcaagtgct taaagaattt 1380
attaattatt tactcggagt tgcaaacgga aatatggcaa acaatgagaa aaataacata 1440
agagaaatat ctatatttaa aaacggtgta acactataa 1479
<210> 157
<211> 492
<212> PRT
<213> 丙酮丁醇梭菌
<400> 157
Met Lys Asn Val Ile Lys Ile Asn Glu Lys Asp Asn Val Val Val Ala
1 5 10 15
Leu Asn Asp Leu Asn Lys Gly Asp Val Ile Glu Ile Asp Gly Lys Val
20 25 30
Ile Thr Ala Glu Glu Pro Val Lys Lys Gly His Lys Ile Ala Ile Thr
35 40 45
Asp Ile Gln Lys Asn Ser Asn Ile Tyr Lys Tyr Gly Phe Pro Ile Gly
50 55 60
His Ala Leu Glu Glu Ile Lys Lys Gly Gln Trp Val His Thr His Asn
65 70 75 80
Ile Lys Thr Asn Leu Asp Gly Ile Lys Asp Tyr Glu Tyr Asn Lys Gln
85 90 95
Thr Phe Glu Asn Pro Phe Lys Asn Glu Asn Leu Thr Phe Lys Gly Tyr
100 105 110
Arg Arg Glu Asp Gly Thr Val Gly Ile Arg Asn Glu Leu Trp Ile Val
115 120 125
Pro Thr Val Gly Cys Val Asn Gly Thr Ala Asp Leu Ile Ala Glu Arg
130 135 140
Phe Lys Ser Glu Thr Glu Phe Lys Asp Val His Val Phe Lys His Asn
145 150 155 160
Phe Gly Cys Ser Gln Leu Gly Asp Asp His Asn Asn Thr Arg Thr Ile
165 170 175
Leu Gly Asn Ile Val Lys His Pro Asn Ala Gly Gly Val Leu Val Leu
180 185 190
Gly Leu Gly Cys Glu Asn Asn Thr Met Glu Ser Phe Lys Glu Ser Leu
195 200 205
His Ser Tyr Asn Lys Glu Arg Val Arg Phe Leu Ile Ala Gln Asp Val
210 215 220
Glu Asp Glu Ile Ser Ser Gly Cys Glu Leu Leu Lys Glu Leu Tyr Glu
225 230 235 240
Lys Ile Gln Lys Asp Glu Arg Glu Glu Val Ser Ile Ser Glu Leu Lys
245 250 255
Ile Gly Leu Lys Cys Gly Ala Ser Asp Gly Phe Ser Gly Ile Thr Ala
260 265 270
Asn Pro Leu Leu Gly Lys Leu Ser Asp Phe Leu Ile Ala Gln Gly Gly
275 280 285
Thr Thr Ile Leu Thr Glu Val Pro Glu Met Phe Gly Ala Glu Thr Ile
290 295 300
Leu Met Asn Arg Ala Lys Asp Glu Lys Val Phe Ala Lys Thr Val Asn
305 310 315 320
Leu Ile Asn Asp Phe Lys Lys Tyr Phe Met Ser Tyr Asn Gln Pro Val
325 330 335
Tyr Glu Asn Pro Ser Pro Gly Asn Lys Ala Gly Gly Ile Thr Thr Leu
340 345 350
Glu Asp Lys Ser Leu Gly Cys Thr Gln Lys Ser Gly Ser Ser Glu Val
355 360 365
Val Gly Val Leu Lys Tyr Gly Glu Thr Leu Glu Asn Lys Gly Leu Asn
370 375 380
Leu Leu Ser Ala Pro Gly Asn Asp Leu Val Ala Ser Thr Ala Leu Ala
385 390 395 400
Ser Ala Gly Cys His Met Val Leu Phe Thr Thr Gly Arg Gly Thr Pro
405 410 415
Phe Gly Thr Phe Val Pro Thr Val Lys Ile Ser Thr Asn Ser Asp Ile
420 425 430
Tyr Asn Lys Lys Lys Asn Trp Ile Asp Phe Asn Ala Gly Ala Leu Leu
435 440 445
Glu Asn Gln Ser Met Asp Gln Val Leu Lys Glu Phe Ile Asn Tyr Leu
450 455 460
Leu Gly Val Ala Asn Gly Asn Met Ala Asn Asn Glu Lys Asn Asn Ile
465 470 475 480
Arg Glu Ile Ser Ile Phe Lys Asn Gly Val Thr Leu
485 490
<210> 158
<211> 1530
<212> DNA
<213> 含羞草副伯克霍尔德菌
<400> 158
atgaagacct caacagcaga cgcagatgtc accgtgcgac tgcatcccga tgacgatgtg 60
atcatcgcgc tgcagcagct tgtgggtggc acaacggtgg acggtggccg cgtaaccgtc 120
cgggggctgg tggcccctgg tcacaaggtc gcctcacgtg ccatccagca aggcgagccg 180
gttcgacggt acgggcagat catcggcttt gcctcgtgcg acatcggctg cggcgaccac 240
gtccacacgc ataacctgtc gatgggcagc ttcgaccgcg actatgcgtt tggaaaggac 300
gcaaagccag cgcccgtcgc gagccgtgcg agcttcatgg gtatcgtccg tcctgatgga 360
cgcgtcgcga cgcgcaatta catcggcatt cttacgtcag tgaactgctc tgcgaccgtc 420
gcacgtgcca ttgcagacca cttcaaacct gaagtgctac gcgactatcc aaacgtggac 480
ggggtcgtcg cactcacgca tggccagggt tgcgcgatcg actcacaagg agaaggcctg 540
gcactgatcc gtcgaacgct cggtgggtac gcacgacatc ctaatttcgc ggcggtgctc 600
atcgtcggac tcgggtgcga gaccaaccag attgggggcc tgctcgaaag tcagggtctt 660
gaggcgggca gcacgctgaa ggtcatgacc attcaggcta ccggtggcac gtcgcgcaca 720
gtggcggctg gcatcgacgc tgtgcaggcg ctgctgccag aggccaatcg cgtgaagcgg 780
gaggcagtcg acgcaagtaa tctcgtggtt ggtttgcagt gcggcgggtc cgacggctat 840
tcgggtatct cggcgaatcc ggcgcttggt gctgcggtcg accgtcttgt tgcccacggc 900
ggcgttgcca ttctgtccga gacaccggaa atatacggcg cggagcacct gctcacccgc 960
cgcgccattt cacgcgcggt cggagaaaag ctgatcgagc gcattcactg gtgggaaact 1020
tattgtgcac gcatgggcgc agacctgaat aacaacccgt cggcgggtaa taaggcaggt 1080
ggcctgacga caattctcga gaagtcgttg ggcgctgtgg ccaaaggcgg cacgacgaac 1140
ctgattgctg tttatcgcta tgcggagccg gtatcggcac acggtctggt tttcatggac 1200
accccgggct atgaccccgt ctcggcaacc ggccaggtag caggcggagc caacctgatc 1260
tgctttacaa cggggcgcgg ctctgcatac ggctgcgcgc cttcgccctc gctgaaactg 1320
ggcaccaaca gcgaactatg gcgcagacag gaagaagata tcgacctgaa ctgcggagag 1380
atcctcgacg gaacgaggag cgtcgacgaa atgggccagg agattttcga gctcatgctg 1440
gcaaccgcat cgggtatgcg atcaaagagt gagcttcacg gctacggaca aaatgagttc 1500
gtaccctggc agatcggcgc gatcacctga 1530
<210> 159
<211> 509
<212> PRT
<213> 含羞草副伯克霍尔德菌
<400> 159
Met Lys Thr Ser Thr Ala Asp Ala Asp Val Thr Val Arg Leu His Pro
1 5 10 15
Asp Asp Asp Val Ile Ile Ala Leu Gln Gln Leu Val Gly Gly Thr Thr
20 25 30
Val Asp Gly Gly Arg Val Thr Val Arg Gly Leu Val Ala Pro Gly His
35 40 45
Lys Val Ala Ser Arg Ala Ile Gln Gln Gly Glu Pro Val Arg Arg Tyr
50 55 60
Gly Gln Ile Ile Gly Phe Ala Ser Cys Asp Ile Gly Cys Gly Asp His
65 70 75 80
Val His Thr His Asn Leu Ser Met Gly Ser Phe Asp Arg Asp Tyr Ala
85 90 95
Phe Gly Lys Asp Ala Lys Pro Ala Pro Val Ala Ser Arg Ala Ser Phe
100 105 110
Met Gly Ile Val Arg Pro Asp Gly Arg Val Ala Thr Arg Asn Tyr Ile
115 120 125
Gly Ile Leu Thr Ser Val Asn Cys Ser Ala Thr Val Ala Arg Ala Ile
130 135 140
Ala Asp His Phe Lys Pro Glu Val Leu Arg Asp Tyr Pro Asn Val Asp
145 150 155 160
Gly Val Val Ala Leu Thr His Gly Gln Gly Cys Ala Ile Asp Ser Gln
165 170 175
Gly Glu Gly Leu Ala Leu Ile Arg Arg Thr Leu Gly Gly Tyr Ala Arg
180 185 190
His Pro Asn Phe Ala Ala Val Leu Ile Val Gly Leu Gly Cys Glu Thr
195 200 205
Asn Gln Ile Gly Gly Leu Leu Glu Ser Gln Gly Leu Glu Ala Gly Ser
210 215 220
Thr Leu Lys Val Met Thr Ile Gln Ala Thr Gly Gly Thr Ser Arg Thr
225 230 235 240
Val Ala Ala Gly Ile Asp Ala Val Gln Ala Leu Leu Pro Glu Ala Asn
245 250 255
Arg Val Lys Arg Glu Ala Val Asp Ala Ser Asn Leu Val Val Gly Leu
260 265 270
Gln Cys Gly Gly Ser Asp Gly Tyr Ser Gly Ile Ser Ala Asn Pro Ala
275 280 285
Leu Gly Ala Ala Val Asp Arg Leu Val Ala His Gly Gly Val Ala Ile
290 295 300
Leu Ser Glu Thr Pro Glu Ile Tyr Gly Ala Glu His Leu Leu Thr Arg
305 310 315 320
Arg Ala Ile Ser Arg Ala Val Gly Glu Lys Leu Ile Glu Arg Ile His
325 330 335
Trp Trp Glu Thr Tyr Cys Ala Arg Met Gly Ala Asp Leu Asn Asn Asn
340 345 350
Pro Ser Ala Gly Asn Lys Ala Gly Gly Leu Thr Thr Ile Leu Glu Lys
355 360 365
Ser Leu Gly Ala Val Ala Lys Gly Gly Thr Thr Asn Leu Ile Ala Val
370 375 380
Tyr Arg Tyr Ala Glu Pro Val Ser Ala His Gly Leu Val Phe Met Asp
385 390 395 400
Thr Pro Gly Tyr Asp Pro Val Ser Ala Thr Gly Gln Val Ala Gly Gly
405 410 415
Ala Asn Leu Ile Cys Phe Thr Thr Gly Arg Gly Ser Ala Tyr Gly Cys
420 425 430
Ala Pro Ser Pro Ser Leu Lys Leu Gly Thr Asn Ser Glu Leu Trp Arg
435 440 445
Arg Gln Glu Glu Asp Ile Asp Leu Asn Cys Gly Glu Ile Leu Asp Gly
450 455 460
Thr Arg Ser Val Asp Glu Met Gly Gln Glu Ile Phe Glu Leu Met Leu
465 470 475 480
Ala Thr Ala Ser Gly Met Arg Ser Lys Ser Glu Leu His Gly Tyr Gly
485 490 495
Gln Asn Glu Phe Val Pro Trp Gln Ile Gly Ala Ile Thr
500 505
<210> 160
<211> 1488
<212> DNA
<213> 大肠杆菌
<400> 160
atgcaataca tcaagatcca tgcgctggat aacgtcgcgg tcgctttagc agatttggct 60
gaaggcacag aagtcagtgt cgataaccag actgttacgc tgcgccagga tgttgctcgt 120
ggacataaat ttgcgttaac ggatatcgca aaaggggcca atgtcatcaa atatggcctg 180
ccgattggtt atgcattggc ggatattgcg gcaggtgttc acgttcacgc ccacaatacg 240
cgcacgaatc tgagcgatct ggatcagtat cgctatcaac ctgattttca ggatctgcct 300
gcgcaagcgg cagatcgtga agtgcagatc tatcgtcgcg ctaacggcga tgtcggggtg 360
cgtaatgagc tgtggatcct gccaaccgtg ggctgtgtca acggcatcgc gcggcagatc 420
cagaaccgtt ttctgaaaga gaccaacaac gccgaaggta ctgacggcgt gttcctcttc 480
agccacacct acggctgctc acagctgggc gacgatcaca ttaatacccg caccatgctg 540
caaaacatgg tgcgccaccc gaacgcaggc gcagtgctgg tgattggtct gggctgtgaa 600
aacaaccagg ttgccgcatt ccgtgaaacg ctgggcgata tcgatcctga acgcgttcat 660
ttcatgatct gccaacagca ggatgatgag atcgaagccg gaatcgagca tttgcatcag 720
ctgtataacg tgatgcgcaa cgataaacgc gagccaggca aactcagcga actgaagttt 780
ggtctggagt gcggtggttc tgacggtctt tctggtatta ctgctaaccc gatgctgggg 840
cgtttctctg actacgtgat tgctaacggc ggtactaccg tactgaccga agtgccggag 900
atgtttggcg cagagcagtt gctgatggac cattgccgcg acgaagcaac gtttgaaaaa 960
ctggtcacca tggtcaacga cttcaaacag tactttattg cccatgatca gccgatctat 1020
gaaaacccat cgccggggaa caaagcgggc ggtatcacca cgctggaaga caaatcactt 1080
ggctgtaccc agaaagcggg ttccagcgtc gtggttgacg tgctgcgtta cggcgagcgt 1140
ctgaaaacgc cagggctgaa cttgttaagt gcgccgggta acgatgccgt agcgaccagc 1200
gccctggcgg gtgcgggctg ccatatggtg ctgttcagta ctggtcgtgg cacgccgtat 1260
ggtggatttg tgccgacggt gaaaatcgcc accaacagtg aactggcggc gaagaaaaaa 1320
cactggatcg actttgacgc gggtcagttg atccacggta aagcgatgcc gcagttgctg 1380
gaagaattta tcgacaccat cgttgagttt gccaacggta agcaaacctg taacgagcgt 1440
aacgacttcc gcgaactggc gatcttcaaa agcggcgtaa cgctataa 1488
<210> 161
<211> 495
<212> PRT
<213> 大肠杆菌
<400> 161
Met Gln Tyr Ile Lys Ile His Ala Leu Asp Asn Val Ala Val Ala Leu
1 5 10 15
Ala Asp Leu Ala Glu Gly Thr Glu Val Ser Val Asp Asn Gln Thr Val
20 25 30
Thr Leu Arg Gln Asp Val Ala Arg Gly His Lys Phe Ala Leu Thr Asp
35 40 45
Ile Ala Lys Gly Ala Asn Val Ile Lys Tyr Gly Leu Pro Ile Gly Tyr
50 55 60
Ala Leu Ala Asp Ile Ala Ala Gly Val His Val His Ala His Asn Thr
65 70 75 80
Arg Thr Asn Leu Ser Asp Leu Asp Gln Tyr Arg Tyr Gln Pro Asp Phe
85 90 95
Gln Asp Leu Pro Ala Gln Ala Ala Asp Arg Glu Val Gln Ile Tyr Arg
100 105 110
Arg Ala Asn Gly Asp Val Gly Val Arg Asn Glu Leu Trp Ile Leu Pro
115 120 125
Thr Val Gly Cys Val Asn Gly Ile Ala Arg Gln Ile Gln Asn Arg Phe
130 135 140
Leu Lys Glu Thr Asn Asn Ala Glu Gly Thr Asp Gly Val Phe Leu Phe
145 150 155 160
Ser His Thr Tyr Gly Cys Ser Gln Leu Gly Asp Asp His Ile Asn Thr
165 170 175
Arg Thr Met Leu Gln Asn Met Val Arg His Pro Asn Ala Gly Ala Val
180 185 190
Leu Val Ile Gly Leu Gly Cys Glu Asn Asn Gln Val Ala Ala Phe Arg
195 200 205
Glu Thr Leu Gly Asp Ile Asp Pro Glu Arg Val His Phe Met Ile Cys
210 215 220
Gln Gln Gln Asp Asp Glu Ile Glu Ala Gly Ile Glu His Leu His Gln
225 230 235 240
Leu Tyr Asn Val Met Arg Asn Asp Lys Arg Glu Pro Gly Lys Leu Ser
245 250 255
Glu Leu Lys Phe Gly Leu Glu Cys Gly Gly Ser Asp Gly Leu Ser Gly
260 265 270
Ile Thr Ala Asn Pro Met Leu Gly Arg Phe Ser Asp Tyr Val Ile Ala
275 280 285
Asn Gly Gly Thr Thr Val Leu Thr Glu Val Pro Glu Met Phe Gly Ala
290 295 300
Glu Gln Leu Leu Met Asp His Cys Arg Asp Glu Ala Thr Phe Glu Lys
305 310 315 320
Leu Val Thr Met Val Asn Asp Phe Lys Gln Tyr Phe Ile Ala His Asp
325 330 335
Gln Pro Ile Tyr Glu Asn Pro Ser Pro Gly Asn Lys Ala Gly Gly Ile
340 345 350
Thr Thr Leu Glu Asp Lys Ser Leu Gly Cys Thr Gln Lys Ala Gly Ser
355 360 365
Ser Val Val Val Asp Val Leu Arg Tyr Gly Glu Arg Leu Lys Thr Pro
370 375 380
Gly Leu Asn Leu Leu Ser Ala Pro Gly Asn Asp Ala Val Ala Thr Ser
385 390 395 400
Ala Leu Ala Gly Ala Gly Cys His Met Val Leu Phe Ser Thr Gly Arg
405 410 415
Gly Thr Pro Tyr Gly Gly Phe Val Pro Thr Val Lys Ile Ala Thr Asn
420 425 430
Ser Glu Leu Ala Ala Lys Lys Lys His Trp Ile Asp Phe Asp Ala Gly
435 440 445
Gln Leu Ile His Gly Lys Ala Met Pro Gln Leu Leu Glu Glu Phe Ile
450 455 460
