CN110551771B - 一种手性3-氨基-1-丁醇的合成方法 - Google Patents

一种手性3-氨基-1-丁醇的合成方法 Download PDF

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CN110551771B
CN110551771B CN201810552521.6A CN201810552521A CN110551771B CN 110551771 B CN110551771 B CN 110551771B CN 201810552521 A CN201810552521 A CN 201810552521A CN 110551771 B CN110551771 B CN 110551771B
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孙周通
赵强
刘保艳
刘贝贝
闫豪杰
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Abstract

本发明公开了一种手性3‑氨基‑1‑丁醇的合成方法。该方法包括如下步骤：以1，3‑丁二醇为底物，经酶A及其辅酶催化反应生成3‑酮‑1‑丁醇；以3‑酮‑1‑丁醇为底物，经酶B及其辅酶催化反应生成手性3‑氨基‑1‑丁醇；所述酶A选自醇脱氢酶或醇脱氢酶酶的突变体；所述酶B转氨酶或转氨酶的突变体。本发明提供了一条全新的绿色生物合成路线，以廉价的1,3‑丁二醇为原料，通过多酶共表达或级联或分步催化合成手性3‑氨基‑1‑丁醇，即(R)‑3‑氨基‑1‑丁醇和(S)‑3‑氨基‑1‑丁醇。

Description

一种手性3-氨基-1-丁醇的合成方法
技术领域
本发明属于生物技术领域，涉及一种手性3-氨基-1-丁醇的合成方法，特别涉及一种以利用生物酶催化合成手性3-氨基-1-丁醇的方法。
背景技术
含有手性氨基团的化合物在制药领域有着非常广泛的应用，其可以被用来合成多种具有生物活性的化合物，因此，开发一种廉价的、有效的合成手性氨化合物的方法在工业生产中有着非常广泛的需求。
(R)-3-氨基-1-丁醇(结构式如图1中A所示)是一种含有手性氨基团的、重要的手性药物中间体，在有机合成和制药领域有着非常广泛的应用。(R)-3-氨基-1-丁醇可以用来合成抗肿瘤药物4-甲基环磷酰胺和抗艾滋病药物Dolutegravir(2013年在美国上市，商品名Tivicay)，还可衍生为β-内酰胺，用于合成青霉烯类抗生素。目前，(R)-3-氨基-1-丁醇主要通过化学法合成。第一种方法为以手性(R)-丙氨酸为原料，经氨基保护后，以重氮甲烷增长碳链变为β-氨基酸酯，脱保护后还原得到目标产物(Gertzmann et al.，Tetrahedron，1995，51(33)，9031-9044)。该方法的缺点是高手性纯度的(R)-丙氨酸较难得到，重氮甲烷使用危险，不适合规模化工业化生产。第二种方法为巴豆酸酯与(R)-(+)-α-苯乙胺反应生成具有两个手性中心的一组差向异构体，通过硅胶柱层析分离后得到单一的异构体，然后经过酯还原、脱苄基得到(R)-3-氨基-1-丁醇(Kinas et al.，The Journal of organicchemistry，1977，42(9)，1650-1652)。该方法步骤较少，原料易得，是一种有希望实现工业化生产的方法，但是还存在以下问题，由于第一步反应选择性较差，得到几乎等量的两个差向异构体，分离纯化比较困难，常采用色谱柱法分离，洗脱剂用量大、损失大、效率低，同时由于使用了价格昂贵的LiAlH4作为还原剂，原材料成本也显著上升，由于效率与成本原因，柱层析方法不宜规模化工业化生产。第三种方法为以3-酮丁酸乙酯为原料，经生物还原法得到3(S)-羟基丁酸乙酯，并在其后的反应步骤中在Witsunobu试剂催化下与酞酰亚胺反应，发生Walden转化，成功的把3(S)构型改变为所需要的3(R)构型中间体，并最终得到目标物(Besse et al.，Tetrahedron：Asymmetry，1999，10(11)，2213-2224)。但是，冗长的合成路线，使用比较昂贵的Witsunobu试剂和价格既贵又具危险性的四氢铝锂等众多因素使其难以实现产业化。由于化学法合成(R)-3-氨基-1-丁醇的存在许多不足。因此，寻找新的绿色方法来合成(R)-3-氨基-1-丁醇，对促进现有化学工艺的绿色升级和加强对环境的保护具有重要的科学价值和社会意义。
1,3-丁二醇是一种重要的化工原料，具有易于获得、价格低等特点。
发明内容
本发明所要解决的技术问题是合成手性3-氨基-1-丁醇。
为解决上述技术问题，本发明首先提供了一种利用生物酶催化合成手性3-氨基-1-丁醇的方法，可包括如下步骤(反应原理见图2)：
(A)以1，3-丁二醇为底物，经酶A及其辅酶催化反应生成3-酮-1-丁醇；
(B)以步骤(A)生成的3-酮-1-丁醇为底物，经酶B及其辅酶催化反应生成手性3-氨基-1-丁醇；
所述酶A选自如下任一种：醇脱氢酶、所述醇脱氢酶的突变体；
所述酶B选自如下任一种：转氨酶、所述转氨酶的突变体。
本发明所提供的方法通过多酶共表达或级联或分步催化的方法实现。
进一步地，所述醇脱氢酶均可来源于如下任一微生物：短小乳杆菌、高温厌氧杆菌、高加索酸奶乳杆菌、威吉利热厌氧杆菌、酒球菌、致黑脱硫肠状菌、一氧化碳嗜热窦菌、烷嗜热厌氧杆菌、厚壁菌门细菌、甲烷八叠球菌、拜氏梭菌、未培养梭菌、丝状孢子梭菌、脱硫肠状菌、二醇梭菌、匙形梭菌、笃斯梭菌、梭菌属细菌、近平滑假丝酵母；
进一步地，所述转氨酶来源于如下任一微生物：巨大芽胞杆菌、铜绿假单胞菌、紫色杆菌、土曲霉、费希新萨托菌、分支杆菌、节细菌属。
更进一步地，所述醇脱氢酶具体可为如下(a1)-(a20)中任一：
(a1)来源于短小乳杆菌(Lactobacillus brevis)的醇脱氢酶，氨基酸序列为SEQID No.2；
(a2)来源于高温厌氧杆菌(Thermoanaerobacter brockii)的醇脱氢酶，氨基酸序列为SEQ ID No.4；
(a3)来源于高加索酸奶乳杆菌(Lactobacillus kefiri DSM 20587)的醇脱氢酶，氨基酸序列为SEQ ID No.6；
(a4)来源于威吉利热厌氧杆菌(Thermoanaerobacter wiegelii Rt8.B1)的醇脱氢酶，氨基酸序列为SEQ ID No.8；
(a5)来源于酒球菌(Oenococcus alcoholitolerans)的醇脱氢酶，氨基酸序列为SEQ ID No.10；
(a6)来源于致黑脱硫肠状菌(Desulfotomaculum nigrificans)的醇脱氢酶，氨基酸序列为SEQ ID No.12；
(a7)来源于一氧化碳嗜热窦菌(Thermosinus carboxydivorans)的醇脱氢酶，氨基酸序列为SEQ ID No.14；
(a8)来源于烷嗜热厌氧杆菌(Thermoanaerobacter mathranii)的醇脱氢酶，氨基酸序列为SEQ ID No.16；
(a9)来源于厚壁菌门细菌(Firmicutes bacterium CAG:137)的醇脱氢酶，氨基酸序列为SEQ ID No.18；
(a10)来源于甲烷八叠球菌(Methanosarcina thermophila)的醇脱氢酶，氨基酸序列为SEQ ID No.20；
(a11)来源于拜氏梭菌(Clostridium beijerinckii)的醇脱氢酶，氨基酸序列为SEQ ID No.22；
(a12)来源于未培养梭菌(uncultured Clostridium sp.)的醇脱氢酶，氨基酸序列为SEQ ID No.24；
(a13)来源于丝状孢子梭菌(Clostridium taeniosporum)的醇脱氢酶，氨基酸序列为SEQ ID No.26；
(a14)来源于脱硫肠状菌(Desulfotomaculum putei)的醇脱氢酶，氨基酸序列为SEQ ID No.28；
(a15)来源于二醇梭菌(Clostridium diolis)的醇脱氢酶，氨基酸序列为SEQ IDNo.30；
(a16)来源于匙形梭菌(Clostridium cochlearium)的醇脱氢酶，氨基酸序列为SEQ ID No.32；
(a17)来源于笃斯梭菌(Clostridium uliginosum)的醇脱氢酶，氨基酸序列为SEQID No.34；
(a18)来源于梭菌属细菌(Clostridiales bacterium SK-Y3)的醇脱氢酶，氨基酸序列为SEQ ID No.36；
(a19)来源于近平滑假丝酵母(Candida parapsilosis)的醇脱氢酶，氨基酸序列为SEQ ID No.38；
(a20)在(a1)-(a19)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
更进一步地，所述转氨酶具体可为如下(b1)-(b8)中任一：
(b1)来源于巨大芽胞杆菌(Bacillus megaterium)的转氨酶，氨基酸序列为SEQID No.40；
(b2)来源于铜绿假单胞菌(P.aeruginosa PAO2)的转氨酶，氨基酸序列为SEQ IDNo.42；
(b3)来源于紫色杆菌(Chromobacterium violaceum 2025)的转氨酶，氨基酸序列为SEQ ID No.44；
(b4)来源于土曲霉(Aspergillus terreus)的转氨酶，氨基酸序列为SEQ IDNo.46；
(b5)来源于费希新萨托菌(Neosartorya fischeri)的转氨酶，氨基酸序列为SEQID No.48；
(b6)来源于分支杆菌(Mycobacterium vanbaalenii)的转氨酶，氨基酸序列为SEQID No.50；
(b7)来源于节细菌属(Arthrobacter sp.KNK168)的转氨酶，氨基酸序列为SEQ IDNo.52；
(b8)在(b1)-(b7)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
进一步地，所述醇脱氢酶的突变体具体可为如下(c1)-(c5)：
(c1)与SEQ ID No.2所示来源于短小乳杆菌的醇脱氢酶相比，存在或仅存在如下突变中的至少一种：I11V、G37D；
(c2)与SEQ ID No.4所示来源于高温厌氧杆菌的醇脱氢酶相比，存在或仅存在如下突变中的至少一种：A85G、I86A、W110A、G198D；
进一步地，所述醇脱氢酶的突变体为与SEQ ID No.4所示来源于高温厌氧杆菌的醇脱氢酶相比，存在或仅存在如下突变：A85G/I86A或者A85G/I86A/W110A或者A85G/I86A/G198D；
(c3)与SEQ ID No.6所示来源于高加索酸奶乳杆菌的醇脱氢酶相比，存在或仅存在如下突变：G37D；
(c4)与SEQ ID No.8所示来源于威吉利热厌氧杆菌的醇脱氢酶相比，存在或仅存在如下突变：G198D；
(c5)在(c1)-(c4)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
在本发明中，对于氨基酸取代，使用下述命名法：原始氨基酸，位置，取代氨基酸。如，在SEQ ID No.2的第11位用缬氨酸(V)取代原有的异亮氨酸(I)命名为“I11V”。包含多重改变的变体由斜杠符号(“/”)分隔。
进一步地，在所述方法中，所述酶A和所述酶B均可以粗酶液、粗酶液冻干粉、纯酶或全细胞的形式发生催化作用的。
进一步，所述粗酶液、粗酶液冻干粉和纯酶均按照包括如下步骤的方法制备得到：在宿主细胞中表达所述酶A和/或所述酶B，得到重组细胞；裂解所述重组细胞获得所述粗酶液、粗酶液冻干粉或纯酶。所述全细胞可按照包括如下步骤的方法制备得到：在宿主细胞中表达所述酶A和/或所述酶B，得到的重组细胞即为所述酶A和/或所述酶B的全细胞；
再进一步，所述重组细胞具体可按照包括如下步骤的方法制备获得的：向所述宿主细胞到导入能够表达所述酶A和/或所述酶B的核酸分子，经诱导培养后获得表达所述酶A和/或所述酶B的所述重组细胞。
更进一步，所述“能够表达所述酶A和/或所述酶B的核酸分子”是通过重组载体的形式导入到所述宿主细胞中的。其中，所述重组载体可为携带有所述酶A和/或所述酶B的编码基因的细菌质粒(如在细菌中表达的基于T7启动子的表达载体，具体如pET-28a等)、噬菌体、酵母质粒(如YEp系列载体等)或逆转录病毒包装质粒。
在本发明的一个实施例中，所述重组载体具体为将所述酶A或所述酶B的编码基因替换pET-22b载体的酶切位点NdeⅠ和XhoⅠ之间的小片段后得到的重组质粒。
在本发明的另一个实施例中，所述重组载体为将所述酶A的编码基因插入到pETDuet-1载体的酶切位点酶切位点EcoRⅠ和HindIII之间，同时将所述酶B的编码基因插入到pETDuet-1载体的酶切位点NdeⅠ和XhoⅠ之间后得到的重组质粒。
进一步地，所述宿主细胞可为原核细胞或低等真核细胞。
更进一步地，所述原核细胞具体可为细菌。所述低等真核细胞具体可为酵母细胞。
在本发明的一个实施例中，所述宿主细胞具体为大肠杆菌，更加具体的为E.coliBL21(DE3)。相应的，所述诱导培养为向培养体系中加IPTG至终浓度0.1-0.5mM(具体如0.1mM)，20-37℃诱导培养12-24h(具体如16h)。
所述来源于短小乳杆菌的醇脱氢酶的编码基因的序列为SEQ ID No.1或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于高温厌氧杆菌的醇脱氢酶的编码基因的序列为SEQ ID No.3或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于高加索酸奶乳杆菌的醇脱氢酶的编码基因的序列为SEQ ID No.5或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于威吉利热厌氧杆菌的醇脱氢酶的编码基因的序列为SEQ ID No.7或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于酒球菌的醇脱氢酶的编码基因的序列为SEQ ID No.9或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于致黑脱硫肠状菌的醇脱氢酶的编码基因的序列为SEQ ID No.11或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于一氧化碳嗜热窦菌的醇脱氢酶的编码基因的序列为SEQ ID No.13或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于烷嗜热厌氧杆菌的醇脱氢酶的编码基因的序列为SEQ ID No.15或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于厚壁菌门细菌的醇脱氢酶的编码基因的序列为SEQ ID No.17或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于甲烷八叠球菌的醇脱氢酶的编码基因的序列为SEQ ID No.19或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于拜氏梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.21或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于未培养梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.23或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于丝状孢子梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.25或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于脱硫肠状菌的醇脱氢酶的编码基因的序列为SEQ ID No.27或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于二醇梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.29或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于匙形梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.31或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于笃斯梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.33或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于梭菌属细菌的醇脱氢酶的编码基因的序列为SEQ ID No.35或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于近平滑假丝酵母的醇脱氢酶的编码基因的序列为SEQ ID No.37或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于巨大芽胞杆菌的转氨酶的编码基因的序列为SEQ ID No.39或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于铜绿假单胞菌的转氨酶的编码基因的序列为SEQ ID No.41或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于紫色杆菌的转氨酶的编码基因的序列为SEQ ID No.43或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于土曲霉的转氨酶的编码基因的序列为SEQ ID No.45或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于费希新萨托菌的转氨酶的编码基因的序列为SEQ ID No.47或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于分支杆菌的转氨酶的编码基因的序列为SEQ ID No.49或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于节细菌属的转氨酶的编码基因的序列为SEQ ID No.51或在其5’端和/或3’端连接标签编码序列后得到的融合序列或保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于短小乳杆菌的醇脱氢酶的突变体的编码基因的序列为如下(d1)-(d3)中任一：(d1)与SEQ ID No.1相比，存在或仅存在如下突变中的至少一种：A31G/T33G、G110A/C111T；(d2)在(d1)所限定序列的5’端和/或3’端连接标签编码序列后得到的融合序列；(d3)与(d1)或(d2)所限定的序列相比保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于高温厌氧杆菌的醇脱氢酶的突变体的编码基因的序列为如下(e1)-(e3)中任一：(e1)与SEQ ID No.3相比，存在或仅存在如下突变中的至少一种：C254G/T255C、A256G/T257C/T258G、T328G/G329C、G593A/C594T；(e2)在(e1)所限定序列的5’端和/或3’端连接标签编码序列后得到的融合序列；(e3)与(e1)或(e2)所限定的序列相比保有功能且编码相同蛋白的随机或/和定点诱变序列。
进一步，所述(e1)为：与SEQ ID No.3相比，存在或仅存在如下突变中的任一种：C254G/T255C/A256G/T257C/T258G、C254G/T255C/A256G/T257C/T258G/T328G/G329C、C254G/T255C/A256G/T257C/T258G/G593A/C594T。
所述来源于高加索酸奶乳杆菌的醇脱氢酶的突变体的编码基因的序列为如下(f1)-(f3)中任一：(f1)与SEQ ID No.5相比，存在或仅存在如下突变：G110A/C111T；(f2)在(f1)所限定序列的5’端和/或3’端连接标签编码序列后得到的融合序列；(f3)与(f1)或(f2)所限定的序列相比保有功能且编码相同蛋白的随机或/和定点诱变序列。
所述来源于威吉利热厌氧杆菌的醇脱氢酶的突变体的编码基因的序列为如下(g1)-(g3)中任一：(g1)与SEQ ID No.7相比，存在或仅存在如下突变中的至少一种：G593A/C594T；(g2)在(g1)所限定序列的5’端和/或3’端连接标签编码序列后得到的融合序列；(g3)与(g1)或(g2)所限定的序列相比保有功能且编码相同蛋白的随机或/和定点诱变序列。
在本发明中，对于碱基取代，使用下述命名法：原始碱基，位置(即在W1或W2或W3或W4核苷酸序列中的位置)，取代碱基。相应的，在SEQ ID No.1的第31位用A取代原有的G命名为“A31G”。包含多重改变的变体由斜杠符号(“/”)分隔。
在步骤(A)和步骤(B)中，所述催化反应的温度均可为25～37℃，如30～37℃，具体如30℃或37℃。
在步骤(A)和步骤(B)中，所述催化反应的时间均可为4～48h，如24h。
当所述酶A和所述酶B是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，步骤(A)中，所述催化反应可在如下(h1)所示缓冲液中进行；步骤(B)中，所述催化反应可在如下(h2)所示缓冲液中进行；当所述酶A和所述酶B以共表达所述酶A和所述酶B的全细胞的形式发生催化作用时，步骤(A)和步骤(B)的所述催化反应均可在如下(h1)所示缓冲液中进行。(h1)浓度为50～100mM，pH值为6.5～8.0的磷酸盐缓冲液；具体如：浓度为100mM，pH值为8.0的磷酸盐缓冲液。(h2)浓度为50～100mM，pH值为7.5～8.5的磷酸盐缓冲液；具体如：浓度为100mM，pH值为7.5～8.5的磷酸盐缓冲液。
当所述酶A和所述酶B是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，步骤(A)和步骤(B)中，所述酶A和所述酶B在各自反应体系中的浓度均可为0.1g/L～10g/L，如10g/L。当所述酶A和所述酶B以共表达所述酶A和所述酶B的全细胞的形式发生催化作用时，步骤(A)和步骤(B)在一个反应体系中完成，所述反应体系中所述全细胞(共表达所述酶A和所述酶B)的浓度为100g/L(每升反应体系中含有所述全细胞的湿重为100g)。
在本发明中，所述酶A的辅酶具体可为氧化型辅酶I(即NAD+)或氧化型辅酶II(即NADP+)。所述酶B为转氨酶或所述转氨酶的突变体时，所述酶B的辅酶具体为磷酸吡哆醛(PLP)。
在本发明中，当所述酶A和所述酶B是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，所述酶A和所述酶B的辅酶在其各自的反应体系中的浓度均可为0.1～3mM(具体如1mM)。
当所述酶A和所述酶B是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，步骤(A)中，所述催化反应的反应体系中除了含有1，3-丁二醇和所述酶A及其辅酶外，还含有丙酮。
具体的，步骤(A)中，所述催化反应的反应体系组成如下：浓度为100mM，pH值为8.0的磷酸盐缓冲、终浓度为20mM的1，3-丁二醇、终浓度为1mM的氧化型辅酶I(即NAD+)或氧化型辅酶II(即NADP+)、终浓度为5％的丙酮(v/v)、终浓度为10g/L的所述酶A的粗酶液、粗酶液冻干粉或纯酶。
当所述酶A和所述酶B是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，在步骤(B)中，当所述酶B为转氨酶时，所述催化反应的反应体系中除了含有3-酮-1-丁醇、所述酶B及其辅酶磷酸吡哆醛外，还含有异丙胺或丙氨酸；