Asp Thr Ile Val Glu Phe Ala Asn Gly Lys Gln Thr Cys Asn Glu Arg
465 470 475 480
Asn Asp Phe Arg Glu Leu Ala Ile Phe Lys Ser Gly Val Thr Leu
485 490 495
<210> 162
<211> 939
<212> DNA
<213> 人工序列
<220>
<223> Ec.Mdh突变体的序列
<400> 162
atgaaagtcg cagtcctcgg cgccgctggc ggtgtcggcc aggcgcttgc actactgtta 60
aaaacccaac tgccttcagg ttcagaactc tctctgtatg atatcgctcc agtgactccc 120
ggtgtggctg tcgatctgag ccatatccct actgctgtga aaatcaaagg tttttctggt 180
gaagatgcga ctccggcgct ggaaggcgca gatgtcgttc ttatctctgc aggcgtagcg 240
gctaaaccgg gccagtctcg ttccgacctg tttaacgtta acgccggcat cgtgaaaaac 300
ctggtacagc aagttgcgaa aacctgcccg aaagcgtgca ttggtattat cactaacccg 360
gttaacacca cagttgcaat tgctgctgaa gtgctgaaaa aagccggtgt ttatgacaaa 420
aacaaactgt tcggcgttac cacgctggat atcattcgtt ccaacacctt tgttgcggaa 480
ctgaaaggca aacagccagg cgaagttgaa gtgccggtta ttggcggcca ctctgatgtt 540
accattctgc cgctgctgtc acaggttcct ggcgttagtt ttaccgagca ggaagtggct 600
gatctgacca aacgcatcca gaacgcgggt actgaagtgg ttgaagcgaa ggccggtggc 660
gggtctgcaa ccctgtctat gggccaggca gctgcacgtt ttggtctgtc tctggttcgt 720
gcactgcagg gcgaacaagg cgttgtcgaa tgtgcctacg ttgaaggcga cggtcagtac 780
gcccgtttct tctctcaacc gctgctgctg ggtaaaaacg gcgtggaaga gcgtaaatct 840
atcggtaccc tgagcgcatt tgaacagaac gcgctggaag gtatgctgga tacgctgaag 900
aaagatatcg ccctgggcga agagttcgtt aataagtaa 939
<210> 163
<211> 312
<212> PRT
<213> 人工序列
<220>
<223> 具有突变I12V R81A M85Q D86S G179D的Ec.Mdh突变体
<400> 163
Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly Val Gly Gln Ala Leu
1 5 10 15
Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu
20 25 30
Tyr Asp Ile Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His
35 40 45
Ile Pro Thr Ala Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr
50 55 60
Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala
65 70 75 80
Ala Lys Pro Gly Gln Ser Arg Ser Asp Leu Phe Asn Val Asn Ala Gly
85 90 95
Ile Val Lys Asn Leu Val Gln Gln Val Ala Lys Thr Cys Pro Lys Ala
100 105 110
Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala
115 120 125
Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe
130 135 140
Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu
145 150 155 160
Leu Lys Gly Lys Gln Pro Gly Glu Val Glu Val Pro Val Ile Gly Gly
165 170 175
His Ser Asp Val Thr Ile Leu Pro Leu Leu Ser Gln Val Pro Gly Val
180 185 190
Ser Phe Thr Glu Gln Glu Val Ala Asp Leu Thr Lys Arg Ile Gln Asn
195 200 205
Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr
210 215 220
Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg
225 230 235 240
Ala Leu Gln Gly Glu Gln Gly Val Val Glu Cys Ala Tyr Val Glu Gly
245 250 255
Asp Gly Gln Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys
260 265 270
Asn Gly Val Glu Glu Arg Lys Ser Ile Gly Thr Leu Ser Ala Phe Glu
275 280 285
Gln Asn Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala
290 295 300
Leu Gly Glu Glu Phe Val Asn Lys
305 310
<210> 164
<211> 984
<212> DNA
<213> 钩虫贪铜菌
<400> 164
atgcagattt ctatcaaacg cgccatcgag cgcgtgcccg gcggcatgat gatcgtgccg 60
ctgctgatcg gttcgctcgt tgccacgttc gcgccggatg ggccgaagtt ttttggatcg 120
tttaccggcg cgctttttac cggtgccctg cccatcctcg cggtgttcta tgtctgcatg 180
ggggcaagca tcaacatcaa agccacgcct tacattctca agaagggcgg cgtgttgttc 240
ggcaccaagg tcggcacggc aatggtgttg ggcgtgatca tggggcactt cctgggcgag 300
gcgccgatca gtgccgggat gtttgccggc ctgtcgacgc ttgccgtggt ggccgcgatg 360
aacgacacca acggcggcct gtacatggcg ctgatgggcc agtacggcaa gcctgaagac 420
gtgggcgcct acagcatcat gtcgctcgag tccggcccgt tcctgacgat ggtgacgctg 480
ggcgtggcgg gtttgtccgc gttcccatgg ccgacgatgg tcggcagcat tcttccgctc 540
atgctcggca tgttgctggg caacctggat cgggagatgc gcgatttcct gggtaaggcc 600
gtgccggtca tgattccctt tttcgcactg gcgctgggcg caggcctgga cctgcacaag 660
gtctggcagg cgggcatgct gggcctcggc cttggtgttg ccgtggtggc cgtcaccggc 720
tgcgcgctgt acttcgctga ccgcctcacc ggcggcacgg gtgtggcagg tgtcgcagcc 780
gccagcaccg ccggcaatgc cgccgccgtg ccgacgctgg tagcggcggc taatccggtc 840
tacgcagaat cggccaagag cgccacgatc ctggtggccg cctgcgtggt ggtcaccgcc 900
gtactgacgc cattggtgac cgcctggatt gcccgccgcg tggcgggcaa ggaggccgag 960
gcgttggtga ggaccgccgc atga 984
<210> 165
<211> 327
<212> PRT
<213> 钩虫贪铜菌
<400> 165
Met Gln Ile Ser Ile Lys Arg Ala Ile Glu Arg Val Pro Gly Gly Met
1 5 10 15
Met Ile Val Pro Leu Leu Ile Gly Ser Leu Val Ala Thr Phe Ala Pro
20 25 30
Asp Gly Pro Lys Phe Phe Gly Ser Phe Thr Gly Ala Leu Phe Thr Gly
35 40 45
Ala Leu Pro Ile Leu Ala Val Phe Tyr Val Cys Met Gly Ala Ser Ile
50 55 60
Asn Ile Lys Ala Thr Pro Tyr Ile Leu Lys Lys Gly Gly Val Leu Phe
65 70 75 80
Gly Thr Lys Val Gly Thr Ala Met Val Leu Gly Val Ile Met Gly His
85 90 95
Phe Leu Gly Glu Ala Pro Ile Ser Ala Gly Met Phe Ala Gly Leu Ser
100 105 110
Thr Leu Ala Val Val Ala Ala Met Asn Asp Thr Asn Gly Gly Leu Tyr
115 120 125
Met Ala Leu Met Gly Gln Tyr Gly Lys Pro Glu Asp Val Gly Ala Tyr
130 135 140
Ser Ile Met Ser Leu Glu Ser Gly Pro Phe Leu Thr Met Val Thr Leu
145 150 155 160
Gly Val Ala Gly Leu Ser Ala Phe Pro Trp Pro Thr Met Val Gly Ser
165 170 175
Ile Leu Pro Leu Met Leu Gly Met Leu Leu Gly Asn Leu Asp Arg Glu
180 185 190
Met Arg Asp Phe Leu Gly Lys Ala Val Pro Val Met Ile Pro Phe Phe
195 200 205
Ala Leu Ala Leu Gly Ala Gly Leu Asp Leu His Lys Val Trp Gln Ala
210 215 220
Gly Met Leu Gly Leu Gly Leu Gly Val Ala Val Val Ala Val Thr Gly
225 230 235 240
Cys Ala Leu Tyr Phe Ala Asp Arg Leu Thr Gly Gly Thr Gly Val Ala
245 250 255
Gly Val Ala Ala Ala Ser Thr Ala Gly Asn Ala Ala Ala Val Pro Thr
260 265 270
Leu Val Ala Ala Ala Asn Pro Val Tyr Ala Glu Ser Ala Lys Ser Ala
275 280 285
Thr Ile Leu Val Ala Ala Cys Val Val Val Thr Ala Val Leu Thr Pro
290 295 300
Leu Val Thr Ala Trp Ile Ala Arg Arg Val Ala Gly Lys Glu Ala Glu
305 310 315 320
Ala Leu Val Arg Thr Ala Ala
325
<210> 166
<211> 1191
<212> DNA
<213> 大肠杆菌
<400> 166
atgtttgaga acattaccgc cgctcctgcc gacccgattc tgggcctggc cgatctgttt 60
cgtgccgatg aacgtcccgg caaaattaac ctcgggattg gtgtctataa agatgagacg 120
ggcaaaaccc cggtactgac cagcgtgaaa aaggctgaac agtatctgct cgaaaatgaa 180
accaccaaaa attacctcgg cattgacggc atccctgaat ttggtcgctg cactcaggaa 240
ctgctgtttg gtaaaggtag cgccctgatc aatgacaaac gtgctcgcac ggcacagact 300
ccggggggca ctggcgcact acgcgtggct gccgatttcc tggcaaaaaa taccagcgtt 360
aagcgtgtgt gggtgagcaa cccaagctgg ccgaaccata agagcgtctt taactctgca 420
ggtctggaag ttcgtgaata cgcttattat gatgcggaaa atcacactct tgacttcgat 480
gcactgatta acagcctgaa tgaagctcag gctggcgacg tagtgctgtt ccatggctgc 540
tgccataacc caaccggtat cgaccctacg ctggaacaat ggcaaacact ggcacaactc 600
tccgttgaga aaggctggtt accgctgttt gacttcgctt accagggttt tgcccgtggt 660
ctggaagaag atgctgaagg actgcgcgct ttcgcggcta tgcataaaga gctgattgtt 720
gccagttcct actctaaaaa ctttggcctg tacaacgagc gtgttggcgc ttgtactctg 780
gttgctgccg acagtgaaac cgttgatcgc gcattcagcc aaatgaaagc ggcgattcgc 840
gctaactact ctaacccacc agcacacggc gcttctgttg ttgccaccat cctgagcaac 900
gatgcgttac gtgcgatttg ggaacaagag ctgactgata tgcgccagcg tattcagcgt 960
atgcgtcagt tgttcgtcaa tacgctgcag gaaaaaggcg caaaccgcga cttcagcttt 1020
atcatcaaac agaacggcat gttctccttc agtggcctga caaaagaaca agtgctgcgt 1080
ctgcgcgaag agtttggcgt atatgcggtt gcttctggtc gcgtaaatgt ggccgggatg 1140
acaccagata acatggctcc gctgtgcgaa gcgattgtgg cagtgctgta a 1191
<210> 167
<211> 396
<212> PRT
<213> 大肠杆菌
<400> 167
Met Phe Glu Asn Ile Thr Ala Ala Pro Ala Asp Pro Ile Leu Gly Leu
1 5 10 15
Ala Asp Leu Phe Arg Ala Asp Glu Arg Pro Gly Lys Ile Asn Leu Gly
20 25 30
Ile Gly Val Tyr Lys Asp Glu Thr Gly Lys Thr Pro Val Leu Thr Ser
35 40 45
Val Lys Lys Ala Glu Gln Tyr Leu Leu Glu Asn Glu Thr Thr Lys Asn
50 55 60
Tyr Leu Gly Ile Asp Gly Ile Pro Glu Phe Gly Arg Cys Thr Gln Glu
65 70 75 80
Leu Leu Phe Gly Lys Gly Ser Ala Leu Ile Asn Asp Lys Arg Ala Arg
85 90 95
Thr Ala Gln Thr Pro Gly Gly Thr Gly Ala Leu Arg Val Ala Ala Asp
100 105 110
Phe Leu Ala Lys Asn Thr Ser Val Lys Arg Val Trp Val Ser Asn Pro
115 120 125
Ser Trp Pro Asn His Lys Ser Val Phe Asn Ser Ala Gly Leu Glu Val
130 135 140
Arg Glu Tyr Ala Tyr Tyr Asp Ala Glu Asn His Thr Leu Asp Phe Asp
145 150 155 160
Ala Leu Ile Asn Ser Leu Asn Glu Ala Gln Ala Gly Asp Val Val Leu
165 170 175
Phe His Gly Cys Cys His Asn Pro Thr Gly Ile Asp Pro Thr Leu Glu
180 185 190
Gln Trp Gln Thr Leu Ala Gln Leu Ser Val Glu Lys Gly Trp Leu Pro
195 200 205
Leu Phe Asp Phe Ala Tyr Gln Gly Phe Ala Arg Gly Leu Glu Glu Asp
210 215 220
Ala Glu Gly Leu Arg Ala Phe Ala Ala Met His Lys Glu Leu Ile Val
225 230 235 240
Ala Ser Ser Tyr Ser Lys Asn Phe Gly Leu Tyr Asn Glu Arg Val Gly
245 250 255
Ala Cys Thr Leu Val Ala Ala Asp Ser Glu Thr Val Asp Arg Ala Phe
260 265 270
Ser Gln Met Lys Ala Ala Ile Arg Ala Asn Tyr Ser Asn Pro Pro Ala
275 280 285
His Gly Ala Ser Val Val Ala Thr Ile Leu Ser Asn Asp Ala Leu Arg
290 295 300
Ala Ile Trp Glu Gln Glu Leu Thr Asp Met Arg Gln Arg Ile Gln Arg
305 310 315 320
Met Arg Gln Leu Phe Val Asn Thr Leu Gln Glu Lys Gly Ala Asn Arg
325 330 335
Asp Phe Ser Phe Ile Ile Lys Gln Asn Gly Met Phe Ser Phe Ser Gly
340 345 350
Leu Thr Lys Glu Gln Val Leu Arg Leu Arg Glu Glu Phe Gly Val Tyr
355 360 365
Ala Val Ala Ser Gly Arg Val Asn Val Ala Gly Met Thr Pro Asp Asn
370 375 380
Met Ala Pro Leu Cys Glu Ala Ile Val Ala Val Leu
385 390 395
<210> 168
<211> 7978
<212> DNA
<213> 人工序列
<220>
<223> 质粒序列
<400> 168
ggatccgttt aactttaaga aggagatata ccatggaact gtatctggat acttcagacg 60
ttgttgcggt gaaggcgctg tcacgtattt ttccgctggc gggtgtgacc actaacccaa 120
gcattatcgc cgcgggtaaa aaaccgctgg atgttgtgct tccgcaactt catgaagcga 180
tgggcggtca ggggcgtctg tttgcccagg taatggctac cactgccgaa gggatggtta 240
atgacgcgct taagctgcgt tctattattg cggatatcgt ggtgaaagtt ccggtgaccg 300
ccgaggggct ggcagctatt aagatgttaa aagcggaagg gattccgacg tatggaaccg 360
cggtatatgg cgcagcacaa gggctgctgt cggcgctggc aggtgcggaa tatgttggcc 420
cttacgttaa tcgtattgat gctcagggcg gtagcggcat tcagactgtg accgacttac 480
accagttatt gaaaatgcat gcgccgcagg cgaaagtgct ggcagcgagt ttcaaaaccc 540
cgcgtcaggc gctggactgc ttactggcag gatgtgaatc aattactctg ccactggatg 600
tggcacaaca gatgattagc tatccggcgg ttgatgccgc tgtggcgaag tttgagcagg 660
actggcaggg agcgtttggc agaacgtcga tttaaatgtt taactttaag aaggagatat 720
accatgtcta ccgatagttt acaacagttt cgtcgccgtg tggcaattcc gggtccggct 780
ggttttggtg cagcaccgct gggcaatctg tttagccgtg ttaccgatga ggccgcacaa 840
gaaactttaa gtgccgcatg gggtgctggt atccgctact ttgataccgc accgttctat 900
ggcatgggtc tgagtgaacg ccgtctgggt cgcgcactgg ccagctatcc gcgtgatgcc 960
ttcgtgatta gcaccaaagt gggtcgttta ctgatcgccg acgacagtgt gccggaaacc 1020
cagcacggtt atgttggtgg tttaccgtac cgcgttgaat acgactatac tgccgatggc 1080
gcccgccgta gcattgaaag ctctttagaa cgtttaggtt tagatcgtat tgacattgtg 1140
tatattcacg acgtggccga ggatacccat ggtccgctgt ggcgtcagca ttatcgtacc 1200
gccgctggtg gtgcaatgaa agctttaagc cgtttacgcg atgaaggtgt gattggtggc 1260
tggggtttag gcgtgaatcg tgttgaaccg tgtctgatgg ctttagccga tgccgatccg 1320
gacgtctttt taatcgccgg ccgctatact ttactggata ccacagcttt agatgcttta 1380
attccggcat gcgaagcacg cggtgcacgt ttagttgttg gtggtccgta caatagtggt 1440
ttactggctg gtggcgatac ctttgaatat gcccgcgcag atgccgcaat gttagcccgt 1500
cgcgcacgtt tattaagcca ctgcgaacgc tttggtgttg atttaaaggc cgccgcttta 1560