具体的，步骤(B)中，当所述酶B为转氨酶或所述转氨酶的突变体时，所述催化反应的反应体系的组成可如下：浓度为50～100mM，pH值为8.0的磷酸盐缓冲液、终浓度为500mM的异丙胺(或丙氨酸)、终浓度为1mM的磷酸吡哆醛(PLP)、终浓度为10g/L的所述酶B的粗酶液、粗酶液冻干粉或纯酶。
当所述酶A和所述酶B以共表达所述酶A和所述酶B的全细胞的形式发生催化作用时，所述反应体系中可以不加入辅酶，进一步地，所述反应体系中还含有葡萄糖。
具体的，所述反应体系的组成可为如下：浓度为100mM、pH值为8.0的磷酸盐缓冲液；终浓度为50mM的1，3-丁二醇；100mM葡萄糖；终浓度为100g/L的所述全细胞(即每升反应体系中含有所述全细胞的湿重为100g)。
在所述方法中，所述手性3-氨基-1-丁醇为(R)-3-氨基-1-丁醇和/或(S)-3-氨基-1-丁醇。
上文中，来源于费希新萨托菌的转氨酶、来源于分支杆菌的转氨酶、来源于土曲霉的转氨酶或来源于节细菌属的转氨酶均可用于(R)-3-氨基-1-丁醇的合成。来源于巨大芽胞杆菌的转氨酶、来源于铜绿假单胞菌的转氨酶、来源于紫色杆菌的转氨酶均可用于(S)-3-氨基-1-丁醇的合成。
本发明还提供了一种酶系统及其相关产品。
本发明所提供的酶系统包括前文所述酶A和所述酶B。当然，也可以包括所述酶A和所述酶B各自的辅酶。
所述相关产品可为能够表达所述酶系统中各酶的核酸分子，或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
所述酶系统或所述相关产品在合成手性3-氨基-1-丁醇中的应用也属于本发明的保护范围。
本发明所提供的合成手性3-氨基-1-丁醇的方法中，存在辅因子再生系统。所述辅因子再生系统为醇脱氢酶催化丙酮还原或异丙醇氧化促进辅因子再生。在本发明合成手性3-氨基-1-丁醇的方法中，醇脱氢酶催化1，3-丁二醇氧化成3-酮-1-丁醇，NAD(P)+被还原成NAD(P)H，同时，醇脱氢酶催化丙酮还原成异丙醇，NAD(P)H被重新氧化成NAD(P)+，生成的NAD(P)+重新参与到1，3-丁二醇生成3-酮-1-丁醇的氧化。
本发明提供了一条全新的绿色生物合成路线，以廉价的1,3-丁二醇为原料，通过多酶共表达或级联或分步催化合成手性3-氨基-1-丁醇，即(R)-3-氨基-1-丁醇和/或(S)-3-氨基-1-丁醇。
附图说明
图1为(R)-3-氨基-1-丁醇和(S)-3-氨基-1-丁醇的结构式。
图2为醇脱氢酶偶联转氨酶制备(R)-3-氨基-1-丁醇或者(S)-3-氨基-1-丁醇的反应原理图。
图3为3-酮-1-丁醇气相色谱(GC)鉴定结果。
图4为3-氨基-1-丁醇标准品液相色谱结果。A为混旋型3-氨基-1-丁醇标准品；B为(R)-3-氨基-1-丁醇标准品。
图5为反应液液相色谱图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、醇脱氢酶偶联转氨酶制备手性3-氨基-1-丁醇
一、醇脱氢酶或其突变体、转氨酶的工程菌的制备
将相关酶的编码基因分别进行全基因合成(根据需要以大肠杆菌为宿主进行密码子优化)，将合成的基因连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体。本发明所述载体具体为pET22b(+)，将全基因合成后的相关酶的编码基因插入到pET22b(+)的酶切位点NdeⅠ和XhoⅠ之间后，并经测序验证正确后得到重组载体。并利用定点突变方法得到其相关基因突变体。
将上述测序验证正确的重组表达载体转化至合适的微生物宿主中。所述宿主微生物为本领域常规的各种宿主微生物，只要能满足上述重组表达载体稳定的自行复制以及醇脱氢酶或转氨酶基因可被有效表达即可。其中，较佳的所述宿主微生物为大肠杆菌(Escherichia coli)，优选大肠杆菌BL21(DE3)，将前述重组表达质粒转化入E.coli BL21(DE3)中，可得到本发明的基因工程菌株。其中所述转化方法为本领域常规转化方法，较佳的为电转法或化学转化法。
本实施例中所涉及的醇脱氢酶或其突变体、转氨酶详见表1。
表1.涉及的酶及其突变体
Figure BDA0001680735120000071
Figure BDA0001680735120000081
Figure BDA0001680735120000091
注：W1表示来源于短小乳杆菌(Lactobacillus brevis)的醇脱氢酶；W2表示来源于高温厌氧杆菌(Thermoanaerobacter brockii)的醇脱氢酶；W3表示来源于高加索酸奶乳杆菌(Lactobacillus kefiri DSM 20587)的醇脱氢酶；W4表示来源于威吉利热厌氧杆菌(Thermoanaerobacter wiegelii Rt8.B1)的醇脱氢酶；W5表示来源于酒球菌(Oenococcusalcoholitolerans)的醇脱氢酶；W6表示来源于致黑脱硫肠状菌(Desulfotomaculumnigrificans)的醇脱氢酶；W7表示来源于一氧化碳嗜热窦菌(Thermosinuscarboxydivorans)的醇脱氢酶；W8表示来源于烷嗜热厌氧杆菌(Thermoanaerobactermathranii)的醇脱氢酶；W9表示来源于厚壁菌门细菌(Firmicutes bacterium CAG:137)的醇脱氢酶；W10表示来源于甲烷八叠球菌(Methanosarcina thermophila)的醇脱氢酶；W11表示来源于拜氏梭菌(Clostridium beijerinckii)的醇脱氢酶；W12表示来源于未培养梭菌(uncultured Clostridium sp.)的醇脱氢酶；W13表示来源于丝状孢子梭菌(Clostridium taeniosporum)的醇脱氢酶；W14表示来源于脱硫肠状菌(Desulfotomaculumputei)的醇脱氢酶；W15表示来源于二醇梭菌(Clostridium diolis)的醇脱氢酶；W16表示来源于匙形梭菌(Clostridium cochlearium)的醇脱氢酶；W17表示来源于笃斯梭菌(Clostridium uliginosum)的醇脱氢酶；W18表示来源于梭菌属细菌(Clostridialesbacterium SK-Y3)的醇脱氢酶；W19表示来源于近平滑假丝酵母(Candida parapsilosis)的醇脱氢酶；W20表示来源于巨大芽胞杆菌(Bacillus megaterium)的转氨酶；W21表示来源于铜绿假单胞菌(P.aeruginosa PAO2)的转氨酶；W22表示来源于紫色杆菌(Chromobacterium violaceum 2025)的转氨酶；W23表示来源于土曲霉(Aspergillusterreus)的转氨酶；W24表示来源于费希新萨托菌(Neosartorya fischeri)的转氨酶；W25表示来源于分支杆菌(Mycobacterium vanbaalenii)的转氨酶；W26表示来源于节细菌属(Arthrobacter sp.KNK168)的转氨酶。Wn-Mn表示Wn(n为自然数)的突变体。蛋白取代的编号是从Wn(n为自然数)所示野生型氨基酸序列的N端为起始的；基因取代的编号是从Wn(n为自然数)所示野生型核苷酸序列的5’端为起始的。表中，对于氨基酸取代，使用下述命名法：原始氨基酸，位置(即在Wn氨基酸序列中的位置)，取代氨基酸。相应地，在SEQ ID No.2的第11位用缬氨酸(V)取代原有的异亮氨酸(I)命名为“I11V”。对于碱基取代，使用下述命名法：原始碱基，位置(即在Wn核苷酸序列中的位置)，取代碱基。相应的，在SEQ ID No.1的第31位用A取代原有的G命名为“A31G”。包含多重改变的变体由斜杠符号(“/”)分隔。
二、醇脱氢酶或其突变体、转氨酶的表达及粗酶制备
将步骤一构建的重组表达载体转入大肠杆菌BL21(DE3)感受态细胞中，37℃培养12-16h，待转化子长出，将单个转化子接种到含有相应抗生素的5mL LB培养基中，37℃、220rmp培养过夜(12-16h)。然后按照1％(体积百分含量)的比例接种到TB培养基中，37℃、220rmp培养到OD600nm为0.6左右，添加终浓度为0.1mM的IPTG，20-37℃诱导培养16h，然后4000rpm，4℃离心10min收集细胞，用100mM，pH值为8.0的磷酸盐缓冲重悬清洗一次，之后超声破菌并制备酶冻干粉。
三、手性3-氨基-1-丁醇的制备
第一步反应：在反应体系中依次加入浓度为100mM，pH值为8.0的磷酸盐缓冲液、终浓度为20mM的1，3-丁二醇、终浓度为1mM的氧化型辅酶I(即NAD+)或氧化型辅酶II(即NADP+)、终浓度为5％(v/v)的丙酮和终浓度为10g/L的醇脱氢酶冻干粉或酶液组成反应体系。将该反应体系在30℃的条件下反应24h后，对产物进行气相色谱(GC)检测。
气相色谱(GC)的检测条件如下：进样量：2μL；色谱柱：HP-5；分流比：20:1；分流流量：40mL/min；升温程序：40℃，5分钟；5℃/min升温到60℃，2分钟；30℃/min升温到200℃，2.333分钟。运行时间：18分钟。
结果参考图3，证明该步反应得到3-酮-1-丁醇。
第二步反应：以上一步反应生成的3-酮-1-丁醇为底物，在反应体系中依次加入浓度为100mM，pH值为8.0的磷酸盐缓冲液、终浓度为500mM的异丙胺(或丙氨酸)、终浓度为1mM的磷酸吡哆醛(PLP)、终浓度为10g/L的转氨酶冻干粉或酶液组成反应体系。将该反应体系在30℃的条件下反应24h，得到的反应液用邻苯二甲醛衍生后液相色谱检测。同时将混旋型3-氨基-1-丁醇标准品和(R)-3-氨基-1-丁醇标准品进行液相色谱检测。
HPLC检测条件如下：Agilent SB-Aq C18柱(4.6mm*250mm,5μLm)；检测波长334nm；柱温：35℃；流速：1mL/min；梯度洗脱程序如表2所示。
表2.HPLC的梯度洗脱程序
时间(min) 流动相(甲醇)％ 流动相(0.05M乙酸钠)％
0 30 70
6 30 70
7 45 55
15 45 55
15.5 30 70
20 30 70
注：表中的％表示体积百分含量。
ee1＝(AS-AR)/(AS+AR)×100％；ee2＝(AR-AS)/(AS+AR)×100％；AS为液相色谱分析获得的(S)-3-氨基-1-丁醇的峰面积值；AR为液相色谱分析获得的(R)-3-氨基-1-丁醇的峰面积值；ee1大于95％说明产物构型是S；ee2大于95％说明产物构型是R。
底物转化效率＝C转/C总×100％；C转为反应体系中转化为(R)-3-氨基-1-丁醇或(S)-3-氨基-1-丁醇的底物的摩尔数；C总为反应体系中底物的总摩尔数。
混旋型3-氨基-1-丁醇标准品的液相色谱结果见图4中A。(R)-3-氨基-1-丁醇标准品的液相色谱结果见图4中B。部分反应液液相色谱图见图5(A为混旋型3-氨基-1-丁醇标准品；B为阴性对照反应液；C为实验组1反应液(产物为(R)-3-氨基-1-丁醇)；D为实验组2反应液(产物为(S)-3-氨基-1-丁醇)。其中，阴性对照反应体系中只含有空表达载体的表达宿主粗酶粉或酶液或全细胞，其它成分与实验组相同)。底物转化效率为40-60％。具体结果参见表3。结果表明，醇脱氢酶或其突变体偶联转氨酶W20、W21或W22可以制备(S)-3-氨基-1-丁醇，醇脱氢酶或其突变体偶联转氨酶W23、W24、W25或W26可以制备(R)-3-氨基-1-丁醇。
表3-1.醇脱氢酶或其突变体偶联转氨酶制备(S)-3-氨基-1-丁醇的结果
Figure BDA0001680735120000101
Figure BDA0001680735120000111
注：表中第一步反应和第二步反应中Wn和Wn-Mn(n为自然数)所代表的含义与表1相同。
表3-2.醇脱氢酶或其突变体偶联转氨酶制备(R)-3-氨基-1-丁醇的结果
Figure BDA0001680735120000112
Figure BDA0001680735120000121
Figure BDA0001680735120000131
注：表中第一步反应和第二步反应中Wn和Wn-Mn(n为自然数)所代表的含义与表1相同。
实施例2、酶A和酶B共表达全细胞制备手性3-氨基-1-丁醇
酶A为表1所示的醇脱氢酶或其突变体。酶B为表1所示的转氨酶。
一、酶A和酶B共表达工程菌的制备
将相关酶的编码基因分别进行全基因合成(根据需要以大肠杆菌为宿主进行密码子优化)，将合成的基因连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体。本发明所述载体具体pETDuet-1，将全基因合成后酶A的DNA片段插入到pETDuet-1的酶切位点EcoRI和HindIII之间，将全基因合成后酶B的DNA片段插入到pETDuet-1的酶切位点NdeI和XhoI之间。将重组载体转入大肠杆菌DH5α感受态细胞；挑取阳性转化子并测序鉴定后，得到正确的重组表达载体。
将上述测序验证正确的重组表达载体转化至合适的微生物宿主中。所述宿主微生物为本领域常规的各种宿主微生物，只要能满足上述重组表达载体稳定的自行复制以及醇脱氢酶和转氨酶基因同时被有效表达即可。其中所述宿主微生物较佳地为：大肠杆菌(Escherichia coli)，优选地为大肠杆菌BL21(DE3)，将前述重组表达质粒转化入E.coliBL21(DE3)中，可得到本发明的基因工程菌株。其中所述转化方法为本领域常规转化方法，较佳地为电转法或化学转化法。
二、酶A和酶B的共表达
将步骤一构建的重组表达载体转入大肠杆菌BL21(DE3)感受态细胞中，37℃培养12-16h，待转化子长出，将单个转化子接种到含有相应抗生素的5mL LB培养基中，37℃、220rmp培养过夜(12-16h)。然后按照1％(体积百分含量)的比例接种到TB培养基中，37℃、220rmp培养到OD600nm为0.6左右，添加终浓度为0.1mM的IPTG，20-37℃诱导培养16h，然后4℃、4000rpm离心10min收集细胞。
三、手性3-氨基-1-丁醇的制备
在反应体系中依次加入浓度为100mM、pH值为8.0的磷酸盐缓冲液，终浓度为50mM的1，3-丁二醇，100mM葡萄糖，终浓度为100g/L的能够共表达酶A和酶B的全细胞(湿菌重)组成反应体系。将该反应体系在30℃的条件下反应24h后，得到的发酵液用邻苯二甲醛衍生后液相色谱检测。同时将混旋型3-氨基-1-丁醇标准品和(R)-3-氨基-1-丁醇标准品进行液相色谱检测。
HPLC检测条件如实施例1中步骤三所示。
ee1值、ee2值和底物转化效率的具体计算方法同实施例1中步骤三。
结果表明，底物转化效率15-30％。具体结果参见表4。
表4-1.酶A和B共表达全细胞催化制备(S)-3-氨基-1-丁醇的结果
Figure BDA0001680735120000141
Figure BDA0001680735120000151
注：表中Wn和Wn-Mn(n为自然数)所代表的含义与表1相同。
表4-2.酶A和B共表达全细胞催化制备(R)-3-氨基-1-丁醇的结果
Figure BDA0001680735120000152
Figure BDA0001680735120000161
注：表中Wn和Wn-Mn(n为自然数)所代表的含义与表1相同。
<110> 中国科学院天津工业生物技术研究所
<120> 一种手性3-氨基-1-丁醇的合成方法
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Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 7
<211> 1059
<212> DNA
<213> 威吉利热厌氧杆菌（Thermoanaerobacter wiegelii Rt8.B1）
<400> 7
atgaaaggtt ttgcaatgct cagtatcggt aaggttggct ggattgaggt agaaaagcct 60
aatccaggac cctttgatgc tatcgtaaga cccctagctg tggccccttg ctcttcggac 120
attcacactg tttttgaagg aggccttggt gaacttcaca acgcagtgct aggtcacgaa 180
gctgtaggtg aagtagtcga agtcggtagt gaagtaaaag actttaaacc tggtgataag 240
gtggtcattc ctgctatcac tcctgattgg agaacgttag atgttcaacg tggttatcat 300
cagcagtccg gaggtatgct tgctggttac aagttcacag cccagaaacc tggtgtgttc 360
gccgagtaca tctacgttaa cgatgcagac atgaatcttg ctcatttacc tgacggcatc 420
tctttagaag cggccgtcat gatcacagat atgatgacta ccggttttca cggagccgaa 480
ctggcagaaa tagaattagg tgcaacagta gcggttttgg gtattggtcc agtaggtctt 540
atggcagtcg ctggtgccaa attgcggggt gctggaagaa ttattgcagt aggcagtaga 600
cctgtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaaaat 660
ggtcctatcg acagtcagat tatggattta acgaaaggca aaggtgttga tgctgccatc 720
atcgctggag gaaatgttga catcatggct acagcagtta agattgttaa acctggtggc 780
accattgcta atgtaaatta ctttggcgaa ggagatgttt tgcctgttcc tcgtcttgaa 840
tggggttgcg gcatggctca taaagctata aaaggcggtt tatgccctgg tggacgtcta 900
agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc caaactcgtc 960
actcatgttt ttcaaggatt tgataatatt gaaaaagctc taatgctgat gaaagataaa 1020
ccaaaggacc taatcaaacc tgttgtaata ttagcataa 1059
<210> 8
<211> 352
<212> PRT
<213>威吉利热厌氧杆菌（Thermoanaerobacter wiegelii Rt8.B1）
<400> 8
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Val Glu Lys Pro Asn Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Ser Ser Asp Ile His Thr Val Phe Glu Gly Gly
35 40 45
Leu Gly Glu Leu His Asn Ala Val Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Lys
65 70 75 80
Val Val Ile Pro Ala Ile Thr Pro Asp Trp Arg Thr Leu Asp Val Gln
85 90 95
Arg Gly Tyr His Gln Gln Ser Gly Gly Met Leu Ala Gly Tyr Lys Phe
100 105 110
Thr Ala Gln Lys Pro Gly Val Phe Ala Glu Tyr Ile Tyr Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Asp Gly Ile Ser Leu Glu Ala
130 135 140
Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Glu Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asn Gly Pro Ile Asp
210 215 220
Ser Gln Ile Met Asp Leu Thr Lys Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Val Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Asp
260 265 270
Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Ala Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
290 295 300
Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
305 310 315 320
Thr His Val Phe Gln Gly Phe Asp Asn Ile Glu Lys Ala Leu Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
<210> 9
<211> 762
<212> DNA
<213> 酒球菌（Oenococcus alcoholitolerans）
<400> 9
atggttgatc gcctgaaagg taaagttgcc attgttaccg gcggcaccct gggtattggt 60
ctgagcattg ttgatctgta tctgaaagaa ggcgcaaaag ttgtttttac cggccgccgt 120
gaaaatgtgg gcaaaaaagc atatcaggat ctgggtagtc cgaaaaatgc aaaatttgtt 180
gtgcatgatg cagccgatga agaaggctgg aaaaaactgt ttgcagatac cattgccgaa 240
tttggtaaag tggatattct ggtgaataat gcaggtattg gcgttccggg taatgtggaa 300
aataccgatt atgcacagtg gcgtcagaca atggatgtta atctggatgg tgtttatctg 360
ggcacccatt atggcgttat taatatgaaa aacccgcaga gtggcgatgc cagcattatt 420
aatatgagca gcattgaagg tctggtgggc gatccgaatc tgtttgccta taatgccacc 480
aaaggtgccc tgcgtattat gagtaaaagt gcagccatct attgcgcaaa tcaggattat 540
aatctgcgca ttaataccat tcatccgggt tatattaaga ccccgctggt ggataaaatt 600
cagggcgccg aagaaatgat gagccagcgc accaaaaccc cgatgggcca tgttggcgtg 660
ccggatgata ttggctggct ggcagtttat ctgggtagcg aagaaagtaa atttgcaacc 720
ggcgccgagt ttactgtgga tggtggctat accgcccagt aa 762
<210> 10
<211> 253
<212> PRT
<213> 酒球菌（Oenococcus alcoholitolerans）
<400> 10
Met Val Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ser Ile Val Asp Leu Tyr Leu Lys Glu Gly Ala
20 25 30
Lys Val Val Phe Thr Gly Arg Arg Glu Asn Val Gly Lys Lys Ala Tyr
35 40 45
Gln Asp Leu Gly Ser Pro Lys Asn Ala Lys Phe Val Val His Asp Ala
50 55 60
Ala Asp Glu Glu Gly Trp Lys Lys Leu Phe Ala Asp Thr Ile Ala Glu
65 70 75 80
Phe Gly Lys Val Asp Ile Leu Val Asn Asn Ala Gly Ile Gly Val Pro
85 90 95
Gly Asn Val Glu Asn Thr Asp Tyr Ala Gln Trp Arg Gln Thr Met Asp
100 105 110
Val Asn Leu Asp Gly Val Tyr Leu Gly Thr His Tyr Gly Val Ile Asn
115 120 125
Met Lys Asn Pro Gln Ser Gly Asp Ala Ser Ile Ile Asn Met Ser Ser
130 135 140
Ile Glu Gly Leu Val Gly Asp Pro Asn Leu Phe Ala Tyr Asn Ala Thr
145 150 155 160
Lys Gly Ala Leu Arg Ile Met Ser Lys Ser Ala Ala Ile Tyr Cys Ala
165 170 175
Asn Gln Asp Tyr Asn Leu Arg Ile Asn Thr Ile His Pro Gly Tyr Ile
180 185 190
Lys Thr Pro Leu Val Asp Lys Ile Gln Gly Ala Glu Glu Met Met Ser
195 200 205
Gln Arg Thr Lys Thr Pro Met Gly His Val Gly Val Pro Asp Asp Ile
210 215 220
Gly Trp Leu Ala Val Tyr Leu Gly Ser Glu Glu Ser Lys Phe Ala Thr
225 230 235 240
Gly Ala Glu Phe Thr Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 11
<211> 1059
<212> DNA
<213> 致黑脱硫肠状菌（Desulfotomaculum nigrificans）
<400> 11
atgaagggtt ttgcaatgat tcagattggc aaagccggct ggattgaaaa agaaaaaccg 60
gtggccggtc cgtatgatgc aatttgtcgc ccgctggcac tggccccgtg taccagtgat 120
attcataccg tgtttgaagg tgcaattggt gaacgctata atatgattct gggtcatgaa 180
gccgttggcg aagttgttga agtgggcagt gaagtgcgcg attttaaagt tggcgatcgt 240
gtggttgttc cggcaattac cccggattgg catacccgtg aaattcagga aggttatcat 300
cagcatagcg gtggtatgct ggccggttgg aaattttcaa atattaagga tggcagtatg 360
gccgaatatt ttcatgttaa tgatgccgat atgaacctgg cactgctgcc ggatggtatt 420
ccgctggaaa ccgccgccat gattccggat atgatgacca ccggttttca tggcgcagaa 480
aatggtgaca ttcagtttgg tgacaccgtg gccgttctgg gtattggccc ggttggcctg 540
atggcagtgg caggtgccaa actgcgtggt gccggccgta ttattgttgt gggtagccgc 600
ccggtttgcg tggatgttgc caaattttat ggcgcaaccg atgttgtgaa ttatcgcgat 660
ggcgatattg ccgatcagat tctgaaaatt accgatggta aaggtgtgga tgttgtgatt 720
attgcaggcg gtagtgtgga tgtgatggcc agtgcagtta aaattgcaaa accgggcggt 780
cgtattagta atattaatta tttcggcgag ggcgaatttc tgccggttcc gcgcgtggaa 840
tggggttgcg gcatggccca taaaaccatt cgcggcggtc tgtgtccggg cggccgtgtg 900
agaatggaaa aactgattga actggttaaa tacaaccgtg ttgatccggg caaactggtt 960
acccatgtgt ttaaaggcat tgaaaatgtt gaaaaggcac tgatgctgat gaaagataaa 1020
ccgaaagatt tgattaagcc ggtggttctg gttgattaa 1059
<210> 12
<211> 352
<212> PRT
<213> 致黑脱硫肠状菌（Desulfotomaculum nigrificans）
<400> 12
Met Lys Gly Phe Ala Met Ile Gln Ile Gly Lys Ala Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Val Ala Gly Pro Tyr Asp Ala Ile Cys Arg Pro Leu
20 25 30
Ala Leu Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg Tyr Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Arg Asp Phe Lys Val Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp His Thr Arg Glu Ile Gln
85 90 95
Glu Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Ile Lys Asp Gly Ser Met Ala Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala Leu Leu Pro Asp Gly Ile Pro Leu Glu Thr
130 135 140
Ala Ala Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Asn Gly Asp Ile Gln Phe Gly Asp Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Val Val Gly Ser Arg Pro Val Cys Val Asp Val Ala Lys
195 200 205
Phe Tyr Gly Ala Thr Asp Val Val Asn Tyr Arg Asp Gly Asp Ile Ala
210 215 220
Asp Gln Ile Leu Lys Ile Thr Asp Gly Lys Gly Val Asp Val Val Ile
225 230 235 240
Ile Ala Gly Gly Ser Val Asp Val Met Ala Ser Ala Val Lys Ile Ala
245 250 255
Lys Pro Gly Gly Arg Ile Ser Asn Ile Asn Tyr Phe Gly Glu Gly Glu
260 265 270
Phe Leu Pro Val Pro Arg Val Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Arg Gly Gly Leu Cys Pro Gly Gly Arg Val Arg Met Glu Lys
290 295 300
Leu Ile Glu Leu Val Lys Tyr Asn Arg Val Asp Pro Gly Lys Leu Val
305 310 315 320
Thr His Val Phe Lys Gly Ile Glu Asn Val Glu Lys Ala Leu Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Leu Val Asp
340 345 350
<210> 13
<211> 1059
<212> DNA
<213> 一氧化碳嗜热窦菌（Thermosinus carboxydivorans）
<400> 13
atgaagggct ttgcaatgct gaaaattggt gaagttggtt ggattgaaat tgaaaaaccg 60
aaagcaggcc cgtatgatgc cattgttcgt ccgctggccg tggcaccgtg taccagtgat 120
attcataccg tgtttgaagg cgcaattggt gaacgtcata atctgattct gggccatgaa 180
gccgttggcg aagtggttga agttggcagt gaagtgaaag attttaaacc gggcgatcgt 240
gtggttgtgc cggcaattac cccggattgg cgtaccctgg aagcacagcg cggctttcat 300
cagcatagtg gcggcatgct ggcaggctgg aaattttcaa atattaagga tggcgttttc 360
gcagaatatt ttcatgtgaa tgatgcagat atgaacctgg cccatctgcc gaaagaaatt 420
ccgctggaaa aagcagtgat gctgccggat atgatgacca ccggcctgca tggtgccgaa 480
ctggccgata ttggtctggg cgataccgtg gcagtgattg gcattggccc ggtgggtctg 540
atggccgttg caggcgccaa actgcgtggc gcagcacgca ttattggcgt gggtagtcgt 600
ccggccctga ttgaagcagc aaaatattat ggtgtgaccg atattgtgaa ttataaacat 660
ggcgatatcg ttgaacaggt tatgaaactg accgataata agggtgttga tgcagttatt 720
attgccggtg ccggtgcaga tgccattgat aaagcagtta aaatggttaa accgggtggc 780
accgttagta atgtgaatta ttttggtgaa ggcggtattc tgccgattcc gcgcctggat 840
tggggttgcg gcatgagcca taaaagtatt cgtggcggcc tgtgtccggg tggtcgtctg 900
cgtatggaaa aactggtgga tctggtgcgt tataatcgta ttgatccggg taaactggtt 960
agtcatgttt ttgaaggttt tgaacatatc gaaaaggccc tgctgatgat gaaagaaaaa 1020
agtagtgatg tgatcaagcc ggtggttatt attaagtaa 1059
<210> 14
<211> 352
<212> PRT
<213> 一氧化碳嗜热窦菌（Thermosinus carboxydivorans）
<400> 14
Met Lys Gly Phe Ala Met Leu Lys Ile Gly Glu Val Gly Trp Ile Glu
1 5 10 15
Ile Glu Lys Pro Lys Ala Gly Pro Tyr Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Leu Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Leu Glu Ala Gln
85 90 95
Arg Gly Phe His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Ile Lys Asp Gly Val Phe Ala Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ile Pro Leu Glu Lys
130 135 140
Ala Val Met Leu Pro Asp Met Met Thr Thr Gly Leu His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Gly Leu Gly Asp Thr Val Ala Val Ile Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Ala
180 185 190
Arg Ile Ile Gly Val Gly Ser Arg Pro Ala Leu Ile Glu Ala Ala Lys
195 200 205
Tyr Tyr Gly Val Thr Asp Ile Val Asn Tyr Lys His Gly Asp Ile Val
210 215 220
Glu Gln Val Met Lys Leu Thr Asp Asn Lys Gly Val Asp Ala Val Ile
225 230 235 240
Ile Ala Gly Ala Gly Ala Asp Ala Ile Asp Lys Ala Val Lys Met Val
245 250 255
Lys Pro Gly Gly Thr Val Ser Asn Val Asn Tyr Phe Gly Glu Gly Gly
260 265 270
Ile Leu Pro Ile Pro Arg Leu Asp Trp Gly Cys Gly Met Ser His Lys
275 280 285
Ser Ile Arg Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Lys
290 295 300
Leu Val Asp Leu Val Arg Tyr Asn Arg Ile Asp Pro Gly Lys Leu Val
305 310 315 320
Ser His Val Phe Glu Gly Phe Glu His Ile Glu Lys Ala Leu Leu Met
325 330 335
Met Lys Glu Lys Ser Ser Asp Val Ile Lys Pro Val Val Ile Ile Lys
340 345 350
<210> 15
<211> 1059
<212> DNA
<213> 烷嗜热厌氧杆菌（Thermoanaerobacter mathranii）
<400> 15
atgaagggct ttgcaatgct gagtattggt aaagttggtt ggattgaaaa agaaaaaccg 60
accccgggcc cgtttgatgc aattgtgcgc ccgctggcag ttgcaccgtg caccagtgat 120
attcataccg tgtttgaagg cgcaattggt gaacgtcata atatgattct gggccatgaa 180
gcaattggcg aagtggtgga agttggtagt gaagtgaaag attttaaacc gggtgaccgt 240
gttgttgttc cggcaattac cccggattgg cgtaccagcg aagtgcagcg tggctatcat 300
cagcatagtg gcggcatgct ggcaggttgg aaattttcaa atattaagga tggtgtgttc 360
ggcgaatttt tccatgttaa tgatgccgat atgaatctgg cacatctgcc gaaagaaatt 420
ccgctggaag cagccgtgat gattccggat atgatgacca ccggctttca tggtgcagaa 480
ctggccgaaa ttgaactggg tgccagcgtt gcagttctgg gcattggccc ggtgggcctg 540
atggcagtgg caggtgccaa actgcgtggt gccggtcgta ttattgcagt gggtagccgt 600
ccggtttgtg tggatgccgc aaaatattat ggtgcaaccg atattgttaa ctataaaaat 660
ggtccgatcg aaagccagat tatggatctg accaaaggca aaggcgtgga tgcagccatt 720
attgcaggcg gtaatgcaga tattatggcc accgccgtta aaattgtgaa accgggcggc 780
accattgcca atgttaatta ttttggtgaa ggtgaagtgc tgccggttcc gcgcctggaa 840
tggggttgtg gtatggccca taaaaccatt aagggcggtc tgtgcccggg cggtcgctta 900
cgtatggaac gcctgattga tctggttttc tataaacgcg tggaccctag caaactggtt 960
acccatgttt ttcagggttt tgataatatt gagaaggcac tgatgctgat gaaagataaa 1020
ccgaaagatt tgattaagcc ggttgtgatt ctggcataa 1059
<210> 16
<211> 352
<212> PRT
<213> 烷嗜热厌氧杆菌（Thermoanaerobacter mathranii）
<400> 16
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Thr Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Ile Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Ile Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ile Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Glu Ile Glu Leu Gly Ala Ser Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asn Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asp Leu Thr Lys Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
260 265 270
Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
290 295 300
Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
305 310 315 320
Thr His Val Phe Gln Gly Phe Asp Asn Ile Glu Lys Ala Leu Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
<210> 17
<211> 1062
<212> DNA
<213> 厚壁菌门细菌（Firmicutes bacterium）
<400> 17
atgcgcatga aaggttttgc catgctgggc attggcaaaa ccggctggat tgaaaaagaa 60
gtgccggcat gtggtccgct ggatgccatt gttcgtccgc tggccgttag cccgtgcacc 120
agtgatattc ataccgtgtg ggaaggtgca attggcgaac gtaccgatat gattctgggt 180
catgaagccg ttggtgaaat tgttgaagtg ggtagcctgg tgaaaaccct gaaaccgggc 240
gatcgtgtga ttgttccggc aattaccccg gattggggca gtctggaagc acaggcaggc 300
tatagcatgc atagtggtgg tatgctggca ggctggaaat tttctaattt taaagatggt 360
gtgttcgccg aatattttca tgttaatgaa gccgatgcca atctggcacg tctgccggaa 420
cagctggacc ctgccagtgc cgttatgctg agtgatatgg ttccgaccgg ctttcatggt 480
gtggaactgg cagatgttca gtttggcgat accgtgtgtg ttgtgggcat tggcccggtt 540
ggcctgatgg ccgttgcagg cgcagcactg cgcggcgcaa gcagactgtt tgccgttggt 600
agccgtccgg tttgtgtgga agccgccaaa gcctatggcg ccaccgatat tattaattat 660
cgtgaaggtg acatcgtgca gcagattctg gatcagaccc acggtaaagg cgtggatcgt 720
gtgatcctgg ccggtggcga taatgatacc tttcgtcagg cagtggaaat ggtgaaaccg 780
ggcggttgca ttggcaatgt taattatctg ggtagcggcg attatgttcg tattccgcgt 840
gttgaatggg gttgcggcat gggccataaa accattcgcg gcggtctgat gccgggtggc 900
cgtttacgta tggaaaaact ggccagtctg atggaaaccg gccgtctgga taccagccgt 960
ctgctgaccc atcgttttca gggctttgca cacatggaag aagcactgct gctgatgaaa 1020
gataaaccgc gcgatctgat taagccggtg gttattctgt aa 1062
<210> 18
<211> 353
<212> PRT
<213>厚壁菌门细菌（Firmicutes bacterium）
<400> 18
Met Arg Met Lys Gly Phe Ala Met Leu Gly Ile Gly Lys Thr Gly Trp
1 5 10 15
Ile Glu Lys Glu Val Pro Ala Cys Gly Pro Leu Asp Ala Ile Val Arg
20 25 30