cagttttgta aagccccggg cgttgttgca tgcgttattg ccggtgcccg caatgcagat 1620
gaggttcgtc agaactgcgc agcaatgaca gcaccggtgc cgcgcgaatt ttggcaagct 1680
ttaaaggaag agggcttagt tccgcaaggt gcaccggttc cggcataatc tagagtttaa 1740
ctttaagaag gagatatacc atggaaccga gtcagaatcc gagcgaaaag gtggcatgga 1800
ttgaacgcgc agatcccggc ctcgacgatc tgattgctcc agatgcgcaa gtggaagtgc 1860
tggcagaagg ttttgagtgg agcgaaggtc cggtatggat tcccgaaggc ggctacttgc 1920
tgtttagcga tgttccgaag aacaccattt accgctggaa agaaggtcag gggattgaca 1980
tcttcctgaa accgtcggga tataccggct ttcgtgaacg tggtggtgaa tcgggctcaa 2040
acggcttagc cctggatcgc caaggccgcc ttctgttgtg tcagcatggt gatcgtcgtg 2100
tagctcgttg ggagaaaggt tgctttatca cactggcaga ccagtatgag ggcaaaccgc 2160
tgaattcccc gaatgacttg gtagtcaagt cgaacggcga tatctacttc acggatccac 2220
catatggcat gactcctgag gcccaacgcg atcccaatgc gctcggcttt tgcggcgttt 2280
atcgcattag tgcggatggg aagctgacgc tgttagttcg tgacatgact cgccctaacg 2340
ggatcgcctt cagcccagac gagaaaacgc tctatgttgc ccagtcagat ccgcaacgcc 2400
cgttatggat ggcgttcccg gttcgcgaag atgggacctt aggggaaggc aaagtgttct 2460
tcgatgcgaa accgtggcag caaagtggct tgccgggact cccagatggc atgaaagtgg 2520
accagaaagg caatctgttt gcgactggtc cgggtggtgt caacatcttt cgccctgatg 2580
gtacctttct gggccggatt cgggtgaaag tccctaccgc taactgtgcg tttggtgatg 2640
acggatctac gctttacatt accgcggaca tgtatctgct tcgtgtgaaa acgaaaacca 2700
aaggactggg gttctaagtc gacctgcagg catgcaagct tctgttttgg cggatgagag 2760
aagaaattcg tcgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga 2820
cggatggcct ttttgcgttt ctacaaactc ttcctgtcta gcaggtggca cttttcgggg 2880
aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 2940
catgctagaa atattttatc tgattaataa gatgatcttc ttgagatcgt tttggtctgc 3000
gcgtaatctc ttgctctgaa aacgaaaaaa ccgccttgca gggcggtttt tcgaaggttc 3060
tctgagctac caactctttg aaccgaggta actggcttgg aggagcgcag tcaccaaaac 3120
ttgtcctttc agtttagcct taaccggcgc atgacttcaa gactaactcc tctaaatcaa 3180
ttaccagtgg ctgctgccag tggtgctttt gcatgtcttt ccgggttgga ctcaagacga 3240
tagttaccgg ataaggcgca gcggtcggac tgaacggggg gttcgtgcat acagtccagc 3300
ttggagcgaa ctgcctaccc ggaactgagt gtcaggcgtg gaatgagaca aacgcggcca 3360
taacagcgga atgacaccgg taaaccgaaa ggcaggaaca ggagagcgca cgagggagcc 3420
gccaggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacca ctgatttgag 3480
cgtcagattt cgtgatgctt gtcagggggg cggagcctat ggaaaaacgg ctttgccgcg 3540
gccctctcac ttccctgtta agtatcttcc tggcatcttc caggaaatct ccgccccgtt 3600
cgtaagccat ttccgctcgc cgcagtcgaa cgaccgagcg tagcgagtca gtgagcgagg 3660
aagcggaata tatcctgtat cacatattct gctgacgcac cggtgcagcc ttttttctcc 3720
tgccacatga agcacttcac tgacaccctc atcagtgcca acatagtaag ccagtataca 3780
ctccgctagc gctgatgtcc ggcggtgctt ttgccgttac gcaccacccc gtcagtagct 3840
gaacaggagg gacagctgat agaaacagaa gccactggag cacctcaaaa acaccatcat 3900
acactaaatc agtaagttgg cagcatcacc cgacgcactt tgcgccgaat aaatacctgt 3960
gacggaagat cacttcgcag aataaataaa tcctggtgtc cctgttgata ccgggaagcc 4020
ctgggccaac ttttggcgaa aatgagacgt tgatcggcac gtaagaggtt ccaactttca 4080
ccataatgaa ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc 4140
taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg 4200
gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac 4260
cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta 4320
tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc 4380
aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca 4440
tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt 4500
tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa 4560
agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt 4620
tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata 4680
ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtctg 4740
tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca 4800
gggcggggcg taattttttt aaggcagtta ttggtgccct taaacgcctg gtgctacgcc 4860
tgaataagtg ataataagcg gatgaatggc agaaattcga aagcaaattc gacccggtcg 4920
tcggttcagg gcagggtcgt taaatagccg cttatgtcta ttgctggttt accggtttat 4980
tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc ctgaggccag tttgctcagg 5040
ctctccccgt ggaggtaata attgacgata tgatcattta ttctgcctcc cagagcctga 5100
taaaaacggt tagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca 5160
tcctgcgatg cagatccgga acataatggt gcagggcgct tgtttcggcg tgggtatggt 5220
ggcaggcccc gtggccgggg gactgttggg cgctgccggc acctgtccta cgagttgcat 5280
gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga 5340
gctaccggac agcggtgcgg actgttgtaa ctcagaataa gaaatgaggc cgctcatggc 5400
gttgactctc agtcatagta tcgtggtatc accggttggt tccactctct gttgcgggca 5460
acttcagcag cacgtagggg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa 5520
gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg 5580
ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt 5640
cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat 5700
gcgccgcgtg cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt 5760
ttgcgcattc acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc 5820
gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga 5880
cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc 5940
catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt 6000
aggctggtaa gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc 6060
ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata 6120
atggggaagg ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg 6180
gccaattcgc gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 6240
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 6300
attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt 6360
caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg 6420
aaaatcctgt ttgatggtgg ttgacggcgg gatataacat gagctgtctt cggtatcgtc 6480
gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat 6540
tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt 6600
cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc 6660
tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc 6720
cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag 6780
atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt 6840
ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat 6900
ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag 6960
attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac 7020
gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg 7080
cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg 7140
tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt 7200
tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc 7260
ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact 7320
ctcttccggg cgctatcatg ccataccgcg aaaggttttg caccattcga tggtgtcaac 7380
gtaaatgcat gccgcttcgc cttcgcgcgc gaattggccg ccatgccggc gataatggcc 7440
tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc 7500
aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc 7560
tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag 7620
acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg 7680
ttgaaggctc tcaagggcat cggcggagct tatcgactgc acggtgcacc aatgcttctg 7740
gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat 7800
tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg 7860
gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg tataatgtgt 7920
ggaattgtga gcggataaca atttcacaca ggaaacagaa ttcgagctcg gtacccgg 7978
<210> 169
<211> 8122
<212> DNA
<213> 人工序列
<220>
<223> 质粒序列
<400> 169
ggatccgttt aactttaaga aggagatata ccatggaact gtatctggat acttcagacg 60
ttgttgcggt gaaggcgctg tcacgtattt ttccgctggc gggtgtgacc actaacccaa 120
gcattatcgc cgcgggtaaa aaaccgctgg atgttgtgct tccgcaactt catgaagcga 180
tgggcggtca ggggcgtctg tttgcccagg taatggctac cactgccgaa gggatggtta 240
atgacgcgct taagctgcgt tctattattg cggatatcgt ggtgaaagtt ccggtgaccg 300
ccgaggggct ggcagctatt aagatgttaa aagcggaagg gattccgacg tatggaaccg 360
cggtatatgg cgcagcacaa gggctgctgt cggcgctggc aggtgcggaa tatgttggcc 420
cttacgttaa tcgtattgat gctcagggcg gtagcggcat tcagactgtg accgacttac 480
accagttatt gaaaatgcat gcgccgcagg cgaaagtgct ggcagcgagt ttcaaaaccc 540
cgcgtcaggc gctggactgc ttactggcag gatgtgaatc aattactctg ccactggatg 600
tggcacaaca gatgattagc tatccggcgg ttgatgccgc tgtggcgaag tttgagcagg 660
actggcaggg agcgtttggc agaacgtcga tttaaatgtt taactttaag aaggagatat 720
accatgaata cacgtcgcca attcctgtct gctgccgcgg ccggcaccac ggcgctggcc 780
gcatcgccat tgctggcaca gtcgtccgca cccggaagcg tgatgcccac gcgcggccgc 840
acggcggcct ctgccgtgca aactagcgcg ccggccaagg gcgcacgtta tcggccgacc 900
acgcggttgg gctttggcgg tgttgcaatc ggcaatgggt ttgcgccagc caccgatgcg 960
caaagcgagc agacgctggc ggcggtgtgg gcctcgggcg tgcgctattt cgatacctcg 1020
ccgtggtacg ggctgggcct gtccgaacgc cgcaccggcc atcatctgca taaccatgcc 1080
gccgatagct acgtgctctc gaccaaggtg gggcgcttgc tgaccgcgac cgacaagccg 1140
ccgaagacca tgtggcagca accgtccccg ttcgattacc gctacgacta cagcgccgcc 1200
ggcgtgcgcc gctcgatcga agacagcctg caacggctgg gcgtgtcgca gatcgacatc 1260
gtgtacatcc acgacctctc gccggagaac gaaaaagacc tcggcatgcc ctgggaacag 1320
cgctttgccg aggccgccaa gggcgcgatg ccggagctga ccaagatgcg caaggagggc 1380
ctgatcaagg cctggggctt tggcgtcaat cgcccggaac cggccttgcg tgcgattgaa 1440
gaagccgacc cggatatctt cttgctggcc tgccagtact cgttgctcga ccacgcgcag 1500
gccttgcatg acaccttccc gaagatcgcc agacacggcg cctcggtggt ggtgggcgcg 1560
ccgctgctgg ccggttatct ggccgggcgc gaccgttatc tgtacgaggg cacggtgccg 1620
gagtgggcgc cgggcaaacg ccagaaggcc ctggcaatct gcgacaaaca cggcgtggat 1680
ctgcgtacgg tgtcgctgca atttgctgcc gcaccggagg tggtgtcggc ggtgattccc 1740
ggcgcacgca cggccgaaca ggcgcaggcc aatgcggcct cgatgcgggt ggcgattcct 1800
gccgcgctct gggacgacct caagcgcgcg caggtgatcg aagcggatgc gccggtgccg 1860
gcggctggat gatctagagt ttaactttaa gaaggagata taccatggaa ccgagtcaga 1920
atccgagcga aaaggtggca tggattgaac gcgcagatcc cggcctcgac gatctgattg 1980
ctccagatgc gcaagtggaa gtgctggcag aaggttttga gtggagcgaa ggtccggtat 2040
ggattcccga aggcggctac ttgctgttta gcgatgttcc gaagaacacc atttaccgct 2100
ggaaagaagg tcaggggatt gacatcttcc tgaaaccgtc gggatatacc ggctttcgtg 2160
aacgtggtgg tgaatcgggc tcaaacggct tagccctgga tcgccaaggc cgccttctgt 2220
tgtgtcagca tggtgatcgt cgtgtagctc gttgggagaa aggttgcttt atcacactgg 2280
cagaccagta tgagggcaaa ccgctgaatt ccccgaatga cttggtagtc aagtcgaacg 2340
gcgatatcta cttcacggat ccaccatatg gcatgactcc tgaggcccaa cgcgatccca 2400
atgcgctcgg cttttgcggc gtttatcgca ttagtgcgga tgggaagctg acgctgttag 2460
ttcgtgacat gactcgccct aacgggatcg ccttcagccc agacgagaaa acgctctatg 2520
ttgcccagtc agatccgcaa cgcccgttat ggatggcgtt cccggttcgc gaagatggga 2580
ccttagggga aggcaaagtg ttcttcgatg cgaaaccgtg gcagcaaagt ggcttgccgg 2640
gactcccaga tggcatgaaa gtggaccaga aaggcaatct gtttgcgact ggtccgggtg 2700
gtgtcaacat ctttcgccct gatggtacct ttctgggccg gattcgggtg aaagtcccta 2760
ccgctaactg tgcgtttggt gatgacggat ctacgcttta cattaccgcg gacatgtatc 2820
tgcttcgtgt gaaaacgaaa accaaaggac tggggttcta agtcgacctg caggcatgca 2880
agcttctgtt ttggcggatg agagaagaaa ttcgtcgccc gccataaact gccaggcatc 2940
aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttcctg 3000
tctagcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 3060