Pro Leu Ala Val Ser Pro Cys Thr Ser Asp Ile His Thr Val Trp Glu
35 40 45
Gly Ala Ile Gly Glu Arg Thr Asp Met Ile Leu Gly His Glu Ala Val
50 55 60
Gly Glu Ile Val Glu Val Gly Ser Leu Val Lys Thr Leu Lys Pro Gly
65 70 75 80
Asp Arg Val Ile Val Pro Ala Ile Thr Pro Asp Trp Gly Ser Leu Glu
85 90 95
Ala Gln Ala Gly Tyr Ser Met His Ser Gly Gly Met Leu Ala Gly Trp
100 105 110
Lys Phe Ser Asn Phe Lys Asp Gly Val Phe Ala Glu Tyr Phe His Val
115 120 125
Asn Glu Ala Asp Ala Asn Leu Ala Arg Leu Pro Glu Gln Leu Asp Pro
130 135 140
Ala Ser Ala Val Met Leu Ser Asp Met Val Pro Thr Gly Phe His Gly
145 150 155 160
Val Glu Leu Ala Asp Val Gln Phe Gly Asp Thr Val Cys Val Val Gly
165 170 175
Ile Gly Pro Val Gly Leu Met Ala Val Ala Gly Ala Ala Leu Arg Gly
180 185 190
Ala Ser Arg Leu Phe Ala Val Gly Ser Arg Pro Val Cys Val Glu Ala
195 200 205
Ala Lys Ala Tyr Gly Ala Thr Asp Ile Ile Asn Tyr Arg Glu Gly Asp
210 215 220
Ile Val Gln Gln Ile Leu Asp Gln Thr His Gly Lys Gly Val Asp Arg
225 230 235 240
Val Ile Leu Ala Gly Gly Asp Asn Asp Thr Phe Arg Gln Ala Val Glu
245 250 255
Met Val Lys Pro Gly Gly Cys Ile Gly Asn Val Asn Tyr Leu Gly Ser
260 265 270
Gly Asp Tyr Val Arg Ile Pro Arg Val Glu Trp Gly Cys Gly Met Gly
275 280 285
His Lys Thr Ile Arg Gly Gly Leu Met Pro Gly Gly Arg Leu Arg Met
290 295 300
Glu Lys Leu Ala Ser Leu Met Glu Thr Gly Arg Leu Asp Thr Ser Arg
305 310 315 320
Leu Leu Thr His Arg Phe Gln Gly Phe Ala His Met Glu Glu Ala Leu
325 330 335
Leu Leu Met Lys Asp Lys Pro Arg Asp Leu Ile Lys Pro Val Val Ile
340 345 350
Leu
<210> 19
<211> 1062
<212> DNA
<213> 甲烷八叠球菌（Methanosarcina thermophila）
<400> 19
atgaagggtt ttgccatgct ggaaattggt aaagtgggct ggattgatgc cgaaaaaccg 60
agtgcaggtc cgtatgatgc cattgttcgt ccgctggcag ttgccccgtg taccagtgat 120
attcataccg tgtgggaagg cgcactgggt gaccgcaaaa atatgattct gggtcatgaa 180
gcagttggta ttattgaaga agttggcagt gaagttaaag attttaaacc gggtgacaaa 240
gttattgttc cggccattac cccggattgg cgcagcatgg aagcccagga tggtgttccg 300
atgcatagta atggtatgct gagcggttgg aaattttcaa attttaaaaa cggcgtgttc 360
gcagaatatt ttcatgttaa tgatgccgat atgaacctgg cactgctgcc ggaaggcatg 420
ccgctggaac aggccgttat gctgagcgat atggcaacca ccggcattca gggtgccgaa 480
atggcaaata ttaagaccgg tagtaccgtg gttgtgattg gcattggccc ggttggcctg 540
atggccgttg ccggtgccag tattctgggc gccggccgtc tgattgcagt gggtagccgc 600
aaagttagta ttgatctggc actggaatat ggcgccagtg atattgttga ttatcgtaaa 660
ggcggtattg ttgaacagat tctggaaatg accaatggta aaggtgtgga aagtgtgatt 720
attgccggtg gtgacgaaaa taccattagt gatgcagtta aaatcgttaa accgggtggc 780
accgtgagca atgttaatta ttttggtaca ggtaacaccc tgccgattcc gcgtattgaa 840
tggggcagcg gcatggcaca taaagatatt cgcggcggtc tgaccaccgg cggtcgttta 900
cgcatggaac gtatggccgc actgtgtacc tatggtcgca ttaagccgga aaaaatggca 960
acccatgtgt ttaaaggctt tgataaaatt gagaaggcac tgatgctgat gaaagaaaaa 1020
ccgcgcgatc tgattaagcc ggttgttatt atggaagaat aa 1062
<210> 20
<211> 353
<212> PRT
<213> 甲烷八叠球菌（Methanosarcina thermophila）
<400> 20
Met Lys Gly Phe Ala Met Leu Glu Ile Gly Lys Val Gly Trp Ile Asp
1 5 10 15
Ala Glu Lys Pro Ser Ala Gly Pro Tyr Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Trp Glu Gly Ala
35 40 45
Leu Gly Asp Arg Lys Asn Met Ile Leu Gly His Glu Ala Val Gly Ile
50 55 60
Ile Glu Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Lys
65 70 75 80
Val Ile Val Pro Ala Ile Thr Pro Asp Trp Arg Ser Met Glu Ala Gln
85 90 95
Asp Gly Val Pro Met His Ser Asn Gly Met Leu Ser Gly Trp Lys Phe
100 105 110
Ser Asn Phe Lys Asn Gly Val Phe Ala Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala Leu Leu Pro Glu Gly Met Pro Leu Glu Gln
130 135 140
Ala Val Met Leu Ser Asp Met Ala Thr Thr Gly Ile Gln Gly Ala Glu
145 150 155 160
Met Ala Asn Ile Lys Thr Gly Ser Thr Val Val Val Ile Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Ser Ile Leu Gly Ala Gly
180 185 190
Arg Leu Ile Ala Val Gly Ser Arg Lys Val Ser Ile Asp Leu Ala Leu
195 200 205
Glu Tyr Gly Ala Ser Asp Ile Val Asp Tyr Arg Lys Gly Gly Ile Val
210 215 220
Glu Gln Ile Leu Glu Met Thr Asn Gly Lys Gly Val Glu Ser Val Ile
225 230 235 240
Ile Ala Gly Gly Asp Glu Asn Thr Ile Ser Asp Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Val Ser Asn Val Asn Tyr Phe Gly Thr Gly Asn
260 265 270
Thr Leu Pro Ile Pro Arg Ile Glu Trp Gly Ser Gly Met Ala His Lys
275 280 285
Asp Ile Arg Gly Gly Leu Thr Thr Gly Gly Arg Leu Arg Met Glu Arg
290 295 300
Met Ala Ala Leu Cys Thr Tyr Gly Arg Ile Lys Pro Glu Lys Met Ala
305 310 315 320
Thr His Val Phe Lys Gly Phe Asp Lys Ile Glu Lys Ala Leu Met Leu
325 330 335
Met Lys Glu Lys Pro Arg Asp Leu Ile Lys Pro Val Val Ile Met Glu
340 345 350
Glu
<210> 21
<211> 1056
<212> DNA
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 21
atgaagggct ttgcaatgct gggtattaat aaggttggct ggattgaaaa agaaaaaccg 60
gttgcaggta catacgatgc aattgttcgt ccgctggcag ttagtccgtg taccagcgat 120
attcataccg tgtttgaagg cgccctgggt gaccgtcata atatgattct gggtcatgaa 180
gccgtgggcg aagttgtgga agtgggcagt gaagtgaaag attttaaacc gggcgatcgc 240
gttattgtgc cgtgcaccac cccggattgg cgtagtctgg aagtgcaggc cggctttcag 300
cagcatagca atggcatgct ggcaggctgg aaattttcta attttaaaga tggtgtgttc 360
ggcgaatatt ttcatgttaa tgatgccgat atgaacctgg cagcactgcc gaaagatatg 420
ccgctggaaa ccgcagttat gattaccgat atgatgacca ccggttttca tggcgccgaa 480
ctggccgata ttcagatggg tagcagtgtt gtggttattg gtattggcgc cgttggcctg 540
atgggtattg ccggtgccaa actgcgtggt gccggtcgca ttattggtgt gggtagtcgt 600
ccgatttgtg tggaaaccgc aaaattttat ggcgcaaccg atattgtgaa ttataaaaat 660
ggtgacatcg tggatcaggt tatgaaactg accaatggta aaggtgtgga tcgcgttatc 720
atggccggtg gtggcgccga aaccctggca caggcagtga gcatggttaa accgggcggc 780
attattagca atattaatta tcatggcagc ggtgacaccc tgccgattcc gcgtgtggaa 840
tggggttgcg gcatggccca taaaaccatt cgcggtggtc tgtgtccggg tggtcgtctg 900
cgtgccgaaa tgctgcgtga tatggtggtt tataatcgcg ttgatctgag caaactggtg 960
acccatgtgt atcatggctt tgatcatatt gaagaagccc tgctgctgat gaaagataaa 1020
ccgaaagatt tgattaaggc agtggttatt atctaa 1056
<210> 22
<211> 351
<212> PRT
<213>拜氏梭菌（Clostridium beijerinckii）
<400> 22
Met Lys Gly Phe Ala Met Leu Gly Ile Asn Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Val Ala Gly Thr Tyr Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ser Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Leu Gly Asp Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Ile Val Pro Cys Thr Thr Pro Asp Trp Arg Ser Leu Glu Val Gln
85 90 95
Ala Gly Phe Gln Gln His Ser Asn Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Phe Lys Asp Gly Val Phe Gly Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala Ala Leu Pro Lys Asp Met Pro Leu Glu Thr
130 135 140
Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Gln Met Gly Ser Ser Val Val Val Ile Gly Ile Gly
165 170 175
Ala Val Gly Leu Met Gly Ile Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Gly Val Gly Ser Arg Pro Ile Cys Val Glu Thr Ala Lys
195 200 205
Phe Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asn Gly Asp Ile Val
210 215 220
Asp Gln Val Met Lys Leu Thr Asn Gly Lys Gly Val Asp Arg Val Ile
225 230 235 240
Met Ala Gly Gly Gly Ala Glu Thr Leu Ala Gln Ala Val Ser Met Val
245 250 255
Lys Pro Gly Gly Ile Ile Ser Asn Ile Asn Tyr His Gly Ser Gly Asp
260 265 270
Thr Leu Pro Ile Pro Arg Val Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Arg Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Ala Glu Met
290 295 300
Leu Arg Asp Met Val Val Tyr Asn Arg Val Asp Leu Ser Lys Leu Val
305 310 315 320
Thr His Val Tyr His Gly Phe Asp His Ile Glu Glu Ala Leu Leu Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Ala Val Val Ile Ile
340 345 350
<210> 23
<211> 1056
<212> DNA
<213> 未培养梭菌（uncultured Clostridium sp.）
<400> 23
atgaagggtt ttgcaatgct gggcattggt aaaaccggct ggattgaaaa agaacgcccg 60
ctgtgtggcc cgctggatgc aattgtgcgc ccgctggcaa ttagtccgtg taccagcgat 120
attcataccg tttgggaagg tgcaattggc gaacgtcata atatgattct gggtcatgaa 180
gcagtgggtg aagttgtgga aattggcagt ctggtgaaag atattaaggt gggcgataaa 240
gtgattgttc cggccattac cccggattgg ggtagcctgg aagcccaggc aggttatagt 300
atgcatagtg gtggtatgct ggccggctgg aaattttcaa attttaaaga tggtgtgttc 360
ggtgaatatt ttcatgttaa tgaagccgat gcaaatctgg ccgtgctgcc ggaaggcatt 420
gatattgccg atgcagttat gctgagtgat atggttccga ccggttttca tggtgtggaa 480
ctggcagatg ttcagtttgg cgatagcgtt tgcgtgattg gcattggtcc ggttggcctg 540
atggccgtgg caggtgcagc actgcgcggt gcaagcgatc tgtatgcagt tggtagtcgc 600
ccgaattgca ttgaaattgc caaagaatat ggtgccaccg atattattaa ttatcgtgaa 660
ggtgacatcg tggaacagat tatgagcaaa acccacggta aaggcgttga taaagttatt 720
gtggccggtg gtgacgttga tacaatggct caggcaatta gtattgttaa accgggcggt 780
attattggca atgttaatta tctgggtagt ggtgaatatg tgaaaattcc gcgcgtggaa 840
tggggttgcg gtatgggtca taaaaccatt agcggtggtc tgatgccggg tggccgcctg 900
cgtatggaaa aactggcaaa actgctgcag accaatcgtc tggataccag taaactgatt 960
acccatcgct tttatggctt tgataaaatt gaagatgcgc tgatgctgat gaaagataaa 1020
ccgaaagatt tgattaagcc ggttgttatt ctgtaa 1056
<210> 24
<211> 351
<212> PRT
<213>未培养梭菌（uncultured Clostridium sp.）
<400> 24
Met Lys Gly Phe Ala Met Leu Gly Ile Gly Lys Thr Gly Trp Ile Glu
1 5 10 15
Lys Glu Arg Pro Leu Cys Gly Pro Leu Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Ile Ser Pro Cys Thr Ser Asp Ile His Thr Val Trp Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Ile Gly Ser Leu Val Lys Asp Ile Lys Val Gly Asp Lys
65 70 75 80
Val Ile Val Pro Ala Ile Thr Pro Asp Trp Gly Ser Leu Glu Ala Gln
85 90 95
Ala Gly Tyr Ser Met His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Phe Lys Asp Gly Val Phe Gly Glu Tyr Phe His Val Asn Glu
115 120 125
Ala Asp Ala Asn Leu Ala Val Leu Pro Glu Gly Ile Asp Ile Ala Asp
130 135 140
Ala Val Met Leu Ser Asp Met Val Pro Thr Gly Phe His Gly Val Glu
145 150 155 160
Leu Ala Asp Val Gln Phe Gly Asp Ser Val Cys Val Ile Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Ala Leu Arg Gly Ala Ser
180 185 190
Asp Leu Tyr Ala Val Gly Ser Arg Pro Asn Cys Ile Glu Ile Ala Lys
195 200 205
Glu Tyr Gly Ala Thr Asp Ile Ile Asn Tyr Arg Glu Gly Asp Ile Val
210 215 220
Glu Gln Ile Met Ser Lys Thr His Gly Lys Gly Val Asp Lys Val Ile
225 230 235 240
Val Ala Gly Gly Asp Val Asp Thr Met Ala Gln Ala Ile Ser Ile Val
245 250 255
Lys Pro Gly Gly Ile Ile Gly Asn Val Asn Tyr Leu Gly Ser Gly Glu
260 265 270
Tyr Val Lys Ile Pro Arg Val Glu Trp Gly Cys Gly Met Gly His Lys
275 280 285
Thr Ile Ser Gly Gly Leu Met Pro Gly Gly Arg Leu Arg Met Glu Lys
290 295 300
Leu Ala Lys Leu Leu Gln Thr Asn Arg Leu Asp Thr Ser Lys Leu Ile
305 310 315 320
Thr His Arg Phe Tyr Gly Phe Asp Lys Ile Glu Asp Ala Leu Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu
340 345 350
<210> 25
<211> 1056
<212> DNA
<213> 丝状孢子梭菌（Clostridium taeniosporum）
<400> 25
atgaagggtt ttgccatgct gagtattaat aatgttggtt ggattgaaaa ggacaaaccg 60
gtggccggtc cgtatgatgc cattgtgcgc ccgctggccg tgagtccgtg taccagtgat 120
attcataccg tttttgaagg cgcactgggt aatcgtcata atatgattct gggtcatgaa 180
gcagtgggcg aaattgttga aattggtagt gaagtgaaag agtttaaagt tggcgatcgt 240
gtgattgttc cgtgcaccac cccggattgg cgtagtctgg aagttcaggc aggctttcag 300
cagcatagta atggcatgct ggcaggttgg aaattttcaa attttaaaga cggtgtgttc 360
gcagaatatt ttcatgttaa tgatgcagat atgaacctgg cactgctgcc gaaagaaatt 420
ccgctggaaa gcgcagttat gattaccgat atgatgacca ccggctttca tggcgcagaa 480
ctggcagata ttcagatggg cagtagcgtt gtggttattg gcattggtgc agttggcctg 540
atgggtattg ccggtgccaa actgcgcggt gccggcagaa ttattggcgt gggcagccgc 600
ccggtgtgcg ttgaagcagc caaattttat ggtgcaaccg atattgttaa ctataaaaat 660
ggcgatatcg tgaaacagat tatgaatctg accaatggta aaggcgttga tcgtgttatt 720
atggccggtg gtggtagcga aaccctgagt caggccatta gtatgctgaa accgggcggc 780
gttgttagca atattaatta tcatggtagc ggtgacaccc tgccggttcc gcgcgtggaa 840
tggggttgtg gcatggcaca taaaaccatt cgcggcggcc tgtgtccggg tggtcgctta 900
cgcgccgaaa tgctgcgtga tctggttatc tataatcgtg tggatctgag ccgcctggtt 960
acccatgttt ataatggctt tgaacatatt gaggatgcac tgctgctgat gaaagataaa 1020
ccgaaagatt ttatcaaggc agttgtgctg ctgtaa 1056
<210> 26
<211> 351
<212> PRT
<213>丝状孢子梭菌（Clostridium taeniosporum）
<400> 26
Met Lys Gly Phe Ala Met Leu Ser Ile Asn Asn Val Gly Trp Ile Glu
1 5 10 15
Lys Asp Lys Pro Val Ala Gly Pro Tyr Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ser Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Leu Gly Asn Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Ile Val Glu Ile Gly Ser Glu Val Lys Glu Phe Lys Val Gly Asp Arg
65 70 75 80
Val Ile Val Pro Cys Thr Thr Pro Asp Trp Arg Ser Leu Glu Val Gln
85 90 95
Ala Gly Phe Gln Gln His Ser Asn Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Phe Lys Asp Gly Val Phe Ala Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala Leu Leu Pro Lys Glu Ile Pro Leu Glu Ser
130 135 140
Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Gln Met Gly Ser Ser Val Val Val Ile Gly Ile Gly
165 170 175
Ala Val Gly Leu Met Gly Ile Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Gly Val Gly Ser Arg Pro Val Cys Val Glu Ala Ala Lys
195 200 205
Phe Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asn Gly Asp Ile Val
210 215 220
Lys Gln Ile Met Asn Leu Thr Asn Gly Lys Gly Val Asp Arg Val Ile
225 230 235 240
Met Ala Gly Gly Gly Ser Glu Thr Leu Ser Gln Ala Ile Ser Met Leu
245 250 255
Lys Pro Gly Gly Val Val Ser Asn Ile Asn Tyr His Gly Ser Gly Asp
260 265 270
Thr Leu Pro Val Pro Arg Val Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Arg Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Ala Glu Met
290 295 300
Leu Arg Asp Leu Val Ile Tyr Asn Arg Val Asp Leu Ser Arg Leu Val
305 310 315 320
Thr His Val Tyr Asn Gly Phe Glu His Ile Glu Asp Ala Leu Leu Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Phe Ile Lys Ala Val Val Leu Leu
340 345 350
<210> 27
<211> 1059
<212> DNA
<213> 脱硫肠状菌属（Desulfotomaculum putei）
<400> 27
atgaagggtt ttgcaatgct gcgtattggt gaagttggct ggattgaaaa agatcgtccg 60
gtggccggtc cgtatgatgc aatttgccgt ccgctggcca ttgcaccgtg taccagtgat 120
attcataccg tttttgaagg tgcaattggc gaacgccata atatgattct gggtcatgaa 180
gccattggcg aagtggttga agtgggtagc gaagttaaag attttaaacc gggtgaccgc 240
gtggtggtgc cggcaattac cccggattgg cgcaccctgg aagcagccgc cggttttcat 300
cagcatagcg gtggtatgct ggccggctgg aaattttcta atattaagga tggtagcatg 360
ggtgaatatt ttcatgttaa tgatgcagat atgaacctgg cccatctgcc ggatggcatg 420
ccgctggaag ccgcagttat gattccggat atggtgacca ccggtctgca tggcgcagaa 480
aatgccgata ttcagctggg cgccaccgtg gttgtgattg gcattggccc ggttggtctg 540
atgggtgttg caggtgccaa actgtgtggt gccggccgcc tgattgccgt gggtagccgt 600
ccggtttgca ttgaagccgc aaaattttat ggcgccaccg atattgttaa ttatcgcaat 660
ggtgacattg tggaacaggt gctgaaactg accaatggta aaggtgcaga tgccaccatt 720
attgcaggtg gtaaaccgga agttgttgcc caggcaatta agatgaccaa accgggtggc 780
accattagca atattaatta tcatggcgaa ggtgaaatcc tgccgattcc gcgcctggaa 840
tggggcagcg gcatggcaca taaaaccatt aagggcggcc tgtgtccggg tggtcgccgt 900
agaatggaaa tgctgattga tctggtgaaa tataatcgcc tggaccctgg taaactggtt 960
acccatgtgt ttaatggcat tgaaaattgc gaaaaagcac tgctgctgat gaaagataaa 1020
ccgaaagatt tgattaagcc ggtggttctg gttaattaa 1059
<210> 28
<211> 352
<212> PRT
<213> 脱硫肠状菌属（Desulfotomaculum putei）
<400> 28
Met Lys Gly Phe Ala Met Leu Arg Ile Gly Glu Val Gly Trp Ile Glu
1 5 10 15
Lys Asp Arg Pro Val Ala Gly Pro Tyr Asp Ala Ile Cys Arg Pro Leu
20 25 30
Ala Ile Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Ile Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Leu Glu Ala Ala
85 90 95
Ala Gly Phe His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Ile Lys Asp Gly Ser Met Gly Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Asp Gly Met Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Val Thr Thr Gly Leu His Gly Ala Glu
145 150 155 160
Asn Ala Asp Ile Gln Leu Gly Ala Thr Val Val Val Ile Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Gly Val Ala Gly Ala Lys Leu Cys Gly Ala Gly
180 185 190
Arg Leu Ile Ala Val Gly Ser Arg Pro Val Cys Ile Glu Ala Ala Lys
195 200 205
Phe Tyr Gly Ala Thr Asp Ile Val Asn Tyr Arg Asn Gly Asp Ile Val
210 215 220
Glu Gln Val Leu Lys Leu Thr Asn Gly Lys Gly Ala Asp Ala Thr Ile
225 230 235 240
Ile Ala Gly Gly Lys Pro Glu Val Val Ala Gln Ala Ile Lys Met Thr
245 250 255
Lys Pro Gly Gly Thr Ile Ser Asn Ile Asn Tyr His Gly Glu Gly Glu
260 265 270
Ile Leu Pro Ile Pro Arg Leu Glu Trp Gly Ser Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Arg Arg Met Glu Met
290 295 300
Leu Ile Asp Leu Val Lys Tyr Asn Arg Leu Asp Pro Gly Lys Leu Val
305 310 315 320
Thr His Val Phe Asn Gly Ile Glu Asn Cys Glu Lys Ala Leu Leu Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Leu Val Asn
340 345 350
<210> 29
<211> 1056
<212> DNA
<213>二醇梭菌（Clostridium diolis）
<400> 29
atgaagggtt ttgcaatgct gggtattaat aagctgggtt ggattgaaaa agaacgcccg 60
gttgccggca gctatgatgc cattgtgcgc ccgctggccg ttagtccgtg taccagcgat 120
attcataccg tttttgaagg tgcactgggc gatcgcaaaa atatgattct gggccatgaa 180
gcagtgggcg aagttgtgga agttggtagt gaagtgaaag attttaaaac cggcgatcgc 240
gttattgttc cgtgtaccac cccggattgg cgtagtctgg aagttcaggc aggttttcag 300
cagcatagta atggcatgct ggcaggctgg aaattttcaa attttaaaga tggtgtgttc 360
ggtgaatatt ttcatgttaa tgatgccgat atgaacctgg caattctgcc gaaagatatg 420
ccgctggaaa atgccgttat gattaccgat atgatgacca ccggttttca tggcgcagaa 480
ctggccgata ttcagatggg cagcagtgtt gtggttattg gcattggcgc cgtgggtctg 540
atgggcattg ccggtgccaa actgcgtggc gccggtcgta ttattggtgt gggcagccgc 600
ccggtgtgcg tggaagtggc aaaattttat ggcgccaccg atattgtgaa ttataaaaat 660
ggtgacatcg tggatcaggt tatgaaactg accaatggta aaggtgtgga tcgtgttatt 720
atggccggtg gtggcagtga aaccctggcc caggccgtga gtatggttaa accgggtggt 780
attattagca atattaatta tcacggcagc ggcgataccc tgccgattcc gcgtgtggaa 840
tggggttgcg gtatggcaca taaaaccatt aagggtggcc tgtgcccggg tggccgctta 900
cgtgctgaaa tgctgcgtga tatggttgtt tataatcgcg ttgatctgag taaactggtg 960
acccatgtgt atcatggttt tgaacatatt gaagaaaccc tgctgctgat gaaagataaa 1020
ccgaaagatt tgattaaggc agttgtgatt ctgtaa 1056
<210> 30
<211> 351
<212> PRT
<213> 二醇梭菌（Clostridium diolis）
<400> 30
Met Lys Gly Phe Ala Met Leu Gly Ile Asn Lys Leu Gly Trp Ile Glu
1 5 10 15
Lys Glu Arg Pro Val Ala Gly Ser Tyr Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ser Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Leu Gly Asp Arg Lys Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Thr Gly Asp Arg
65 70 75 80
Val Ile Val Pro Cys Thr Thr Pro Asp Trp Arg Ser Leu Glu Val Gln
85 90 95
Ala Gly Phe Gln Gln His Ser Asn Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Phe Lys Asp Gly Val Phe Gly Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala Ile Leu Pro Lys Asp Met Pro Leu Glu Asn
130 135 140
Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Gln Met Gly Ser Ser Val Val Val Ile Gly Ile Gly
165 170 175
Ala Val Gly Leu Met Gly Ile Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Gly Val Gly Ser Arg Pro Val Cys Val Glu Val Ala Lys
195 200 205
Phe Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asn Gly Asp Ile Val
210 215 220
Asp Gln Val Met Lys Leu Thr Asn Gly Lys Gly Val Asp Arg Val Ile
225 230 235 240
Met Ala Gly Gly Gly Ser Glu Thr Leu Ala Gln Ala Val Ser Met Val
245 250 255
Lys Pro Gly Gly Ile Ile Ser Asn Ile Asn Tyr His Gly Ser Gly Asp
260 265 270
Thr Leu Pro Ile Pro Arg Val Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Ala Glu Met
290 295 300
Leu Arg Asp Met Val Val Tyr Asn Arg Val Asp Leu Ser Lys Leu Val
305 310 315 320
Thr His Val Tyr His Gly Phe Glu His Ile Glu Glu Thr Leu Leu Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Ala Val Val Ile Leu
340 345 350
<210> 31
<211> 1062
<212> DNA
<213> 匙形梭菌（Clostridium cochlearium）
<400> 31
atgaagggct ttgcaatgct gggcattggc aaagtgggct ggattgaaaa agaaaaaccg 60
gtggcaggcc tgtatgatgc cattgtgaaa ccgctggcac tggccccgtg caccagcgat 120
attcataccg tttatgaagg cgcactgggt gaacgcgaaa atctgattct gggccatgaa 180
gccgttggtg aaattgttga agtgggtaaa gaagttaaag attttaagcc gggtgaccgt 240
gtggtggtgc cggcaattac cccggattgg cgtagtcgcg aagcacagga ttataatctg 300
gcccagcata gtggcggcat gctggcaggt tggaaatttt caaatattaa ggatggtgtg 360
ttcgccgaac tgtttcatgt gaatgatgca gatatgaatc tggcacatct gccggaaacc 420
attccgctgg aagcagccgt gatgctgccg gatatggtta ccaccggttt tcatggcagt 480
gaactggccg gcgtgaattt tggtgacacc gtggccgtga ttggtattgg tccggttggt 540
ctgatgggta ttgccggcgc aaaactgcgc ggcgcaggca gaattattgg tgccggtagc 600
cgtgatgcac tgattgaagc cgcaaaattt tatggtgcca ccgatattgt gaattatcgc 660
aaaggtccga ttgtggaaca gattatggaa ctgaccaaaa ataatggtgt ggatcgtgtt 720
attgtggcag gcggtaatag tgatattctg gttgatgcag ttaaaattct gaaaccgggc 780
ggcgccattg gcaatattaa ttattttggc gaaggcgagt ttattaagat tccgcgtgtg 840
gattggggta atggcatggg tcataaaaat attaatggtg gcctgacccc gggcggtcgc 900
gctcgtatgg aacgtctgat tgaactggtt aaatataatc gcctggaccc tagcaaactg 960
gtgacccatg tttataaagg ctttgataaa gttgaggaag ccctgaatct gatgaaagat 1020
aaaccgaaag atttgattaa gccggttgtg attctggaat aa 1062
<210> 32
<211> 353
<212> PRT
<213> 匙形梭菌（Clostridium cochlearium）
<400> 32
Met Lys Gly Phe Ala Met Leu Gly Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Val Ala Gly Leu Tyr Asp Ala Ile Val Lys Pro Leu
20 25 30
Ala Leu Ala Pro Cys Thr Ser Asp Ile His Thr Val Tyr Glu Gly Ala
35 40 45
Leu Gly Glu Arg Glu Asn Leu Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Ile Val Glu Val Gly Lys Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Ser Arg Glu Ala Gln