aatacattca aatatgtatc cgctcatgct agaaatattt tatctgatta ataagatgat 3120
cttcttgaga tcgttttggt ctgcgcgtaa tctcttgctc tgaaaacgaa aaaaccgcct 3180
tgcagggcgg tttttcgaag gttctctgag ctaccaactc tttgaaccga ggtaactggc 3240
ttggaggagc gcagtcacca aaacttgtcc tttcagttta gccttaaccg gcgcatgact 3300
tcaagactaa ctcctctaaa tcaattacca gtggctgctg ccagtggtgc ttttgcatgt 3360
ctttccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc ggactgaacg 3420
gggggttcgt gcatacagtc cagcttggag cgaactgcct acccggaact gagtgtcagg 3480
cgtggaatga gacaaacgcg gccataacag cggaatgaca ccggtaaacc gaaaggcagg 3540
aacaggagag cgcacgaggg agccgccagg ggaaacgcct ggtatcttta tagtcctgtc 3600
gggtttcgcc accactgatt tgagcgtcag atttcgtgat gcttgtcagg ggggcggagc 3660
ctatggaaaa acggctttgc cgcggccctc tcacttccct gttaagtatc ttcctggcat 3720
cttccaggaa atctccgccc cgttcgtaag ccatttccgc tcgccgcagt cgaacgaccg 3780
agcgtagcga gtcagtgagc gaggaagcgg aatatatcct gtatcacata ttctgctgac 3840
gcaccggtgc agcctttttt ctcctgccac atgaagcact tcactgacac cctcatcagt 3900
gccaacatag taagccagta tacactccgc tagcgctgat gtccggcggt gcttttgccg 3960
ttacgcacca ccccgtcagt agctgaacag gagggacagc tgatagaaac agaagccact 4020
ggagcacctc aaaaacacca tcatacacta aatcagtaag ttggcagcat cacccgacgc 4080
actttgcgcc gaataaatac ctgtgacgga agatcacttc gcagaataaa taaatcctgg 4140
tgtccctgtt gataccggga agccctgggc caacttttgg cgaaaatgag acgttgatcg 4200
gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt 4260
tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata 4320
taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt 4380
tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt 4440
aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa 4500
tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt 4560
tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga 4620
ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg 4680
tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa 4740
tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc 4800
ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc 4860
gattcaggtt catcatgccg tctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt 4920
acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg 4980
cccttaaacg cctggtgcta cgcctgaata agtgataata agcggatgaa tggcagaaat 5040
tcgaaagcaa attcgacccg gtcgtcggtt cagggcaggg tcgttaaata gccgcttatg 5100
tctattgctg gtttaccggt ttattgacta ccggaagcag tgtgaccgtg tgcttctcaa 5160
atgcctgagg ccagtttgct caggctctcc ccgtggaggt aataattgac gatatgatca 5220
tttattctgc ctcccagagc ctgataaaaa cggttagcgc ttcgttaata cagatgtagg 5280
tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg 5340
cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgctgc 5400
cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt 5460
catgccccgc gcccaccgga aggagctacc ggacagcggt gcggactgtt gtaactcaga 5520
ataagaaatg aggccgctca tggcgttgac tctcagtcat agtatcgtgg tatcaccggt 5580
tggttccact ctctgttgcg ggcaacttca gcagcacgta ggggacttcc gcgtttccag 5640
actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt 5700
gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag 5760
gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg 5820
ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat 5880
ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc 5940
tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag 6000
gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg 6060
cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg 6120
atcagcggtc cagtgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt 6180
ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg 6240
ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc 6300
agcaagacgt agcccagcgc gtcggccaat tcgcgctaac ttacattaat tgcgttgcgc 6360
tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa 6420
cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga 6480
gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc 6540
cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttgacg gcgggatata 6600
acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag 6660
cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat 6720
cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc 6780
actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg 6840
ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat 6900
ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga 6960
gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt 7020
agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag 7080
cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct 7140
tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc 7200
cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa 7260
cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat 7320
cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg 7380
ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt 7440
cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt 7500
tttgcaccat tcgatggtgt caacgtaaat gcatgccgct tcgccttcgc gcgcgaattg 7560
gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg 7620
acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc 7680
gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt 7740
cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc 7800
gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggcgg agcttatcga 7860
ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg gtatggctgt 7920
gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt tctggataat 7980
gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat 8040
taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac 8100
agaattcgag ctcggtaccc gg 8122
<210> 170
<211> 9033
<212> DNA
<213> 人工序列
<220>
<223> 质粒序列
<400> 170
ggtaccgttt aactttaaga aggagatata ccatggctga tacaatgctc gccgccgtcg 60
tccgtgaatt cggcaagccg ctctccatcg agcggctacc catcccggac atcaagcccc 120
accagatcct cgtgaaggtc gatacctgtg gcgtctgcca cactgacctg cacgccgcgc 180
gcggggactg gccgtccaag cccaacccgc cgttcattcc cgggcatgaa ggcgtcggac 240
acatcgtcgc cgtcggcagt caggtcggcg atttcgtcaa gaccggcgat gtcgtgggcg 300
tgccctggct ctactccgcc tgcggtcact gcgaacactg tctgggcggc tgggaaacac 360
tctgcgaaaa gcaggacgac accggctaca ccgtcaatgg ctgcttcgcc gaatatgtcg 420
tggcagaccc gaactacgtc gcacacctgc cctcgaccat cgacccgctt caggcctcgc 480
cggtcctgtg cgcggggctg acggtctata agggcctgaa aatgacggag gcccgccccg 540
gccagtgggt cgcagtctcg ggcgtcggcg gtctcggcca gatggccgtg cagtacgccg 600
tcgccatggg catgaatgtc gtcgcggtgg acatcgatga cgaaaaactc gccacagcca 660
aaaagctcgg cgcatccctg accgtcaacg ccaaggacac ggacccggcc aggttcatcc 720
agcagcagat cggcggcgca catggcgctc tcgtcaccgc tgtcggacgg acggcgtttt 780
cgcaggccat gggctatgcc cgccgcggcg gcaccatcgt cctgaacgga ctgccgcccg 840
gcgatttccc ggtctcgatc ttcgacatgg tcatgaacgg caccaccatc cgtggctcca 900
tcgtcggaac acggctggac atgatcgagg ccatggattt cttcgcccgc ggcaaggtca 960
aatccgtcgt cacccccgga aaacttgaaa acatcaatac gatcttcgac gatctgcaga 1020
atggtcgcct cgaaggccgg acagtgctcg acttccggtc ctaaggatcc gtttaacttt 1080
aagaaggaga tataccatgg aactgtatct ggatacttca gacgttgttg cggtgaaggc 1140
gctgtcacgt atttttccgc tggcgggtgt gaccactaac ccaagcatta tcgccgcggg 1200
taaaaaaccg ctggatgttg tgcttccgca acttcatgaa gcgatgggcg gtcaggggcg 1260
tctgtttgcc caggtaatgg ctaccactgc cgaagggatg gttaatgacg cgcttaagct 1320
gcgttctatt attgcggata tcgtggtgaa agttccggtg accgccgagg ggctggcagc 1380
tattaagatg ttaaaagcgg aagggattcc gacgtatgga accgcggtat atggcgcagc 1440
acaagggctg ctgtcggcgc tggcaggtgc ggaatatgtt ggcccttacg ttaatcgtat 1500
tgatgctcag ggcggtagcg gcattcagac tgtgaccgac ttacaccagt tattgaaaat 1560
gcatgcgccg caggcgaaag tgctggcagc gagtttcaaa accccgcgtc aggcgctgga 1620
ctgcttactg gcaggatgtg aatcaattac tctgccactg gatgtggcac aacagatgat 1680
tagctatccg gcggttgatg ccgctgtggc gaagtttgag caggactggc agggagcgtt 1740
tggcagaacg tcgatttaaa tgtttaactt taagaaggag atataccatg tctaccgata 1800
gtttacaaca gtttcgtcgc cgtgtggcaa ttccgggtcc ggctggtttt ggtgcagcac 1860
cgctgggcaa tctgtttagc cgtgttaccg atgaggccgc acaagaaact ttaagtgccg 1920
catggggtgc tggtatccgc tactttgata ccgcaccgtt ctatggcatg ggtctgagtg 1980
aacgccgtct gggtcgcgca ctggccagct atccgcgtga tgccttcgtg attagcacca 2040
aagtgggtcg tttactgatc gccgacgaca gtgtgccgga aacccagcac ggttatgttg 2100
gtggtttacc gtaccgcgtt gaatacgact atactgccga tggcgcccgc cgtagcattg 2160
aaagctcttt agaacgttta ggtttagatc gtattgacat tgtgtatatt cacgacgtgg 2220
ccgaggatac ccatggtccg ctgtggcgtc agcattatcg taccgccgct ggtggtgcaa 2280
tgaaagcttt aagccgttta cgcgatgaag gtgtgattgg tggctggggt ttaggcgtga 2340
atcgtgttga accgtgtctg atggctttag ccgatgccga tccggacgtc tttttaatcg 2400
ccggccgcta tactttactg gataccacag ctttagatgc tttaattccg gcatgcgaag 2460
cacgcggtgc acgtttagtt gttggtggtc cgtacaatag tggtttactg gctggtggcg 2520
atacctttga atatgcccgc gcagatgccg caatgttagc ccgtcgcgca cgtttattaa 2580
gccactgcga acgctttggt gttgatttaa aggccgccgc tttacagttt tgtaaagccc 2640
cgggcgttgt tgcatgcgtt attgccggtg cccgcaatgc agatgaggtt cgtcagaact 2700
gcgcagcaat gacagcaccg gtgccgcgcg aattttggca agctttaaag gaagagggct 2760
tagttccgca aggtgcaccg gttccggcat aatctagagt ttaactttaa gaaggagata 2820
taccatggaa ccgagtcaga atccgagcga aaaggtggca tggattgaac gcgcagatcc 2880
cggcctcgac gatctgattg ctccagatgc gcaagtggaa gtgctggcag aaggttttga 2940
gtggagcgaa ggtccggtat ggattcccga aggcggctac ttgctgttta gcgatgttcc 3000
gaagaacacc atttaccgct ggaaagaagg tcaggggatt gacatcttcc tgaaaccgtc 3060
gggatatacc ggctttcgtg aacgtggtgg tgaatcgggc tcaaacggct tagccctgga 3120
tcgccaaggc cgccttctgt tgtgtcagca tggtgatcgt cgtgtagctc gttgggagaa 3180
aggttgcttt atcacactgg cagaccagta tgagggcaaa ccgctgaatt ccccgaatga 3240
cttggtagtc aagtcgaacg gcgatatcta cttcacggat ccaccatatg gcatgactcc 3300
tgaggcccaa cgcgatccca atgcgctcgg cttttgcggc gtttatcgca ttagtgcgga 3360
tgggaagctg acgctgttag ttcgtgacat gactcgccct aacgggatcg ccttcagccc 3420
agacgagaaa acgctctatg ttgcccagtc agatccgcaa cgcccgttat ggatggcgtt 3480
cccggttcgc gaagatggga ccttagggga aggcaaagtg ttcttcgatg cgaaaccgtg 3540
gcagcaaagt ggcttgccgg gactcccaga tggcatgaaa gtggaccaga aaggcaatct 3600
gtttgcgact ggtccgggtg gtgtcaacat ctttcgccct gatggtacct ttctgggccg 3660
gattcgggtg aaagtcccta ccgctaactg tgcgtttggt gatgacggat ctacgcttta 3720
cattaccgcg gacatgtatc tgcttcgtgt gaaaacgaaa accaaaggac tggggttcta 3780
agtcgacctg caggcatgca agcttctgtt ttggcggatg agagaagaaa ttcgtcgccc 3840
gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc 3900
gtttctacaa actcttcctg tctagcaggt ggcacttttc ggggaaatgt gcgcggaacc 3960
cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgct agaaatattt 4020
tatctgatta ataagatgat cttcttgaga tcgttttggt ctgcgcgtaa tctcttgctc 4080
tgaaaacgaa aaaaccgcct tgcagggcgg tttttcgaag gttctctgag ctaccaactc 4140
tttgaaccga ggtaactggc ttggaggagc gcagtcacca aaacttgtcc tttcagttta 4200
gccttaaccg gcgcatgact tcaagactaa ctcctctaaa tcaattacca gtggctgctg 4260
ccagtggtgc ttttgcatgt ctttccgggt tggactcaag acgatagtta ccggataagg 4320
cgcagcggtc ggactgaacg gggggttcgt gcatacagtc cagcttggag cgaactgcct 4380
acccggaact gagtgtcagg cgtggaatga gacaaacgcg gccataacag cggaatgaca 4440
ccggtaaacc gaaaggcagg aacaggagag cgcacgaggg agccgccagg ggaaacgcct 4500