85 90 95
Asp Tyr Asn Leu Ala Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys
100 105 110
Phe Ser Asn Ile Lys Asp Gly Val Phe Ala Glu Leu Phe His Val Asn
115 120 125
Asp Ala Asp Met Asn Leu Ala His Leu Pro Glu Thr Ile Pro Leu Glu
130 135 140
Ala Ala Val Met Leu Pro Asp Met Val Thr Thr Gly Phe His Gly Ser
145 150 155 160
Glu Leu Ala Gly Val Asn Phe Gly Asp Thr Val Ala Val Ile Gly Ile
165 170 175
Gly Pro Val Gly Leu Met Gly Ile Ala Gly Ala Lys Leu Arg Gly Ala
180 185 190
Gly Arg Ile Ile Gly Ala Gly Ser Arg Asp Ala Leu Ile Glu Ala Ala
195 200 205
Lys Phe Tyr Gly Ala Thr Asp Ile Val Asn Tyr Arg Lys Gly Pro Ile
210 215 220
Val Glu Gln Ile Met Glu Leu Thr Lys Asn Asn Gly Val Asp Arg Val
225 230 235 240
Ile Val Ala Gly Gly Asn Ser Asp Ile Leu Val Asp Ala Val Lys Ile
245 250 255
Leu Lys Pro Gly Gly Ala Ile Gly Asn Ile Asn Tyr Phe Gly Glu Gly
260 265 270
Glu Phe Ile Lys Ile Pro Arg Val Asp Trp Gly Asn Gly Met Gly His
275 280 285
Lys Asn Ile Asn Gly Gly Leu Thr Pro Gly Gly Arg Ala Arg Met Glu
290 295 300
Arg Leu Ile Glu Leu Val Lys Tyr Asn Arg Leu Asp Pro Ser Lys Leu
305 310 315 320
Val Thr His Val Tyr Lys Gly Phe Asp Lys Val Glu Glu Ala Leu Asn
325 330 335
Leu Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu
340 345 350
Glu
<210> 33
<211> 1056
<212> DNA
<213> 笃斯梭菌（Clostridium uliginosum）
<400> 33
atgaagggtt ttgcaatgct gagtattaat aatgtgggtt ggattgaaaa ggataaaccg 60
gttgccagcc cgtatgatgc cattgtgcgc ccgctggccg tgagtccgtg taccagcgat 120
attcataccg tttttgaagg cgccctgggt aatcgtcata atatgattct gggtcatgaa 180
gcagttggcg aaattgtgga aattggtagt gaagttaaag agtttaaagt gggcgatcgc 240
gtgattgtgc cgtgtaccac cccggattgg cgtagcctgg aagtgcaggc aggttttcag 300
cagcatagca atggtatgct ggcaggttgg aaattttcaa attttaaaga cggtgtgttc 360
gccgaatatt ttcatgttaa tgatgccgat atgaacctgg cactgctgcc gagcgaaatt 420
agcctggaaa gcgccgtgat gattaccgat atgatgacca ccggttttca tggtgcagaa 480
ctggccgata ttaagatggg cagcagtgtg gttgttattg gcattggtgc cgttggtctg 540
atgggcattg caggcgcccg cctgcgcggt gcaggtcgta ttattggtgt gggcagccgt 600
ccgatttgcg ttgaaaccgc caaattttat ggtgcaaccg atattgttaa ctataaaaat 660
ggtgacatcg tgaaacagat tatgaaactg accaatggta aaggcgtgga tcgtgtgatt 720
atggccggcg gcggcccgga aaccctgggt caggcaatta gtatggttaa accgggtggt 780
gttgtgagca atattaatta tcatggcagc ggtgacaccc tgccgattcc gcgtgttgat 840
tggggttgtg gtatggcaca taaaaccatt cgtggcggtc tgtgcccggg cggtcgttta 900
cgcgcagaaa tgctgcgtga tctggttatc tatgatcgta ttgatctgag tcgtctggtg 960
acccatgtgt ataatggctt tgaacatatt gaagaagccc tgctgctgat gaaagataaa 1020
ccgaaagatt tgattaaggc agttgtgatt gtttaa 1056
<210> 34
<211> 351
<212> PRT
<213> 笃斯梭菌（Clostridium uliginosum）
<400> 34
Met Lys Gly Phe Ala Met Leu Ser Ile Asn Asn Val Gly Trp Ile Glu
1 5 10 15
Lys Asp Lys Pro Val Ala Ser Pro Tyr Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ser Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Leu Gly Asn Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Ile Val Glu Ile Gly Ser Glu Val Lys Glu Phe Lys Val Gly Asp Arg
65 70 75 80
Val Ile Val Pro Cys Thr Thr Pro Asp Trp Arg Ser Leu Glu Val Gln
85 90 95
Ala Gly Phe Gln Gln His Ser Asn Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Phe Lys Asp Gly Val Phe Ala Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala Leu Leu Pro Ser Glu Ile Ser Leu Glu Ser
130 135 140
Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Lys Met Gly Ser Ser Val Val Val Ile Gly Ile Gly
165 170 175
Ala Val Gly Leu Met Gly Ile Ala Gly Ala Arg Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Gly Val Gly Ser Arg Pro Ile Cys Val Glu Thr Ala Lys
195 200 205
Phe Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asn Gly Asp Ile Val
210 215 220
Lys Gln Ile Met Lys Leu Thr Asn Gly Lys Gly Val Asp Arg Val Ile
225 230 235 240
Met Ala Gly Gly Gly Pro Glu Thr Leu Gly Gln Ala Ile Ser Met Val
245 250 255
Lys Pro Gly Gly Val Val Ser Asn Ile Asn Tyr His Gly Ser Gly Asp
260 265 270
Thr Leu Pro Ile Pro Arg Val Asp Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Arg Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Ala Glu Met
290 295 300
Leu Arg Asp Leu Val Ile Tyr Asp Arg Ile Asp Leu Ser Arg Leu Val
305 310 315 320
Thr His Val Tyr Asn Gly Phe Glu His Ile Glu Glu Ala Leu Leu Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Ala Val Val Ile Val
340 345 350
<210> 35
<211> 1059
<212> DNA
<213> 梭菌属细菌（Clostridiales bacterium SK-Y3）
<400> 35
atgaagggct ttgcaatgct gcgcattggc gaagtgggtt ggattgaaaa agaaaaaccg 60
gttgccggcc cgtatgatgc cattgtgcgc ccgctggcag ttgccccgtg cacaagcgat 120
attcataccg tttttgaagg tgcaattggt gaacgtcata atatgattct gggtcatgaa 180
gcagttggtg aagtggttga agttggtagc gaagtgaaag attttaaacc gggcgatcgt 240
gtggtggttc cggccattac cccggattgg cgcagcattg aagttcagga aggttatcat 300
cagcatagcg aaggcatgct ggcaggttgg aaattttcta atgttaaaga tggcgtgttc 360
ggtgaatatt ttcatgttaa tgatgccgat atgaacctgg cactgctgcc ggatgaaatt 420
agcctggaaa aagccgcaat gattccggat atgatgacca ccggctttca tggtgcagaa 480
ctggcagata ttaagattgg cagcaccgtg gccgttattg gcattggtcc ggttggcctg 540
atgggcgttg ccggtgccga actgtgcggt gccggtcgta ttattgcagt gggtagccgt 600
ccgaattgcg ttgaagttgc aaaaagttat ggtgccaccg atattgttaa ttatcgtaat 660
ggcgatatcg ttacccagat tattgatctg accaatggta aaggcgttga tagcaccatt 720
attgcaggcg gcaatccgga tgtgatggaa agcgcagttc gcatgaccaa accgggcggc 780
accattgcca atattaatta ttttggtgag ggtaaaatcc tgccgattcc gcgcagtgat 840
tggggttgcg gtatggccca taaaaatatt cgcggtggtc tgtgcccggg cggtcgctta 900
cgcatggaac gtctgattaa tattgtgaaa tacaaccgcg ttgatccggg taaactggtg 960
acccatgttt atcatggctt tgaaaatatg gaaaaggccc tgctgctgat gaaagataaa 1020
ccgaaagatt tgattaagcc ggtggttatt ctggattaa 1059
<210> 36
<211> 352
<212> PRT
<213>梭菌属细菌（Clostridiales bacterium SK-Y3）
<400> 36
Met Lys Gly Phe Ala Met Leu Arg Ile Gly Glu Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Val Ala Gly Pro Tyr Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Ser Ile Glu Val Gln
85 90 95
Glu Gly Tyr His Gln His Ser Glu Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Val Lys Asp Gly Val Phe Gly Glu Tyr Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala Leu Leu Pro Asp Glu Ile Ser Leu Glu Lys
130 135 140
Ala Ala Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Lys Ile Gly Ser Thr Val Ala Val Ile Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Gly Val Ala Gly Ala Glu Leu Cys Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Asn Cys Val Glu Val Ala Lys
195 200 205
Ser Tyr Gly Ala Thr Asp Ile Val Asn Tyr Arg Asn Gly Asp Ile Val
210 215 220
Thr Gln Ile Ile Asp Leu Thr Asn Gly Lys Gly Val Asp Ser Thr Ile
225 230 235 240
Ile Ala Gly Gly Asn Pro Asp Val Met Glu Ser Ala Val Arg Met Thr
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Ile Asn Tyr Phe Gly Glu Gly Lys
260 265 270
Ile Leu Pro Ile Pro Arg Ser Asp Trp Gly Cys Gly Met Ala His Lys
275 280 285
Asn Ile Arg Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
290 295 300
Leu Ile Asn Ile Val Lys Tyr Asn Arg Val Asp Pro Gly Lys Leu Val
305 310 315 320
Thr His Val Tyr His Gly Phe Glu Asn Met Glu Lys Ala Leu Leu Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Asp
340 345 350
<210> 37
<211> 1011
<212> DNA
<213> 近平滑假丝酵母（Candida parapsilosis）
<400> 37
atgagcattc cgagtagtca gtatggcttt gtttttaata agcagagcgg tctgaatctg 60
cgtaatgatc tgccggtgca taaaccgaaa gcaggccagc tgctgctgaa agtggatgcc 120
gttggtctgt gccatagcga tctgcatgtg atctatgaag gcctggattg tggcgataat 180
tatgtgatgg gccatgaaat tgccggtaca gtggccgcag ttggcgatga tgtgattaat 240
tataaagtgg gcgatcgtgt ggcctgcgtt ggtccgaatg gctgcggtgg ttgcaaatat 300
tgtcgtggcg ccattgataa tgtttgtaaa aatgcattcg gcgattggtt tggcctgggc 360
tatgatggcg gctatcagca gtatctgctg gtgacccgtc cgcgcaatct gagtcgtatt 420
ccggataatg ttagcgcaga tgtggccgcc gccagcaccg atgcagtgct gaccccgtat 480
catgcaatta agatggccca ggtgagtccg accagcaata ttctgctgat tggtgcaggc 540
ggcctgggtg gtaatgccat tcaggttgca aaagcctttg gtgcaaaagt gaccgttctg 600
gataaaaaga aagaagcacg tgatcaggcc aaaaaactgg gtgcagatgc cgtttatgaa 660
accctgccgg aaagcattag cccgggtagc tttagcgcct gttttgattt tgttagcgtt 720
caggcaacct ttgatgtttg tcagaaatat gttgaaccga aaggtgttat tatgccggtt 780
ggcctgggtg caccgaatct gagctttaat ctgggtgacc tggcactgcg tgaaattcgt 840
attctgggta gtttttgggg caccaccaat gatctggatg atgttctgaa actggttagc 900
gaaggcaaag ttaaaccggt tgtgcgtagt gcaaaactga aagaactgcc ggaatatatt 960
gaaaaactgc gcaataatgc ctatgaaggt cgtgttgttt ttaatccgta a 1011
<210> 38
<211> 336
<212> PRT
<213> 近平滑假丝酵母（Candida parapsilosis）
<400> 38
Met Ser Ile Pro Ser Ser Gln Tyr Gly Phe Val Phe Asn Lys Gln Ser
1 5 10 15
Gly Leu Asn Leu Arg Asn Asp Leu Pro Val His Lys Pro Lys Ala Gly
20 25 30
Gln Leu Leu Leu Lys Val Asp Ala Val Gly Leu Cys His Ser Asp Leu
35 40 45
His Val Ile Tyr Glu Gly Leu Asp Cys Gly Asp Asn Tyr Val Met Gly
50 55 60
His Glu Ile Ala Gly Thr Val Ala Ala Val Gly Asp Asp Val Ile Asn
65 70 75 80
Tyr Lys Val Gly Asp Arg Val Ala Cys Val Gly Pro Asn Gly Cys Gly
85 90 95
Gly Cys Lys Tyr Cys Arg Gly Ala Ile Asp Asn Val Cys Lys Asn Ala
100 105 110
Phe Gly Asp Trp Phe Gly Leu Gly Tyr Asp Gly Gly Tyr Gln Gln Tyr
115 120 125
Leu Leu Val Thr Arg Pro Arg Asn Leu Ser Arg Ile Pro Asp Asn Val
130 135 140
Ser Ala Asp Val Ala Ala Ala Ser Thr Asp Ala Val Leu Thr Pro Tyr
145 150 155 160
His Ala Ile Lys Met Ala Gln Val Ser Pro Thr Ser Asn Ile Leu Leu
165 170 175
Ile Gly Ala Gly Gly Leu Gly Gly Asn Ala Ile Gln Val Ala Lys Ala
180 185 190
Phe Gly Ala Lys Val Thr Val Leu Asp Lys Lys Lys Glu Ala Arg Asp
195 200 205
Gln Ala Lys Lys Leu Gly Ala Asp Ala Val Tyr Glu Thr Leu Pro Glu
210 215 220
Ser Ile Ser Pro Gly Ser Phe Ser Ala Cys Phe Asp Phe Val Ser Val
225 230 235 240
Gln Ala Thr Phe Asp Val Cys Gln Lys Tyr Val Glu Pro Lys Gly Val
245 250 255
Ile Met Pro Val Gly Leu Gly Ala Pro Asn Leu Ser Phe Asn Leu Gly
260 265 270
Asp Leu Ala Leu Arg Glu Ile Arg Ile Leu Gly Ser Phe Trp Gly Thr
275 280 285
Thr Asn Asp Leu Asp Asp Val Leu Lys Leu Val Ser Glu Gly Lys Val
290 295 300
Lys Pro Val Val Arg Ser Ala Lys Leu Lys Glu Leu Pro Glu Tyr Ile
305 310 315 320
Glu Lys Leu Arg Asn Asn Ala Tyr Glu Gly Arg Val Val Phe Asn Pro
325 330 335
<210> 39
<211> 1431
<212> DNA
<213> 巨大芽胞杆菌（Bacillus megaterium）
<400> 39
atgagcctga ccgtgcaaaa aattaactgg gaacaggtta aggagtggga tcgtaaatat 60
ctgatgcgta cctttagcac ccagaatgaa tatcagccgg ttccgattga aagtaccgaa 120
ggcgattatc tgatcatgcc ggatggtaca cgcctgctgg atttctttaa tcagctgtat 180
tgcgtgaacc tgggtcagaa aaatcagaaa gttaacgcag ccatcaagga agcactggat 240
cgctatggct ttgtttggga tacctatgcc accgattata aagccaaagc agcaaaaatc 300