ggtatcttta tagtcctgtc gggtttcgcc accactgatt tgagcgtcag atttcgtgat 4560
gcttgtcagg ggggcggagc ctatggaaaa acggctttgc cgcggccctc tcacttccct 4620
gttaagtatc ttcctggcat cttccaggaa atctccgccc cgttcgtaag ccatttccgc 4680
tcgccgcagt cgaacgaccg agcgtagcga gtcagtgagc gaggaagcgg aatatatcct 4740
gtatcacata ttctgctgac gcaccggtgc agcctttttt ctcctgccac atgaagcact 4800
tcactgacac cctcatcagt gccaacatag taagccagta tacactccgc tagcgctgat 4860
gtccggcggt gcttttgccg ttacgcacca ccccgtcagt agctgaacag gagggacagc 4920
tgatagaaac agaagccact ggagcacctc aaaaacacca tcatacacta aatcagtaag 4980
ttggcagcat cacccgacgc actttgcgcc gaataaatac ctgtgacgga agatcacttc 5040
gcagaataaa taaatcctgg tgtccctgtt gataccggga agccctgggc caacttttgg 5100
cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga 5160
tcactaccgg gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg 5220
gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat 5280
tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt 5340
acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac 5400
attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag 5460
ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg 5520
ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca catatattcg 5580
caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat 5640
atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc 5700
aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac 5760
aaggtgctga tgccgctggc gattcaggtt catcatgccg tctgtgatgg cttccatgtc 5820
ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt 5880
ttttaaggca gttattggtg cccttaaacg cctggtgcta cgcctgaata agtgataata 5940
agcggatgaa tggcagaaat tcgaaagcaa attcgacccg gtcgtcggtt cagggcaggg 6000
tcgttaaata gccgcttatg tctattgctg gtttaccggt ttattgacta ccggaagcag 6060
tgtgaccgtg tgcttctcaa atgcctgagg ccagtttgct caggctctcc ccgtggaggt 6120
aataattgac gatatgatca tttattctgc ctcccagagc ctgataaaaa cggttagcgc 6180
ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc 6240
cggaacataa tggtgcaggg cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc 6300
gggggactgt tgggcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc 6360
ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctacc ggacagcggt 6420
gcggactgtt gtaactcaga ataagaaatg aggccgctca tggcgttgac tctcagtcat 6480
agtatcgtgg tatcaccggt tggttccact ctctgttgcg ggcaacttca gcagcacgta 6540
ggggacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt 6600
gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat 6660
tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc 6720
acgatcatgc gcacccgtgg ccaggaccca acgctgcccg agatgcgccg cgtgcggctg 6780
ctggagatgg cggacgcgat ggatatgttc tgccaagggt tggtttgcgc attcacagtt 6840
ctccgcaaga attgattggc tccaattctt ggagtggtga atccgttagc gaggtgccgc 6900
cggcttccat tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc 6960
agacaaggta tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg 7020
cggcataaat cgccgtgacg atcagcggtc cagtgatcga agttaggctg gtaagagccg 7080
cgagcgatcc ttgaagctgt ccctgatggt cgtcatctac ctgcctggac agcatggcct 7140
gcaacgcggg catcccgatg ccgccggaag cgagaagaat cataatgggg aaggccatcc 7200
agcctcgcgt cgcgaacgcc agcaagacgt agcccagcgc gtcggccaat tcgcgctaac 7260
ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 7320
tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg 7380
gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga 7440
gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg 7500
gtggttgacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag 7560
atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc 7620
tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt 7680
tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga 7740
ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat 7800
gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt 7860
cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca 7920
agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc 7980
agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct 8040
ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga 8100
tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag 8160
gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga 8220
atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg 8280
ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca 8340
tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat 8400
catgccatac cgcgaaaggt tttgcaccat tcgatggtgt caacgtaaat gcatgccgct 8460
tcgccttcgc gcgcgaattg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac 8520
gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg 8580
caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc 8640
agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg 8700
cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg 8760
gcatcggcgg agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc 8820
ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc 8880
gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc 8940
tgaaatgagc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat 9000
aacaatttca cacaggaaac agaattcgag ctc 9033
<210> 171
<211> 8592
<212> DNA
<213> 人工序列
<220>
<223> 质粒序列
<400> 171
gagctcgttt aactttaaga aggagatata ccatgaaagt cgcagtcctc ggcgccgctg 60
gcggtgtcgg ccaggcgctt gcactactgt taaaaaccca actgccttca ggttcagaac 120
tctctctgta tgatatcgct ccagtgactc ccggtgtggc tgtcgatctg agccatatcc 180
ctactgctgt gaaaatcaaa ggtttttctg gtgaagatgc gactccggcg ctggaaggcg 240
cagatgtcgt tcttatctct gcaggcgtag cggctaaacc gggccagtct cgttccgacc 300
tgtttaacgt taacgccggc atcgtgaaaa acctggtaca gcaagttgcg aaaacctgcc 360
cgaaagcgtg cattggtatt atcactaacc cggttaacac cacagttgca attgctgctg 420
aagtgctgaa aaaagccggt gtttatgaca aaaacaaact gttcggcgtt accacgctgg 480
atatcattcg ttccaacacc tttgttgcgg aactgaaagg caaacagcca ggcgaagttg 540
aagtgccggt tattggcggc cactctgatg ttaccattct gccgctgctg tcacaggttc 600
ctggcgttag ttttaccgag caggaagtgg ctgatctgac caaacgcatc cagaacgcgg 660
gtactgaagt ggttgaagcg aaggccggtg gcgggtctgc aaccctgtct atgggccagg 720
cagctgcacg ttttggtctg tctctggttc gtgcactgca gggcgaacaa ggcgttgtcg 780
aatgtgccta cgttgaaggc gacggtcagt acgcccgttt cttctctcaa ccgctgctgc 840
tgggtaaaaa cggcgtggaa gagcgtaaat ctatcggtac cctgagcgca tttgaacaga 900
acgcgctgga aggtatgctg gatacgctga agaaagatat cgccctgggc gaagagttcg 960
ttaataagta aggatccgtt taactttaag aaggagatat accatgaaag ccaactctcc 1020
cgtcatccgc ctgaacccgg tcgatgatgt cgtcatcgcc cgccagcagc tcatctccgg 1080
caccgtgctg caggacgaag gcggcctcaa agtccagggc ctcattcccg ccggccacaa 1140
gatggcgacc cgcgccatct ctgcgggcga gccggtcaag cgctatggcc agatcatcgg 1200
cacggccagc caggacatcg cgcccggaca gcacgtgcac acgcacaacc tggccatggc 1260
cgagttctcg cgtgaacacc atttcggcgc tgacgtgaag ccggtcgatt tcgtcgccga 1320
gcccgcaacc ttcatgggca tcgtgcgtcc cgatggccgc gtggccacgc gcaactacat 1380
cggcgtactg acctcggtga actgctcggc caccgcggca cgcgccatcg ccgattactt 1440
ccgccgcgac atccatcccg aagtgctggc cgactatccc aacatcgatg gcgtggtggc 1500
cctgacccat ggccagggtt gcgccacgga ttcgcagggc gagccgctgc agatcctgcg 1560
ccgcacgctg gcgggttacg ccacgcatcc caacttcgct gcggtgctgg tggtgggcct 1620
gggctgcgag accaaccaga tctcgggcct gatggaaagc cacaacctga aggaaggcga 1680
atacttccac accttcacca tccagggcac cggcggcacc gccaagaccg tggcgctggg 1740
gatcgagaag atcaagaaga tgctgcccaa ggccaatgac atcaagcgcg agccggtgtc 1800
ggccaagcat ctgacgctgg gcctgcagtg cggtggttcg gatggctatt cgggcatcac 1860
ggccaatccg gcgctgggtg cggcagtgga cttgctggtg cggcatggcg gcacggccat 1920
cctttccgaa acgccggaaa tctatggggc cgaacacctg ctcacccgcc gcgcggtctc 1980
gccggaagtc ggtgagaagc tgctggcccg catcgcctgg tgggaggagt attgcgccaa 2040
gaacgatgcc gagatgaaca acaatccctc agcgggcaac aaggccggtg gcttgaccac 2100
catcctcgag aagtcgctgg gcgcggttgc caagggtggc actaccaatc tggtggatgt 2160
ctacaagtat gcggagacgg tcaccgcacg cggcttcgtc ttcatggata cgccgggcta 2220
cgacccgatc tccgccaccg gccaggtcgc cggaggggcc aacatgatct gcttcaccac 2280
gggccggggt tctgcctatg gctgcgcacc ggcaccgtcg ctcaagctgg ccaccaatac 2340
tgcgctgtgg caacgtcagg aagaagacat ggatatcaac tgcggcgaga ttgccgacag 2400
caatgtcacc ccgcaggagg tcggcgaacg cttcttccag atgatcctcg atacggcatc 2460
gggcaagaag accaagagcg agctgcatgg ctacgggcaa gacgaattcg tgccctggca 2520
catcggcgtc tacacctgat ctagagtcga cctgcaggca tgcaagcttc tgttttggcg 2580
gatgagagaa gaaattcgtc gcccgccata aactgccagg catcaaatta agcagaaggc 2640
catcctgacg gatggccttt ttgcgtttct acaaactctt cctgtctagc aggtggcact 2700
tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 2760
tatccgctca tgagcttcac gctgccgcaa gcactcaggg cgcaagggct gctaaaggaa 2820
gcggaacacg tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta 2880
ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg 2940
gcttacatgg cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc 3000
agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt 3060
gccgccaagg atctgatggc gcaggggatc aagatctgat caagagacag gatgaggatc 3120
gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 3180
gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 3240
gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 3300
tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 3360
agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 3420
ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 3480
tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 3540
acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 3600
ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 3660
gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 3720
ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 3780
tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 3840
ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 3900
ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 3960
cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 4020
ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 4080
ttcttcgccc accccgatat tgattacgtg tttgaatcat tgcatcgcct gtaaattttt 4140
aacttgtcct atttttgtca ttaccacgta tatacacatg tataacaatt cagatatcgt 4200
taccaggata tgccgcatca gcggcatgga aggcggcact ctgttgtttc atatgataca 4260
ggagtaaaac cgccgaagcc cggcgtaagc cggtactgat tgatagattt caccttaccc 4320
atccccagcc ctgccagacc atacccgctt tcagccatga gagagcttct gtgcgcggtc 4380
ggagtggtcc cgacgagggt ttacccgaag tcggggcgta tctccgcgtt agcgggccgt 4440
cagggccgct tacgagcgtg tactgagaac ttccagcgag aagactgaca gcgatgatga 4500
agtagttaca acattcataa ttaaaagcga ctctgttccg gcccttcggg ccggggcggg 4560
gccgcttttc agttatgagg gaggggcttt gtggtttcag ttctgcgctg gaccggggtt 4620
tttctggtgg ttgtgtctga gtgttgtaac taaagcggct ccggtagggg cccgccgctt 4680
gcggtgggag gtgcatatct gtctgtccac aggacaggca gtgaataggt tttcttttta 4740
aatgaatgta attaagtagt ttaaaggaga tataaacagg tgtttaaaag atacattgca 4800
ccctgtaacg ctggcggatg gcgctttatg acatgaacgg ttgtaacctt atggggaagt 4860
cccttgcagt ttaatgtgga taagcaaaat tccccgtctg tgaggcgtgt tttgtatcga 4920
aaacaggggg ggccggatgc accagaagct ggatgatgag gttgtttttt gtatgtggtg 4980
ctgatttttt gtgcgctgtc gggcttcaga cgtgcggaag cctccggcgc aggccggatt 5040
attcagagga ggtcactttc agggggaagc tgtggccagc cggctgtaat tacggttacg 5100
tgacagaatc atgcgctcct tcacacgacg ctcaacttcg cgttttaccg cctcacgatt 5160
ggcagtgaag cgcccttccg cgatttcgcg cgtcagctgc cgtttcacca gggtgacaat 5220
atcctgacgt tccctgtccg catcacgacg cgcacgggca cgctttattc cccgggactt 5280
aagctctgtc tgataactgc gaaaacgctc acgaacaaaa cgccaggctt tcgctatcag 5340
ttcatccatg cccagggtat ccagcccctg ctttttgcgt tgtttgtttt cccataccac 5400
acggctgcgg cgcgcggcgg ccactgcctc ctctgataca tcgagggcag caaacagtgc 5460
agatgtgaac gtgatatcgg tcggaatgta gcacccgata agcgggtcat attccgtctg 5520
gtaggtaatc agtcccagct ctgacaggaa cgtcagggcc cgggtggcac gggtgatgga 5580
gagttttccg gcagcagact ccgtcgccag tccgcactca atggccagcg tggtgatgga 5640
gcactggacg cggttggcca gcgggtcata gtggaaacac agcccctgca ggagcgcatc 5700
aatagcccgt cgacgcagca ctggtggcat gcgtcgacgc agaccacgcg aacgggcatg 5760
cgccacatga atggcgaaat caaaacggga agtgaagccc accgcctttt ccatcagttt 5820
ttcgcagaac ttcagcgttc ctgcaccttc acggggtgta aacaccggat tcgggttctt 5880
tacctggcgg taatacgttt ggtgaagatc agtcacacca tcctgcactt acaatgcgca 5940
gaaggagcga gcacagaaag aagtcttgaa cttttccggg catataacta tactccccgc 6000
atagctgaat tgttggctat acggtttaag tgggccccgg taatcttttc gtactcgcaa 6060
aagttgaaga agattatcgg ggtttttgct tttctggctc ctgtaaatcc acatcagaac 6120
cagttccttg ccaccttacg gcgtggccag ccacaaaatt ccttaaacga tcagtaatct 6180
agcactaatc ttctgaacac tcaagaatgt aagcccatca tcacatgcat cgttttgcgc 6240
ttcacttttt atcagtgcgg tcagaacttc agcctgagtc agaccatctt catgacacat 6300
ttgcatgagc atggccttat actttggttc aagaaatact tttacttcct tgaacgaagc 6360
tcttttacgg gccactgata atctttgttt ctctgcatca gaaagcggat tcccctttct 6420
gtatgctcgt tttgcgccag atgaggaagt cactgcattt tctgtctgcg acatctcgcc 6480
tcctcaatac tcaaacaggg atcgtttcgc agaggatact acagtttttt gaaatcagcg 6540
acttgagaat tgtgacgaag atctgtgttg gcgcacaaat caacggggat tactgtcgtt 6600
taatgtgatt taaactgtga aatagtatgg ttttcagtta ttgaaacgcc gggagcgggg 6660
aaaacttgct ttttcccgtt tccggggttg gacaactgag caacgcgaag gcgtcagcta 6720
cgatgttccg gggactgctg atcagcccct gttgtattca gccattctcc ggtcatccga 6780
acgccagcaa gacgtagccc agcgcgtcgg ccaattcgcg ctaacttaca ttaattgcgt 6840
tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 6900
gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc 6960
agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag 7020
cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt tgacggcggg 7080
atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg 7140
cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc 7200
agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac 7260
atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat 7320
ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc 7380
gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca 7440
tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga 7500
acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg 7560
atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg 7620
ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta 7680
atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc 7740
agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc 7800
gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc 7860
acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact 7920
ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga 7980
aaggttttgc accattcgat ggtgtcaacg taaatgcatg ccgcttcgcc ttcgcgcgcg 8040
aattggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac 8100
cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga 8160
tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca 8220
cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc 8280
cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggcggagctt 8340
atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg 8400
gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg 8460
ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg 8520
acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag 8580
gaaacagaat tc 8592
<210> 172
<211> 939
<212> DNA
<213> 人工序列
<220>
<223> Ec.Mdh突变体的序列
<400> 172
atgaaagtcg cagtcctcgg cgccgctggc ggtgtcggcc aggcgcttgc actactgtta 60
aaaacccaac tgccttcagg ttcagaactc tctctgtatg gcatcgctcc agtgactccc 120
ggtgtggctg tcgatctgag ccatatccct actgctgtga aaatcaaagg tttttctggt 180
gaagatgcga ctccggcgct ggaaggcgca gatgtcgttc ttatctctgc aggcgtagcg 240
gctaaaccgg gccagtctcg ttccgacctg tttaacgtta acgccggcat cgtgaaaaac 300
ctggtacagc aagttgcgaa aacctgcccg aaagcgtgca ttggtattat cactaacccg 360
gttaacacca cagttgcaat tgctgctgaa gtgctgaaaa aagccggtgt ttatgacaaa 420
aacaaactgt tcggcgttac cacgctggat atcattcgtt ccaacacctt tgttgcggaa 480
ctgaaaggca aacagccagg cgaagttgaa gtgccggtta ttggcggcca ctctgatgtt 540
accattctgc cgctgctgtc acaggttcct ggcgttagtt ttaccgagca ggaagtggct 600
gatctgacca aacgcatcca gaacgcgggt actgaagtgg ttgaagcgaa ggccggtggc 660
gggtctgcaa ccctgtctat gggccaggca gctgcacgtt ttggtctgtc tctggttcgt 720
gcactgcagg gcgaacaagg cgttgtcgaa tgtgcctacg ttgaaggcga cggtcagtac 780
gcccgtttct tctctcaacc gctgctgctg ggtaaaaacg gcgtggaaga gcgtaaatct 840
atcggtaccc tgagcgcatt tgaacagaac gcgctggaag gtatgctgga tacgctgaag 900
aaagatatcg ccctgggcga agagttcgtt aataagtaa 939
<210> 173
<211> 312
<212> PRT
<213> 人工序列
<220>
<223> 具有突变I12V R81A M85Q D86S G179D D34G的Ec.Mdh
突变体
<400> 173
Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly Val Gly Gln Ala Leu
1 5 10 15
Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu
20 25 30
Tyr Gly Ile Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His
35 40 45
Ile Pro Thr Ala Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr
50 55 60
Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala
65 70 75 80
Ala Lys Pro Gly Gln Ser Arg Ser Asp Leu Phe Asn Val Asn Ala Gly
85 90 95
Ile Val Lys Asn Leu Val Gln Gln Val Ala Lys Thr Cys Pro Lys Ala
100 105 110
Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala
115 120 125
Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe
130 135 140
Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu
145 150 155 160
Leu Lys Gly Lys Gln Pro Gly Glu Val Glu Val Pro Val Ile Gly Gly
165 170 175
His Ser Asp Val Thr Ile Leu Pro Leu Leu Ser Gln Val Pro Gly Val
180 185 190
Ser Phe Thr Glu Gln Glu Val Ala Asp Leu Thr Lys Arg Ile Gln Asn
195 200 205
Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr
210 215 220
Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg
225 230 235 240
Ala Leu Gln Gly Glu Gln Gly Val Val Glu Cys Ala Tyr Val Glu Gly
245 250 255
Asp Gly Gln Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys
260 265 270
Asn Gly Val Glu Glu Arg Lys Ser Ile Gly Thr Leu Ser Ala Phe Glu
275 280 285
Gln Asn Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala
290 295 300
Leu Gly Glu Glu Phe Val Asn Lys
305 310
<210> 174
<211> 939
<212> DNA
<213> 人工序列
<220>
<223> Ec.Mdh突变体的序列
<400> 174
atgaaagtcg cagtcctcgg cgctgctggc ggtgtcggcc aggcgcttgc actactgtta 60
aaaacccaac tgccttcagg ttcagaactc tctctgtatg attctgctcc agtgactccc 120
ggtgtggctg tcgatctgag ccatatccct actgctgtga aaatcaaagg tttttctggt 180
gaagatgcga ctccggcgct ggaaggcgca gatgtcgttc ttatctctgc aggcgtagcg 240
gctaaaccgg gtcagtctcg ttccgacctg tttaacgtta acgccggcat cgtgaaaaac 300
ctggtacagc aagttgcgaa aacctgcccg aaagcgtgca ttggtattat cactaacccg 360
gttaacacca cagttgcaat tgctgctgaa gtgctgaaaa aagccggtgt ttatgacaaa 420
aacaaactgt tcggcgttac cacgctggat atcattcgtt ccaacacctt tgttgcggaa 480
ctgaaaggca aacagccagg cgaagttgaa gtgccggtta ttggcggtca ctctgatgtt 540
accattctgc cgctgctgtc acaggttcct ggcgttagtt ttaccgagca ggaagtggct 600
gatctgacca aacgcatcca gaacgcgggt actgaagtgg ttgaagcgaa ggccggtggc 660
gggtctgcaa ccctgtctat gggccaggca gctgcacgtt ttggtctgtc tctggttcgt 720
gcactgcagg gcgaacaagg cgttgtcgaa tgtgcctacg ttgaaggcga cggtcagtac 780
gcccgtttct tctctcaacc gctgctgctg ggtaaaaacg gcgtggaaga gcgtaaatct 840
atcggtaccc tgagcgcatt tgaacagaac gcgctggaag gtatgctgga tacgctgaag 900
aaagatatcg ccctgggcga agagttcgtt aataagtaa 939
<210> 175
<211> 312
<212> PRT
<213> 人工序列
<220>
<223> 具有突变I12V R81A M85Q D86S G179D I35S的Ec.Mdh
突变体
<400> 175
Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly Val Gly Gln Ala Leu
1 5 10 15
Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu
20 25 30
Tyr Asp Ser Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His
35 40 45
Ile Pro Thr Ala Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr
50 55 60
Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala
65 70 75 80
Ala Lys Pro Gly Gln Ser Arg Ser Asp Leu Phe Asn Val Asn Ala Gly
85 90 95
Ile Val Lys Asn Leu Val Gln Gln Val Ala Lys Thr Cys Pro Lys Ala
100 105 110
Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala
115 120 125
Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe
130 135 140
Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu
145 150 155 160
Leu Lys Gly Lys Gln Pro Gly Glu Val Glu Val Pro Val Ile Gly Gly
165 170 175
His Ser Asp Val Thr Ile Leu Pro Leu Leu Ser Gln Val Pro Gly Val
180 185 190
Ser Phe Thr Glu Gln Glu Val Ala Asp Leu Thr Lys Arg Ile Gln Asn
195 200 205
Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr
210 215 220
Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg
225 230 235 240
Ala Leu Gln Gly Glu Gln Gly Val Val Glu Cys Ala Tyr Val Glu Gly
245 250 255
Asp Gly Gln Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys
260 265 270
Asn Gly Val Glu Glu Arg Lys Ser Ile Gly Thr Leu Ser Ala Phe Glu
275 280 285
Gln Asn Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala
290 295 300
Leu Gly Glu Glu Phe Val Asn Lys
305 310
<210> 176
<211> 939
<212> DNA
<213> 人工序列
<220>
<223> Ec.Mdh突变体的序列
<400> 176
atgaaagtcg cagtcctcgg cgccgctggc ggtgtcggcc aggcgcttgc actactgtta 60
aaaacccaac tgccttcagg ttcagaactc tctctgtatg gcaaagctcc agtgactccc 120
ggtgtggctg tcgatctgag ccatatccct actgctgtga aaatcaaagg tttttctggt 180
gaagatgcga ctccggcgct ggaaggcgca gatgtcgttc ttatctctgc aggcgtagcg 240
gctaaaccgg gccagtctcg ttccgacctg tttaacgtta acgccggcat cgtgaaaaac 300
ctggtacagc aagttgcgaa aacctgcccg aaagcgtgca ttggtattat cactaacccg 360
gttaacacca cagttgcaat tgctgctgaa gtgctgaaaa aagccggtgt ttatgacaaa 420
aacaaactgt tcggcgttac cacgctggat atcattcgtt ccaacacctt tgttgcggaa 480
ctgaaaggca aacagccagg cgaagttgaa gtgccggtta ttggcggcca ctctgatgtt 540
accattctgc cgctgctgtc acaggttcct ggcgttagtt ttaccgagca ggaagtggct 600
gatctgacca aacgcatcca gaacgcgggt actgaagtgg ttgaagcgaa ggccggtggc 660
gggtctgcaa ccctgtctat gggccaggca gctgcacgtt ttggtctgtc tctggttcgt 720
gcactgcagg gcgaacaagg cgttgtcgaa tgtgcctacg ttgaaggcga cggtcagtac 780
gcccgtttct tctctcaacc gctgctgctg ggtaaaaacg gcgtggaaga gcgtaaatct 840
atcggtaccc tgagcgcatt tgaacagaac gcgctggaag gtatgctgga tacgctgaag 900
aaagatatcg ccctgggcga agagttcgtt aataagtaa 939
<210> 177
<211> 312
<212> PRT
<213> 人工序列
<220>
<223> 具有突变I12V R81A M85Q D86S G179D D34G I35K的Ec.Mdh
突变体
<400> 177
Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly Val Gly Gln Ala Leu
1 5 10 15
Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu
20 25 30
Tyr Gly Lys Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His
35 40 45
Ile Pro Thr Ala Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr
50 55 60
Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala
65 70 75 80
Ala Lys Pro Gly Gln Ser Arg Ser Asp Leu Phe Asn Val Asn Ala Gly
85 90 95
Ile Val Lys Asn Leu Val Gln Gln Val Ala Lys Thr Cys Pro Lys Ala
100 105 110
Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala
115 120 125
Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe
130 135 140
Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu
145 150 155 160
Leu Lys Gly Lys Gln Pro Gly Glu Val Glu Val Pro Val Ile Gly Gly
165 170 175
His Ser Asp Val Thr Ile Leu Pro Leu Leu Ser Gln Val Pro Gly Val
180 185 190
Ser Phe Thr Glu Gln Glu Val Ala Asp Leu Thr Lys Arg Ile Gln Asn
195 200 205
Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr
210 215 220
Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg
225 230 235 240
Ala Leu Gln Gly Glu Gln Gly Val Val Glu Cys Ala Tyr Val Glu Gly
245 250 255
Asp Gly Gln Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys
260 265 270
Asn Gly Val Glu Glu Arg Lys Ser Ile Gly Thr Leu Ser Ala Phe Glu
275 280 285
Gln Asn Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala
290 295 300
Leu Gly Glu Glu Phe Val Asn Lys
305 310
<210> 178
<211> 939
<212> DNA
<213> 人工序列
<220>
<223> Ec.Mdh突变体的序列
<400> 178
atgaaagtcg cagtcctcgg cgccgctggc ggtgtcggcc aggcgcttgc actactgtta 60
aaaacccaac tgccttcagg ttcagaactc tctctgtatg gccgtgctcc agtgactccc 120
ggtgtggctg tcgatctgag ccatatccct actgctgtga aaatcaaagg tttttctggt 180
gaagatgcga ctccggcgct ggaaggcgca gatgtcgttc ttatctctgc aggcgtagcg 240
gctaaaccgg gccagtctcg ttccgacctg tttaacgtta acgccggcat cgtgaaaaac 300
ctggtacagc aagttgcgaa aacctgcccg aaagcgtgca ttggtattat cactaacccg 360
gttaacacca cagttgcaat tgctgctgaa gtgctgaaaa aagccggtgt ttatgacaaa 420
aacaaactgt tcggcgttac cacgctggat atcattcgtt ccaacacctt tgttgcggaa 480
ctgaaaggca aacagccagg cgaagttgaa gtgccggtta ttggcggcca ctctgatgtt 540
accattctgc cgctgctgtc acaggttcct ggcgttagtt ttaccgagca ggaagtggct 600
gatctgacca aacgcatcca gaacgcgggt actgaagtgg ttgaagcgaa ggccggtggc 660
gggtctgcaa ccctgtctat gggccaggca gctgcacgtt ttggtctgtc tctggttcgt 720
gcactgcagg gcgaacaagg cgttgtcgaa tgtgcctacg ttgaaggcga cggtcagtac 780
gcccgtttct tctctcaacc gctgctgctg ggtaaaaacg gcgtggaaga gcgtaaatct 840
atcggtaccc tgagcgcatt tgaacagaac gcgctggaag gtatgctgga tacgctgaag 900
aaagatatcg ccctgggcga agagttcgtt aataagtaa 939
<210> 179
<211> 312
<212> PRT
<213> 人工序列
<220>
<223> 具有突变I12V R81A M85Q D86S G179D D34G I35R的Ec.Mdh
突变体
<400> 179
Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly Val Gly Gln Ala Leu
1 5 10 15
Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu
20 25 30
Tyr Gly Arg Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His
35 40 45
Ile Pro Thr Ala Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr
50 55 60
Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala
65 70 75 80
Ala Lys Pro Gly Gln Ser Arg Ser Asp Leu Phe Asn Val Asn Ala Gly
85 90 95
Ile Val Lys Asn Leu Val Gln Gln Val Ala Lys Thr Cys Pro Lys Ala
100 105 110
Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala
115 120 125
Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe
130 135 140
Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu
145 150 155 160
Leu Lys Gly Lys Gln Pro Gly Glu Val Glu Val Pro Val Ile Gly Gly
165 170 175
His Ser Asp Val Thr Ile Leu Pro Leu Leu Ser Gln Val Pro Gly Val
180 185 190
Ser Phe Thr Glu Gln Glu Val Ala Asp Leu Thr Lys Arg Ile Gln Asn
195 200 205
Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr
210 215 220
Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg
225 230 235 240
Ala Leu Gln Gly Glu Gln Gly Val Val Glu Cys Ala Tyr Val Glu Gly
245 250 255
Asp Gly Gln Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys
260 265 270
Asn Gly Val Glu Glu Arg Lys Ser Ile Gly Thr Leu Ser Ala Phe Glu
275 280 285
Gln Asn Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala
290 295 300
Leu Gly Glu Glu Phe Val Asn Lys
305 310
<210> 180
<211> 939
<212> DNA
<213> 人工序列
<220>
<223> Ec.Mdh突变体的序列
<400> 180
atgaaagtcg cagtcctcgg cgccgctggc ggtgtcggcc aggcgcttgc actactgtta 60
aaaacccaac tgccttcagg ttcagaactc tctctgtatg gctctgctcc agtgactccc 120
ggtgtggctg tcgatctgag ccatatccct actgctgtga aaatcaaagg tttttctggt 180
gaagatgcga ctccggcgct ggaaggcgca gatgtcgttc ttatctctgc aggcgtagcg 240
gctaaaccgg gccagtctcg ttccgacctg tttaacgtta acgccggcat cgtgaaaaac 300
ctggtacagc aagttgcgaa aacctgcccg aaagcgtgca ttggtattat cactaacccg 360
gttaacacca cagttgcaat tgctgctgaa gtgctgaaaa aagccggtgt ttatgacaaa 420
aacaaactgt tcggcgttac cacgctggat atcattcgtt ccaacacctt tgttgcggaa 480
ctgaaaggca aacagccagg cgaagttgaa gtgccggtta ttggcggcca ctctgatgtt 540
accattctgc cgctgctgtc acaggttcct ggcgttagtt ttaccgagca ggaagtggct 600
gatctgacca aacgcatcca gaacgcgggt actgaagtgg ttgaagcgaa ggccggtggc 660
gggtctgcaa ccctgtctat gggccaggca gctgcacgtt ttggtctgtc tctggttcgt 720
gcactgcagg gcgaacaagg cgttgtcgaa tgtgcctacg ttgaaggcga cggtcagtac 780
gcccgtttct tctctcaacc gctgctgctg ggtaaaaacg gcgtggaaga gcgtaaatct 840
atcggtaccc tgagcgcatt tgaacagaac gcgctggaag gtatgctgga tacgctgaag 900
aaagatatcg ccctgggcga agagttcgtt aataagtaa 939
<210> 181
<211> 312
<212> PRT
<213> 人工序列
<220>
<223> 具有突变I12V R81A M85Q D86S G179D D34G I35S的Ec.Mdh
突变体
<400> 181
Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly Val Gly Gln Ala Leu
1 5 10 15
Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu
20 25 30
Tyr Gly Ser Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His
35 40 45
Ile Pro Thr Ala Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr
50 55 60
Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala
65 70 75 80
Ala Lys Pro Gly Gln Ser Arg Ser Asp Leu Phe Asn Val Asn Ala Gly
85 90 95
Ile Val Lys Asn Leu Val Gln Gln Val Ala Lys Thr Cys Pro Lys Ala
100 105 110
Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala
115 120 125
Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe
130 135 140
Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu
145 150 155 160
Leu Lys Gly Lys Gln Pro Gly Glu Val Glu Val Pro Val Ile Gly Gly
165 170 175
His Ser Asp Val Thr Ile Leu Pro Leu Leu Ser Gln Val Pro Gly Val
180 185 190
Ser Phe Thr Glu Gln Glu Val Ala Asp Leu Thr Lys Arg Ile Gln Asn
195 200 205
Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr
210 215 220
Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg
225 230 235 240
Ala Leu Gln Gly Glu Gln Gly Val Val Glu Cys Ala Tyr Val Glu Gly
245 250 255
Asp Gly Gln Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys
260 265 270
Asn Gly Val Glu Glu Arg Lys Ser Ile Gly Thr Leu Ser Ala Phe Glu
275 280 285
Gln Asn Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala
290 295 300
Leu Gly Glu Glu Phe Val Asn Lys
305 310
<210> 182
<211> 939
<212> DNA
<213> 人工序列
<220>
<223> Ec.Mdh突变体的序列
<400> 182
atgaaagtcg cagtcctcgg cgccgctggc ggtgtcggcc aggcgcttgc actactgtta 60
aaaacccaac tgccttcagg ttcagaactc tctctgtatg gcaccgctcc agtgactccc 120
ggtgtggctg tcgatctgag ccatatccct actgctgtga aaatcaaagg tttttctggt 180
gaagatgcga ctccggcgct ggaaggcgca gatgtcgttc ttatctctgc aggcgtagcg 240
gctaaaccgg gccagtctcg ttccgacctg tttaacgtta acgccggcat cgtgaaaaac 300
ctggtacagc aagttgcgaa aacctgcccg aaagcgtgca ttggtattat cactaacccg 360
gttaacacca cagttgcaat tgctgctgaa gtgctgaaaa aagccggtgt ttatgacaaa 420
aacaaactgt tcggcgttac cacgctggat atcattcgtt ccaacacctt tgttgcggaa 480
ctgaaaggca aacagccagg cgaagttgaa gtgccggtta ttggcggcca ctctgatgtt 540
accattctgc cgctgctgtc acaggttcct ggcgttagtt ttaccgagca ggaagtggct 600
gatctgacca aacgcatcca gaacgcgggt actgaagtgg ttgaagcgaa ggccggtggc 660
gggtctgcaa ccctgtctat gggccaggca gctgcacgtt ttggtctgtc tctggttcgt 720
gcactgcagg gcgaacaagg cgttgtcgaa tgtgcctacg ttgaaggcga cggtcagtac 780
gcccgtttct tctctcaacc gctgctgctg ggtaaaaacg gcgtggaaga gcgtaaatct 840
atcggtaccc tgagcgcatt tgaacagaac gcgctggaag gtatgctgga tacgctgaag 900
aaagatatcg ccctgggcga agagttcgtt aataagtaa 939
<210> 183
<211> 312
<212> PRT
<213> 人工序列
<220>
<223> 具有突变I12V R81A M85Q D86S G179D D34G I35T的Ec.Mdh
突变体
<400> 183
Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly Val Gly Gln Ala Leu
1 5 10 15
Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu
20 25 30
Tyr Gly Thr Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His
35 40 45
Ile Pro Thr Ala Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr
50 55 60
Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala
65 70 75 80
Ala Lys Pro Gly Gln Ser Arg Ser Asp Leu Phe Asn Val Asn Ala Gly
85 90 95
Ile Val Lys Asn Leu Val Gln Gln Val Ala Lys Thr Cys Pro Lys Ala
100 105 110
Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala
115 120 125
Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe
130 135 140
Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu
145 150 155 160
Leu Lys Gly Lys Gln Pro Gly Glu Val Glu Val Pro Val Ile Gly Gly
165 170 175
His Ser Asp Val Thr Ile Leu Pro Leu Leu Ser Gln Val Pro Gly Val
180 185 190
Ser Phe Thr Glu Gln Glu Val Ala Asp Leu Thr Lys Arg Ile Gln Asn
195 200 205
Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr
210 215 220
Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg
225 230 235 240
Ala Leu Gln Gly Glu Gln Gly Val Val Glu Cys Ala Tyr Val Glu Gly
245 250 255
Asp Gly Gln Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys
260 265 270
Asn Gly Val Glu Glu Arg Lys Ser Ile Gly Thr Leu Ser Ala Phe Glu
275 280 285
Gln Asn Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala
290 295 300
Leu Gly Glu Glu Phe Val Asn Lys
305 310
<210> 184
<211> 8592
<212> DNA
<213> 人工序列
<220>
<223> 质粒序列
<400> 184
gagctcgttt aactttaaga aggagatata ccatgaaagt cgcagtcctc ggcgccgctg 60
gcggtgtcgg ccaggcgctt gcactactgt taaaaaccca actgccttca ggttcagaac 120
tctctctgta tggccgtgct ccagtgactc ccggtgtggc tgtcgatctg agccatatcc 180
ctactgctgt gaaaatcaaa ggtttttctg gtgaagatgc gactccggcg ctggaaggcg 240
cagatgtcgt tcttatctct gcaggcgtag cggctaaacc gggccagtct cgttccgacc 300
tgtttaacgt taacgccggc atcgtgaaaa acctggtaca gcaagttgcg aaaacctgcc 360
cgaaagcgtg cattggtatt atcactaacc cggttaacac cacagttgca attgctgctg 420
aagtgctgaa aaaagccggt gtttatgaca aaaacaaact gttcggcgtt accacgctgg 480
atatcattcg ttccaacacc tttgttgcgg aactgaaagg caaacagcca ggcgaagttg 540
aagtgccggt tattggcggc cactctgatg ttaccattct gccgctgctg tcacaggttc 600
ctggcgttag ttttaccgag caggaagtgg ctgatctgac caaacgcatc cagaacgcgg 660
gtactgaagt ggttgaagcg aaggccggtg gcgggtctgc aaccctgtct atgggccagg 720
cagctgcacg ttttggtctg tctctggttc gtgcactgca gggcgaacaa ggcgttgtcg 780
aatgtgccta cgttgaaggc gacggtcagt acgcccgttt cttctctcaa ccgctgctgc 840
tgggtaaaaa cggcgtggaa gagcgtaaat ctatcggtac cctgagcgca tttgaacaga 900
acgcgctgga aggtatgctg gatacgctga agaaagatat cgccctgggc gaagagttcg 960
ttaataagta aggatccgtt taactttaag aaggagatat accatgaaag ccaactctcc 1020
cgtcatccgc ctgaacccgg tcgatgatgt cgtcatcgcc cgccagcagc tcatctccgg 1080
caccgtgctg caggacgaag gcggcctcaa agtccagggc ctcattcccg ccggccacaa 1140
gatggcgacc cgcgccatct ctgcgggcga gccggtcaag cgctatggcc agatcatcgg 1200
cacggccagc caggacatcg cgcccggaca gcacgtgcac acgcacaacc tggccatggc 1260
cgagttctcg cgtgaacacc atttcggcgc tgacgtgaag ccggtcgatt tcgtcgccga 1320
gcccgcaacc ttcatgggca tcgtgcgtcc cgatggccgc gtggccacgc gcaactacat 1380
cggcgtactg acctcggtga actgctcggc caccgcggca cgcgccatcg ccgattactt 1440
ccgccgcgac atccatcccg aagtgctggc cgactatccc aacatcgatg gcgtggtggc 1500
cctgacccat ggccagggtt gcgccacgga ttcgcagggc gagccgctgc agatcctgcg 1560
ccgcacgctg gcgggttacg ccacgcatcc caacttcgct gcggtgctgg tggtgggcct 1620
gggctgcgag accaaccaga tctcgggcct gatggaaagc cacaacctga aggaaggcga 1680
atacttccac accttcacca tccagggcac cggcggcacc gccaagaccg tggcgctggg 1740
gatcgagaag atcaagaaga tgctgcccaa ggccaatgac atcaagcgcg agccggtgtc 1800
ggccaagcat ctgacgctgg gcctgcagtg cggtggttcg gatggctatt cgggcatcac 1860
ggccaatccg gcgctgggtg cggcagtgga cttgctggtg cggcatggcg gcacggccat 1920
cctttccgaa acgccggaaa tctatggggc cgaacacctg ctcacccgcc gcgcggtctc 1980
gccggaagtc ggtgagaagc tgctggcccg catcgcctgg tgggaggagt attgcgccaa 2040
gaacgatgcc gagatgaaca acaatccctc agcgggcaac aaggccggtg gcttgaccac 2100
catcctcgag aagtcgctgg gcgcggttgc caagggtggc actaccaatc tggtggatgt 2160
ctacaagtat gcggagacgg tcaccgcacg cggcttcgtc ttcatggata cgccgggcta 2220
cgacccgatc tccgccaccg gccaggtcgc cggaggggcc aacatgatct gcttcaccac 2280
gggccggggt tctgcctatg gctgcgcacc ggcaccgtcg ctcaagctgg ccaccaatac 2340
tgcgctgtgg caacgtcagg aagaagacat ggatatcaac tgcggcgaga ttgccgacag 2400
caatgtcacc ccgcaggagg tcggcgaacg cttcttccag atgatcctcg atacggcatc 2460
gggcaagaag accaagagcg agctgcatgg ctacgggcaa gacgaattcg tgccctggca 2520
catcggcgtc tacacctgat ctagagtcga cctgcaggca tgcaagcttc tgttttggcg 2580
gatgagagaa gaaattcgtc gcccgccata aactgccagg catcaaatta agcagaaggc 2640
catcctgacg gatggccttt ttgcgtttct acaaactctt cctgtctagc aggtggcact 2700
tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 2760
tatccgctca tgagcttcac gctgccgcaa gcactcaggg cgcaagggct gctaaaggaa 2820
gcggaacacg tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta 2880
ctgggctatc tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg 2940
gcttacatgg cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc 3000
agctggggcg ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt 3060
gccgccaagg atctgatggc gcaggggatc aagatctgat caagagacag gatgaggatc 3120
gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag 3180
gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg 3240
gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa 3300
tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc 3360
agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc 3420
ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga 3480
tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa 3540
acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct 3600
ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat 3660
gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt 3720
ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta 3780
tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga 3840
ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg 3900
ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg 3960
cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 4020
ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag 4080
ttcttcgccc accccgatat tgattacgtg tttgaatcat tgcatcgcct gtaaattttt 4140
aacttgtcct atttttgtca ttaccacgta tatacacatg tataacaatt cagatatcgt 4200
taccaggata tgccgcatca gcggcatgga aggcggcact ctgttgtttc atatgataca 4260
ggagtaaaac cgccgaagcc cggcgtaagc cggtactgat tgatagattt caccttaccc 4320
atccccagcc ctgccagacc atacccgctt tcagccatga gagagcttct gtgcgcggtc 4380
ggagtggtcc cgacgagggt ttacccgaag tcggggcgta tctccgcgtt agcgggccgt 4440
cagggccgct tacgagcgtg tactgagaac ttccagcgag aagactgaca gcgatgatga 4500
agtagttaca acattcataa ttaaaagcga ctctgttccg gcccttcggg ccggggcggg 4560
gccgcttttc agttatgagg gaggggcttt gtggtttcag ttctgcgctg gaccggggtt 4620
tttctggtgg ttgtgtctga gtgttgtaac taaagcggct ccggtagggg cccgccgctt 4680
gcggtgggag gtgcatatct gtctgtccac aggacaggca gtgaataggt tttcttttta 4740
aatgaatgta attaagtagt ttaaaggaga tataaacagg tgtttaaaag atacattgca 4800
ccctgtaacg ctggcggatg gcgctttatg acatgaacgg ttgtaacctt atggggaagt 4860
cccttgcagt ttaatgtgga taagcaaaat tccccgtctg tgaggcgtgt tttgtatcga 4920
aaacaggggg ggccggatgc accagaagct ggatgatgag gttgtttttt gtatgtggtg 4980
ctgatttttt gtgcgctgtc gggcttcaga cgtgcggaag cctccggcgc aggccggatt 5040
attcagagga ggtcactttc agggggaagc tgtggccagc cggctgtaat tacggttacg 5100
tgacagaatc atgcgctcct tcacacgacg ctcaacttcg cgttttaccg cctcacgatt 5160
ggcagtgaag cgcccttccg cgatttcgcg cgtcagctgc cgtttcacca gggtgacaat 5220
atcctgacgt tccctgtccg catcacgacg cgcacgggca cgctttattc cccgggactt 5280
aagctctgtc tgataactgc gaaaacgctc acgaacaaaa cgccaggctt tcgctatcag 5340
ttcatccatg cccagggtat ccagcccctg ctttttgcgt tgtttgtttt cccataccac 5400
acggctgcgg cgcgcggcgg ccactgcctc ctctgataca tcgagggcag caaacagtgc 5460
agatgtgaac gtgatatcgg tcggaatgta gcacccgata agcgggtcat attccgtctg 5520
gtaggtaatc agtcccagct ctgacaggaa cgtcagggcc cgggtggcac gggtgatgga 5580
gagttttccg gcagcagact ccgtcgccag tccgcactca atggccagcg tggtgatgga 5640
gcactggacg cggttggcca gcgggtcata gtggaaacac agcccctgca ggagcgcatc 5700
aatagcccgt cgacgcagca ctggtggcat gcgtcgacgc agaccacgcg aacgggcatg 5760
cgccacatga atggcgaaat caaaacggga agtgaagccc accgcctttt ccatcagttt 5820
ttcgcagaac ttcagcgttc ctgcaccttc acggggtgta aacaccggat tcgggttctt 5880
tacctggcgg taatacgttt ggtgaagatc agtcacacca tcctgcactt acaatgcgca 5940
gaaggagcga gcacagaaag aagtcttgaa cttttccggg catataacta tactccccgc 6000
atagctgaat tgttggctat acggtttaag tgggccccgg taatcttttc gtactcgcaa 6060
aagttgaaga agattatcgg ggtttttgct tttctggctc ctgtaaatcc acatcagaac 6120
cagttccttg ccaccttacg gcgtggccag ccacaaaatt ccttaaacga tcagtaatct 6180
agcactaatc ttctgaacac tcaagaatgt aagcccatca tcacatgcat cgttttgcgc 6240
ttcacttttt atcagtgcgg tcagaacttc agcctgagtc agaccatctt catgacacat 6300
ttgcatgagc atggccttat actttggttc aagaaatact tttacttcct tgaacgaagc 6360
tcttttacgg gccactgata atctttgttt ctctgcatca gaaagcggat tcccctttct 6420
gtatgctcgt tttgcgccag atgaggaagt cactgcattt tctgtctgcg acatctcgcc 6480
tcctcaatac tcaaacaggg atcgtttcgc agaggatact acagtttttt gaaatcagcg 6540
acttgagaat tgtgacgaag atctgtgttg gcgcacaaat caacggggat tactgtcgtt 6600
taatgtgatt taaactgtga aatagtatgg ttttcagtta ttgaaacgcc gggagcgggg 6660
aaaacttgct ttttcccgtt tccggggttg gacaactgag caacgcgaag gcgtcagcta 6720
cgatgttccg gggactgctg atcagcccct gttgtattca gccattctcc ggtcatccga 6780
acgccagcaa gacgtagccc agcgcgtcgg ccaattcgcg ctaacttaca ttaattgcgt 6840
tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 6900
gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc 6960
agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag 7020
cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt tgacggcggg 7080
atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg 7140
cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc 7200
agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac 7260
atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat 7320
ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc 7380
gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca 7440
tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga 7500
acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg 7560
atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg 7620
ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta 7680
atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc 7740
agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc 7800
gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc 7860
acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact 7920
ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga 7980
aaggttttgc accattcgat ggtgtcaacg taaatgcatg ccgcttcgcc ttcgcgcgcg 8040
aattggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac 8100
cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga 8160
tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca 8220
cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc 8280
cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggcggagctt 8340
atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg 8400
gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg 8460
ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg 8520
acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag 8580
gaaacagaat tc 8592
Claims (18)
1.一种使用微生物代谢途径生产2,4-二羟基丁酸(DHB)或L-苏氨酸的方法，该方法包括：
使用苏糖醛缩酶将乙醇醛经酶促转化为苏糖的步骤，
使用苏糖脱氢酶将苏糖经酶促转化为苏糖酸-1,4-内酯的步骤，
使用苏糖酸-1,4-内酯酶将苏糖酸-1,4-内酯经酶促转化为苏糖酸的步骤，以及
使用苏糖酸脱水酶将苏糖酸经酶促转化为2-酮基-4-羟基丁酸(OHB)的步骤，
以及进一步包括
使用OHB还原酶将2-酮基-4-羟基丁酸(OHB)经酶促转化为2,4-二羟基丁酸(DHB)的步骤
或
使用L-高丝氨酸转氨酶将2-酮基-4-羟基丁酸经酶促转化为L-高丝氨酸的步骤，然后是使用ATP消耗使用高丝氨酸激酶将L-高丝氨酸经酶促转化为O-磷酸-L-高丝氨酸的步骤，以及使用L-苏氨酸合酶将O-磷酸-高丝氨酸经酶促转化为L-苏氨酸的步骤，
其中该代谢途径在微生物生产菌株中表达，该微生物生产菌株通过将所述酶表达所需的至少一个基因引入该生产菌株中而事先相对于其天然形式(野生型)被修饰。
2.根据权利要求1所述的方法，其特征在于这些基因的表达通过使用质粒或将这些基因整合到基因组中来实现。
3.根据权利要求1或2所述的方法，其特征在于该生产菌株已经具有其天然形式的代谢途径所需的一种或多种酶。
4.根据权利要求3所述的方法，其特征在于使用大肠杆菌种或恶臭假单胞菌种的修饰菌株作为生产菌株。
5.根据权利要求4所述的方法，其特征在于使用该大肠杆菌种的菌株作为在编码醛脱氢酶(AldA)和/或乙醇醛还原酶(YqhD)的基因中具有缺失的生产菌株。
6.根据权利要求1至5中任一项所述的方法，其特征在于为了在该生产菌株中表达D-苏糖脱氢酶，将表达来自石竹副伯克霍尔德菌(Pc.TadH)和/或来自油菜黄单胞菌(Xc.Fdh)的酶D-苏醛糖-1-脱氢酶的遗传信息或表达来自酿酒酵母(Sc.Ara1)或来自燕麦食酸菌(Aa.TadH)的酶D-阿拉伯糖脱氢酶的遗传信息或表达来自多食伯克霍尔德菌(Bm.Fdh)的酶L-岩藻糖脱氢酶的遗传信息引入该生产菌株的基因组中。
7.根据权利要求1至6中任一项所述的方法，其特征在于为了在该生产菌株中表达D-苏糖酸脱水酶，将表达来自燕麦食酸菌(Aa-AraD)和/或赫氏草螺菌(Hh-AraD)和/或含羞草副伯克霍尔德菌(Pm.AraD)的酶D-阿拉伯糖酸脱水酶的遗传信息和/或优化突变体Hh.AraDC434S的遗传信息引入该生产菌株的基因组中。
8.根据权利要求1至7中任一项所述的方法，其特征在于为了在该生产菌株中表达D-苏糖醛缩酶，将表达来自大肠杆菌(Ec.FsaA)的酶D-果糖-6-磷酸醛缩酶的遗传信息和/或突变变体Ec.FsaAL107Y:A129G(Ec.FsaATA)的遗传信息引入该生产菌株的基因组中。
9.根据权利要求1至8中任一项所述的方法，其特征在于为了在该生产菌株中表达苏糖酸-1,4-内酯酶，将表达来自Thermogutta terrifontis(Tt.Lac11)的酶葡糖酸内酯酶的遗传信息和/或该酶的截短变体(Tt.Lac11v1)的遗传信息引入该生产菌株的基因组中。
10.根据权利要求1至9中任一项所述的方法，其特征在于除了该代谢途径的酶之外，苏糖酸导入酶在该生产菌株中表达。
11.根据权利要求10所述的方法，其特征在于来自钩虫贪铜菌(Re.kdgT)的D-苏糖酸导入渗透酶在该生产菌株中表达。
12.根据权利要求1至11中任一项所述的方法，该方法进一步包括从乙二醇、甲醇或木糖进行微生物生产乙醇醛的至少一个先前步骤。
13.根据权利要求1-12中任一项所述的方法，其特征在于将与NADH相比对NADPH具有更高特异性的OHB还原酶用于将2-酮基-4-羟基丁酸(OHB)转化为2,4-二羟基丁酸(DHB)。
14.根据权利要求13所述的方法，其特征在于为了在该生产菌株中表达偏好NADPH的OHB还原酶，将表达来自大肠杆菌的酶L-苹果酸脱氢酶(Ec.Mdh)的突变变体的遗传信息引入该生产菌株的基因组中，其中与野生型酶相比，除了五个点突变I12V、R81A、M85Q、D86S和G179D之外，该突变酶在位置D34和I35的至少一个中具有另一个突变。
15.根据权利要求14所述的方法，其特征在于为了在该生产菌株中表达该偏好NADPH的OHB还原酶，将表达以下酶中一种的遗传信息引入该生产菌株的基因组中：
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:I35S，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35K，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35R(Ec.Mdh7Q)，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35S以及
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35T。
16.一种具有2-酮基-4-羟基丁酸(OHB)还原酶活性的酶，其催化2-酮基-4-羟基丁酸(OHB)转化为2,4-二羟基丁酸(DHB)，其中该酶是来自大肠杆菌的L-苹果酸脱氢酶(Ec.Mdh)的突变体，其中与野生型酶相比，除了五个点突变I12V、R81A、M85Q、D86S和G179D之外，该突变酶在位置D34和I35的至少一个中具有另一个突变。
17.根据权利要求15所述的酶，其特征在于该酶选自以下酶：
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:I35S，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35K，
Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35R(Ec.Mdh7Q)，Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35S以及Ec.Mdh I12V:R81A:M85Q:D86S:G179D:D34G:I35T。
18.根据权利要求16或17所述的酶用于将OHB转化为2,4-DHB的用途。
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