atcatcgagg atattctggg tgacgaagat tggccgggca aagtgcgttt tgtgagtacc 360
ggcagcgaag ccgtggaaac agctttaaat attgcacgcc tgtacaccaa tcgcccgctg 420
gtggtgacac gtgaacatga ttatcatggc tggaccggcg gcgcagcaac cgtgacccgt 480
ctgcgtagct atcgtagcgg tctggtgggt gaaaatagcg aaagttttag tgcccagatc 540
ccgggcagta gctataatag cgcagtgctg atggccccga gccctaacat gtttcaggat 600
agcgatggta atctgctgaa agatgaaaac ggcgaactgc tgagcgttaa atatacccgc 660
cgcatgattg aaaactacgg tccggaacag gtggcagcag ttattaccga agttagccag 720
ggtgccggta gtgctatgcc tccttatgaa tatatcccgc agattcgcaa aatgaccaaa 780
gaactgggcg tgctgtggat taatgatgaa gtgctgaccg gttttggccg caccggtaaa 840
tggtttggtt atcagcatta cggtgtgcag ccggatatta ttacaatggg taaaggtctg 900
agcagcagca gtctgccggc tggtgcagtg ttagtgagca aagaaattgc agcattcatg 960
gataagcacc gttgggaaag cgtgagtacc tatgccggtc atccggttgc aatggctgcc 1020
gtgtgtgcaa atctggaagt gatgatggaa gaaaacttcg ttgagcaggc aaaagatagt 1080
ggtgaatata tccgtagcaa gctggaactg ctgcaggaaa aacataaaag catcggtaac 1140
ttcgacggct atggcctgct gtggattgtt gatattgtta atgccaagac caagaccccg 1200
tatgttaaac tggatcgcaa ttttacccac ggtatgaatc cgaatcagat tccgacccag 1260
attattatga agaaggccct ggaaaagggc gtgctgattg gtggtgtgat gccgaatacc 1320
atgcgcattg gtgcaagcct gaatgtgagt cgcggcgata ttgataaagc aatggatgca 1380
ctggactacg ccctggatta tctggaaagt ggtgaatggc agcagagcta a 1431
<210> 40
<211> 476
<212> PRT
<213>巨大芽胞杆菌（Bacillus megaterium）
<400> 40
Met Ser Leu Thr Val Gln Lys Ile Asn Trp Glu Gln Val Lys Glu Trp
1 5 10 15
Asp Arg Lys Tyr Leu Met Arg Thr Phe Ser Thr Gln Asn Glu Tyr Gln
20 25 30
Pro Val Pro Ile Glu Ser Thr Glu Gly Asp Tyr Leu Ile Met Pro Asp
35 40 45
Gly Thr Arg Leu Leu Asp Phe Phe Asn Gln Leu Tyr Cys Val Asn Leu
50 55 60
Gly Gln Lys Asn Gln Lys Val Asn Ala Ala Ile Lys Glu Ala Leu Asp
65 70 75 80
Arg Tyr Gly Phe Val Trp Asp Thr Tyr Ala Thr Asp Tyr Lys Ala Lys
85 90 95
Ala Ala Lys Ile Ile Ile Glu Asp Ile Leu Gly Asp Glu Asp Trp Pro
100 105 110
Gly Lys Val Arg Phe Val Ser Thr Gly Ser Glu Ala Val Glu Thr Ala
115 120 125
Leu Asn Ile Ala Arg Leu Tyr Thr Asn Arg Pro Leu Val Val Thr Arg
130 135 140
Glu His Asp Tyr His Gly Trp Thr Gly Gly Ala Ala Thr Val Thr Arg
145 150 155 160
Leu Arg Ser Tyr Arg Ser Gly Leu Val Gly Glu Asn Ser Glu Ser Phe
165 170 175
Ser Ala Gln Ile Pro Gly Ser Ser Tyr Asn Ser Ala Val Leu Met Ala
180 185 190
Pro Ser Pro Asn Met Phe Gln Asp Ser Asp Gly Asn Leu Leu Lys Asp
195 200 205
Glu Asn Gly Glu Leu Leu Ser Val Lys Tyr Thr Arg Arg Met Ile Glu
210 215 220
Asn Tyr Gly Pro Glu Gln Val Ala Ala Val Ile Thr Glu Val Ser Gln
225 230 235 240
Gly Ala Gly Ser Ala Met Pro Pro Tyr Glu Tyr Ile Pro Gln Ile Arg
245 250 255
Lys Met Thr Lys Glu Leu Gly Val Leu Trp Ile Asn Asp Glu Val Leu
260 265 270
Thr Gly Phe Gly Arg Thr Gly Lys Trp Phe Gly Tyr Gln His Tyr Gly
275 280 285
Val Gln Pro Asp Ile Ile Thr Met Gly Lys Gly Leu Ser Ser Ser Ser
290 295 300
Leu Pro Ala Gly Ala Val Leu Val Ser Lys Glu Ile Ala Ala Phe Met
305 310 315 320
Asp Lys His Arg Trp Glu Ser Val Ser Thr Tyr Ala Gly His Pro Val
325 330 335
Ala Met Ala Ala Val Cys Ala Asn Leu Glu Val Met Met Glu Glu Asn
340 345 350
Phe Val Glu Gln Ala Lys Asp Ser Gly Glu Tyr Ile Arg Ser Lys Leu
355 360 365
Glu Leu Leu Gln Glu Lys His Lys Ser Ile Gly Asn Phe Asp Gly Tyr
370 375 380
Gly Leu Leu Trp Ile Val Asp Ile Val Asn Ala Lys Thr Lys Thr Pro
385 390 395 400
Tyr Val Lys Leu Asp Arg Asn Phe Thr His Gly Met Asn Pro Asn Gln
405 410 415
Ile Pro Thr Gln Ile Ile Met Lys Lys Ala Leu Glu Lys Gly Val Leu
420 425 430
Ile Gly Gly Val Met Pro Asn Thr Met Arg Ile Gly Ala Ser Leu Asn
435 440 445
Val Ser Arg Gly Asp Ile Asp Lys Ala Met Asp Ala Leu Asp Tyr Ala
450 455 460
Leu Asp Tyr Leu Glu Ser Gly Glu Trp Gln Gln Ser
465 470 475
<210> 41
<211> 1347
<212> DNA
<213> 铜绿假单胞菌（P.aeruginosa PAO2）
<400> 41
atgaaccagc cgctgaatgt ggccccgccg gttagcagcg aactgaatct gcgtgcccat 60
tggatgccgt ttagcgcaaa tcgtaatttt cagaaagatc cgcgtattat tgttgccgca 120
gaaggtagtt ggctgaccga tgataaaggc cgcaaagtgt atgatagtct gagtggcctg 180
tggacctgcg gtgcaggcca tagccgtaaa gaaattcagg aagcagtggc acgccagctg 240
ggcaccctgg attatagccc gggttttcag tatggccatc cgctgagttt tcagctggca 300
gaaaaaattg ccggtctgct gccgggtgaa ctgaatcatg ttttctttac cggtagtggc 360
agcgaatgcg ccgataccag cattaagatg gcccgtgcat attggcgcct gaaaggtcag 420
ccgcagaaaa ccaaactgat tggccgtgca cgcggttatc atggcgtgaa tgttgccggc 480
accagcctgg gcggcattgg tggtaatcgc aaaatgtttg gtcagctgat ggatgtggat 540
catctgccgc ataccctgca gccgggcatg gcattcactc gtggtatggc acagaccggc 600
ggcgttgaac tggcaaatga actgctgaaa ctgattgaac tgcatgatgc cagtaatatt 660
gccgcagtga ttgtggaacc gatgagtggc agtgcaggtg ttctggtgcc gccggtgggt 720
tatctgcagc gtctgcgtga aatttgtgat cagcataata ttctgctgat ttttgatgaa 780
gtgatcaccg catttggccg tctgggtaca tatagcggtg ccgaatattt tggtgtgacc 840
ccggatctga tgaatgtggc aaaacaggtg accaatggtg ccgtgccgat gggcgcagtt 900
attgcaagca gcgaaatcta tgataccttt atgaatcagg ccctgccgga acatgccgtg 960
gaattttctc atggttatac ctatagtgca catccggttg cctgtgccgc cggcctggca 1020
gcactggata ttctggcccg tgataatctg gtgcagcaga gtgcagaact ggcaccgcat 1080
tttgaaaaag gtctgcatgg tctgcagggc gccaaaaatg ttattgatat tcgtaattgc 1140
ggcctggccg gcgccattca gattgcaccg cgtgatggtg acccgaccgt tcgcccgttt 1200
gaagccggca tgaaactgtg gcagcagggt ttttatgtgc gctttggcgg cgataccctg 1260
cagtttggtc cgacctttaa tgcacgcccg gaagaactgg atcgcctgtt tgatgcagtg 1320
ggtgaagcac tgaatggtat tgcctaa 1347
<210> 42
<211> 448
<212> PRT
<213> 铜绿假单胞菌（P.aeruginosa PAO2）
<400> 42
Met Asn Gln Pro Leu Asn Val Ala Pro Pro Val Ser Ser Glu Leu Asn
1 5 10 15
Leu Arg Ala His Trp Met Pro Phe Ser Ala Asn Arg Asn Phe Gln Lys
20 25 30
Asp Pro Arg Ile Ile Val Ala Ala Glu Gly Ser Trp Leu Thr Asp Asp
35 40 45
Lys Gly Arg Lys Val Tyr Asp Ser Leu Ser Gly Leu Trp Thr Cys Gly
50 55 60
Ala Gly His Ser Arg Lys Glu Ile Gln Glu Ala Val Ala Arg Gln Leu
65 70 75 80
Gly Thr Leu Asp Tyr Ser Pro Gly Phe Gln Tyr Gly His Pro Leu Ser
85 90 95
Phe Gln Leu Ala Glu Lys Ile Ala Gly Leu Leu Pro Gly Glu Leu Asn
100 105 110
His Val Phe Phe Thr Gly Ser Gly Ser Glu Cys Ala Asp Thr Ser Ile
115 120 125
Lys Met Ala Arg Ala Tyr Trp Arg Leu Lys Gly Gln Pro Gln Lys Thr
130 135 140
Lys Leu Ile Gly Arg Ala Arg Gly Tyr His Gly Val Asn Val Ala Gly
145 150 155 160
Thr Ser Leu Gly Gly Ile Gly Gly Asn Arg Lys Met Phe Gly Gln Leu
165 170 175
Met Asp Val Asp His Leu Pro His Thr Leu Gln Pro Gly Met Ala Phe
180 185 190
Thr Arg Gly Met Ala Gln Thr Gly Gly Val Glu Leu Ala Asn Glu Leu
195 200 205
Leu Lys Leu Ile Glu Leu His Asp Ala Ser Asn Ile Ala Ala Val Ile
210 215 220
Val Glu Pro Met Ser Gly Ser Ala Gly Val Leu Val Pro Pro Val Gly
225 230 235 240
Tyr Leu Gln Arg Leu Arg Glu Ile Cys Asp Gln His Asn Ile Leu Leu
245 250 255
Ile Phe Asp Glu Val Ile Thr Ala Phe Gly Arg Leu Gly Thr Tyr Ser
260 265 270
Gly Ala Glu Tyr Phe Gly Val Thr Pro Asp Leu Met Asn Val Ala Lys
275 280 285
Gln Val Thr Asn Gly Ala Val Pro Met Gly Ala Val Ile Ala Ser Ser
290 295 300
Glu Ile Tyr Asp Thr Phe Met Asn Gln Ala Leu Pro Glu His Ala Val
305 310 315 320
Glu Phe Ser His Gly Tyr Thr Tyr Ser Ala His Pro Val Ala Cys Ala
325 330 335
Ala Gly Leu Ala Ala Leu Asp Ile Leu Ala Arg Asp Asn Leu Val Gln
340 345 350
Gln Ser Ala Glu Leu Ala Pro His Phe Glu Lys Gly Leu His Gly Leu
355 360 365
Gln Gly Ala Lys Asn Val Ile Asp Ile Arg Asn Cys Gly Leu Ala Gly
370 375 380
Ala Ile Gln Ile Ala Pro Arg Asp Gly Asp Pro Thr Val Arg Pro Phe
385 390 395 400
Glu Ala Gly Met Lys Leu Trp Gln Gln Gly Phe Tyr Val Arg Phe Gly
405 410 415
Gly Asp Thr Leu Gln Phe Gly Pro Thr Phe Asn Ala Arg Pro Glu Glu
420 425 430
Leu Asp Arg Leu Phe Asp Ala Val Gly Glu Ala Leu Asn Gly Ile Ala
435 440 445
<210> 43
<211> 1380
<212> DNA
<213> 紫色杆菌（Chromobacterium violaceum 2025）
<400> 43
atgcagaaac agcgcaccac cagccagtgg cgtgaactgg atgccgcaca tcatctgcat 60
ccgtttaccg ataccgccag cctgaatcag gccggcgcac gcgttatgac ccgcggtgaa 120
ggcgtttatc tgtgggatag tgaaggcaat aagattattg atggtatggc cggcctgtgg 180
tgcgtgaatg ttggctatgg tcgtaaagat tttgccgaag cagcacgtcg ccagatggaa 240
gaactgccgt tttataatac ctttttcaaa accacccacc cggcagttgt tgaactgagt 300
agcctgctgg ccgaagtgac cccggcaggc tttgatcgtg tgttttatac caatagtggt 360
agcgaaagcg ttgataccat gattcgcatg gtgcgccgct attgggatgt tcagggcaaa 420
ccggaaaaga aaaccctgat tggccgctgg aatggttatc atggcagtac cattggcggt 480
gccagcctgg gcggtatgaa atatatgcat gaacagggcg atctgccgat tccgggtatg 540
gcccatattg aacagccgtg gtggtataaa catggtaaag atatgacccc ggatgaattt 600
ggcgtggttg cagcccgctg gctggaagaa aaaattctgg aaattggcgc agataaagtg 660
gccgcctttg ttggtgaacc gattcagggt gccggcggcg ttattgttcc gccggccacc 720
tattggccgg aaattgaacg tatttgtcgc aaatatgatg ttctgctggt tgcagatgaa 780
gttatttgtg gctttggtcg taccggcgaa tggtttggcc atcagcattt tggttttcag 840
ccggatctgt ttaccgcagc caaaggcctg agcagcggtt atctgccgat tggcgccgtt 900
tttgttggta aacgtgtggc agaaggcctg attgcaggtg gtgactttaa tcatggtttt 960
acctatagcg gccatccggt ttgcgccgca gttgcacatg ccaatgttgc agccctgcgt 1020
gatgaaggca ttgttcagcg tgtgaaagat gatattggcc cgtatatgca gaaacgctgg 1080
cgtgaaacct ttagtcgttt tgaacatgtg gatgatgttc gcggtgttgg catggttcag 1140
gcattcactc tggtgaaaaa taaggcaaaa cgcgaactgt ttccggattt tggtgaaatt 1200
ggtacactgt gccgtgatat tttctttcgc aataatctga ttatgcgcgc ctgtggcgat 1260
catattgtta gcgccccgcc gctggttatg acccgtgccg aagttgatga aatgctggcc 1320
gttgcagaac gttgcctgga agaatttgaa cagaccctga aagcccgtgg cctggcctaa 1380
<210> 44
<211> 459
<212> PRT
<213>紫色杆菌（Chromobacterium violaceum 2025）
<400> 44
Met Gln Lys Gln Arg Thr Thr Ser Gln Trp Arg Glu Leu Asp Ala Ala
1 5 10 15
His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly
20 25 30
Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Ser Glu
35 40 45
Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Leu Trp Cys Val Asn Val
50 55 60
Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu
65 70 75 80
Glu Leu Pro Phe Tyr Asn Thr Phe Phe Lys Thr Thr His Pro Ala Val
85 90 95
Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp
100 105 110
Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile
115 120 125
Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys
130 135 140
Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly
145 150 155 160
Ala Ser Leu Gly Gly Met Lys Tyr Met His Glu Gln Gly Asp Leu Pro
165 170 175
Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly
180 185 190
Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu
195 200 205
Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val
210 215 220
Gly Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr
225 230 235 240
Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu
245 250 255
Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe
260 265 270
Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys
275 280 285
Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys
290 295 300
Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe Asn His Gly Phe
305 310 315 320
Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val
325 330 335
Ala Ala Leu Arg Asp Glu Gly Ile Val Gln Arg Val Lys Asp Asp Ile
340 345 350
Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu
355 360 365
His Val Asp Asp Val Arg Gly Val Gly Met Val Gln Ala Phe Thr Leu
370 375 380
Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile
385 390 395 400
Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met Arg
405 410 415
Ala Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg
420 425 430
Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu Arg Cys Leu Glu Glu
435 440 445
Phe Glu Gln Thr Leu Lys Ala Arg Gly Leu Ala
450 455
<210> 45
<211> 978
<212> DNA
<213>土曲霉（Aspergillus terreus）
<400> 45
atggctagta tggataaggt gttcgccggc tatgccgcac gtcaagcaat tctggaaagt 60
accgaaacca ccaatccgtt tgcaaaaggt attgcctggg ttgaaggtga actggtgccg 120
ttagccgaag cacgtattcc gctgctggat cagggcttta tgcatagtga tctgacctat 180
gatgtgccga gtgtttggga tggtcgcttt ttccgcctgg atgatcatat tacccgcctg 240
gaagcaagct gcaccaaact gcgtctgcgc ttaccgctgc ctcgtgacca ggtgaaacag 300
attctggtgg aaatggttgc aaagagcggt attcgcgatg cctttgtgga actgattgtt 360
acccgcggcc tgaaaggtgt gcgcggtacg cgtcctgaag atattgtgaa taatctgtac 420
atgttcgtgc agccgtatgt ttgggttatg gaaccggata tgcagcgtgt gggtggcagt 480
gcagtggttg caagaaccgt gcgtcgcgtt cctcctggtg caattgatcc gaccgttaaa 540
aatctgcagt ggggtgacct ggttcgcggt atgtttgaag ccgccgatcg tggtgccacc 600
tatccttttc tgaccgatgg tgacgcacat ctgaccgaag gcagcggttt taatattgtg 660
ctggttaaag acggcgtgct gtataccccg gatcgcggtg tgttacaggg cgtgaccaga 720
aaaagtgtta ttaatgcagc cgaggccttt ggcattgaag tgcgtgtgga atttgtgccg 780
gtggaactgg cctatcgctg cgacgagatt tttatgtgca ccaccgccgg tggtattatg 840
ccgattacca ccctggatgg tatgccggtg aatggcggtc agattggccc tattaccaaa 900
aagatttggg acggttactg ggccatgcat tatgatgcag catatagctt tgagatcgac 960
tataacgagc gtaattaa 978
<210> 46
<211> 325
<212> PRT
<213> 土曲霉（Aspergillus terreus）
<400> 46
Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala
1 5 10 15
Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala
20 25 30
Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu
35 40 45
Leu Asp Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser
50 55 60
Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu
65 70 75 80
Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp
85 90 95
Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg
100 105 110
Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg
115 120 125
Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln
130 135 140
Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser
145 150 155 160
Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp
165 170 175
Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe
180 185 190
Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp
195 200 205
Ala His Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp
210 215 220
Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg
225 230 235 240
Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val
245 250 255
Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met
260 265 270
Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met
275 280 285
Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp
290 295 300
Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp
305 310 315 320
Tyr Asn Glu Arg Asn
325
<210> 47
<211> 972
<212> DNA
<213> 费希新萨托菌（Neosartorya fischeri）
<400> 47
atggctagta tggataaggt gttcagcggc tatcatgccc gccagaaact gctggaacgt 60
agtgataatc cgtttagtaa gggcattgcc tatgtggaag gtaaactggt gctgccgagt 120
gatgcccgta ttcctctgct ggatgaaggc tttatgcatg gtgacctgac ctatgatgtt 180
accaccgtgt gggatggtcg ctttttccgt ctggatgatc acatgcagcg tattctggaa 240
agctgcgata aaatgcgtct gaaattcccg ctggccccga gtacagttaa aaatattctg 300
gcagagatgg tggcaaagag cggcattcgc gatgcctttg ttgaagtgat tgttacccgt 360
ggtctgaccg gtgttcgtgg tagtaaaccg gaagatttgt ataacaacaa catctacctg 420
ctggtgctgc cttatgtgtg ggttatggca ccggaaaatc agctgctggg cggttcagca 480
attattaccc gcaccgtgcg ccgtacccct cctggtgcat tcgaccctac aattaagaat 540
ctgcagtggg gcgatctgac caaaggctta tttgaagcaa tggatcgcgg cgccacctat 600
ccttttctga ccgatggtga caccaatctg accgaaggta gcggctttaa tattgttctg 660
gtgaaaaacg gcatcatcta caccccggat cgcggtgttc tgcgtggtat tacccgcaaa 720
agtgttattg atgtggcccg cgcaaataat attgatattc gtctggaggt ggtgccggtt 780
gaacaggttt atcatagtga tgaaatcttc atgtgcacca ccgccggcgg tattatgcct 840
attaccctgc tggatggtca gccggttaat gatggtcagg ttggcccgat taccaaaaag 900
atttgggatg gctattggga aatgcattac aatccggcat acagctttcc ggttgattat 960
ggtagcggct aa 972
<210> 48
<211> 323
<212> PRT
<213> 费希新萨托菌（Neosartorya fischeri）
<400> 48
Met Ala Ser Met Asp Lys Val Phe Ser Gly Tyr His Ala Arg Gln Lys
1 5 10 15
Leu Leu Glu Arg Ser Asp Asn Pro Phe Ser Lys Gly Ile Ala Tyr Val
20 25 30
Glu Gly Lys Leu Val Leu Pro Ser Asp Ala Arg Ile Pro Leu Leu Asp
35 40 45
Glu Gly Phe Met His Gly Asp Leu Thr Tyr Asp Val Thr Thr Val Trp
50 55 60
Asp Gly Arg Phe Phe Arg Leu Asp Asp His Met Gln Arg Ile Leu Glu
65 70 75 80
Ser Cys Asp Lys Met Arg Leu Lys Phe Pro Leu Ala Pro Ser Thr Val
85 90 95
Lys Asn Ile Leu Ala Glu Met Val Ala Lys Ser Gly Ile Arg Asp Ala
100 105 110
Phe Val Glu Val Ile Val Thr Arg Gly Leu Thr Gly Val Arg Gly Ser
115 120 125
Lys Pro Glu Asp Leu Tyr Asn Asn Asn Ile Tyr Leu Leu Val Leu Pro
130 135 140
Tyr Val Trp Val Met Ala Pro Glu Asn Gln Leu Leu Gly Gly Ser Ala
145 150 155 160
Ile Ile Thr Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro
165 170 175
Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Lys Gly Leu Phe Glu
180 185 190
Ala Met Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp Thr
195 200 205
Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asn Gly
210 215 220
Ile Ile Tyr Thr Pro Asp Arg Gly Val Leu Arg Gly Ile Thr Arg Lys
225 230 235 240
Ser Val Ile Asp Val Ala Arg Ala Asn Asn Ile Asp Ile Arg Leu Glu
245 250 255
Val Val Pro Val Glu Gln Val Tyr His Ser Asp Glu Ile Phe Met Cys
260 265 270
Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Leu Leu Asp Gly Gln Pro
275 280 285
Val Asn Asp Gly Gln Val Gly Pro Ile Thr Lys Lys Ile Trp Asp Gly
290 295 300
Tyr Trp Glu Met His Tyr Asn Pro Ala Tyr Ser Phe Pro Val Asp Tyr
305 310 315 320
Gly Ser Gly
<210> 49
<211> 1014
<212> DNA
<213> 分支杆菌（Mycobacterium vanbaalenii）
<400> 49
atgggtatcg acaccggtac aagcaatctg gtggccgtgg aaccgggtgc aattagagaa 60
gataccccgg ccggtagcgt gattcagtat agcgattatg aaatcgacta cagcagcccg 120
tttgcaggtg gtgtggcttg gattgaaggc gaatatctgc cggccgaaga tgccaaaatt 180
agcatttttg acaccggttt cggccatagc gatctgacct ataccgttgc acatgtttgg 240
catggcaata ttttccgcct gggcgatcat ctggatcgtc tgttagatgg cgcacgtaaa 300
ctgcgtctgg atagtggcta taccaaagat gaactggcag atattaccaa gaagtgcgtg 360
agcctgagcc agctgcgtga atcatttgtg aatctgacca ttacccgcgg ttatggtaaa 420
cgcaaaggtg aaaaagacct gagtaagctg acccatcagg tgtatatcta tgccattccg 480
tatctgtggg cctttccgcc tgccgagcaa atttttggca ccaccgccgt tgtgccgcgt 540
cacgtgcgtc gtgcaggtcg taacacagtt gatccgacca ttaagaatta ccagtggggt 600
gacctgaccg cagccagctt cgaggcaaaa gatcgcggtg ctcgcaccgc aattctgatg 660
gatgccgata attgtgtggc agaaggcccg ggttttaatg tgtgcattgt taaagacggc 720
aagctggcaa gcccgagtcg taatgcactg cctggtatta cccgtaaaac cgtgtttgaa 780
atcgccggtg caatgggcat tgaagccgca ttacgtgatg ttaccagtca tgaactgtac 840
gatgcagatg aaatcatggc agttaccacc gccggcggtg ttacacctat taataccctg 900
gatggcgttc cgattggtga cggtgaaccg ggtcctgtta ccgttgctat tcgtgatcgc 960
ttttgggcac tgatggatga accgggtccg ttaattgaag ccattcagta ttaa 1014
<210> 50
<211> 337
<212> PRT
<213> 分支杆菌（Mycobacterium vanbaalenii）
<400> 50
Met Gly Ile Asp Thr Gly Thr Ser Asn Leu Val Ala Val Glu Pro Gly
1 5 10 15
Ala Ile Arg Glu Asp Thr Pro Ala Gly Ser Val Ile Gln Tyr Ser Asp
20 25 30
Tyr Glu Ile Asp Tyr Ser Ser Pro Phe Ala Gly Gly Val Ala Trp Ile
35 40 45
Glu Gly Glu Tyr Leu Pro Ala Glu Asp Ala Lys Ile Ser Ile Phe Asp
50 55 60
Thr Gly Phe Gly His Ser Asp Leu Thr Tyr Thr Val Ala His Val Trp
65 70 75 80
His Gly Asn Ile Phe Arg Leu Gly Asp His Leu Asp Arg Leu Leu Asp
85 90 95
Gly Ala Arg Lys Leu Arg Leu Asp Ser Gly Tyr Thr Lys Asp Glu Leu
100 105 110
Ala Asp Ile Thr Lys Lys Cys Val Ser Leu Ser Gln Leu Arg Glu Ser
115 120 125
Phe Val Asn Leu Thr Ile Thr Arg Gly Tyr Gly Lys Arg Lys Gly Glu
130 135 140
Lys Asp Leu Ser Lys Leu Thr His Gln Val Tyr Ile Tyr Ala Ile Pro
145 150 155 160
Tyr Leu Trp Ala Phe Pro Pro Ala Glu Gln Ile Phe Gly Thr Thr Ala
165 170 175
Val Val Pro Arg His Val Arg Arg Ala Gly Arg Asn Thr Val Asp Pro
180 185 190
Thr Ile Lys Asn Tyr Gln Trp Gly Asp Leu Thr Ala Ala Ser Phe Glu
195 200 205
Ala Lys Asp Arg Gly Ala Arg Thr Ala Ile Leu Met Asp Ala Asp Asn
210 215 220
Cys Val Ala Glu Gly Pro Gly Phe Asn Val Cys Ile Val Lys Asp Gly
225 230 235 240
Lys Leu Ala Ser Pro Ser Arg Asn Ala Leu Pro Gly Ile Thr Arg Lys
245 250 255
Thr Val Phe Glu Ile Ala Gly Ala Met Gly Ile Glu Ala Ala Leu Arg
260 265 270
Asp Val Thr Ser His Glu Leu Tyr Asp Ala Asp Glu Ile Met Ala Val
275 280 285
Thr Thr Ala Gly Gly Val Thr Pro Ile Asn Thr Leu Asp Gly Val Pro
290 295 300
Ile Gly Asp Gly Glu Pro Gly Pro Val Thr Val Ala Ile Arg Asp Arg
305 310 315 320
Phe Trp Ala Leu Met Asp Glu Pro Gly Pro Leu Ile Glu Ala Ile Gln
325 330 335
Tyr
<210> 51
<211> 993
<212> DNA
<213> 节细菌属（Arthrobacter sp. KNK168）
<400> 51
atggcatttt ctgcagatac cagcgaaatt gtttataccc atgataccgg tctggattat 60
attacctata gcgattatga actggaccct gccaatccgc tggccggcgg tgctgcttgg 120
attgaaggtg cctttgtgcc gccgagtgaa gcacgcatta gtatttttga tcagggttat 180
ctgcatagtg atgtgaccta taccgtgttt catgtttgga atggtaatgc ctttcgtctg 240
gatgatcata ttgaacgtct gtttagcaat gccgaaagca tgcgcattat tccgccgctg 300
acccaggatg aagttaaaga aattgcactg gaactggttg caaaaaccga actgcgtgaa 360
gcatttgtta gtgttagcat tacccgtggc tatagcagca ccccgggtga acgtgatatt 420
accaaacatc gcccgcaggt ttatatgtat gcagttccgt atcagtggat tgttccgttt 480
gatcgcattc gtgatggtgt gcatgcaatg gttgcacaga gtgttcgtcg caccccgcgt 540
agcagcattg atccgcaggt taaaaatttt cagtggggcg atctgattcg cgccgttcag 600
gaaacccatg atcgcggctt tgaagcaccg ctgctgctgg atggcgatgg tctgctggcc 660
gaaggtagcg gctttaatgt ggtggttatt aaggatggcg ttgttcgcag cccgggtcgt 720
gcagcactgc cgggtattac ccgcaaaacc gtgctggaaa ttgcagaaag cctgggccat 780
gaagccattc tggcagatat taccctggca gaactgctgg atgcagatga agttctgggt 840
tgtaccaccg ccggtggcgt gtggccgttt gttagtgtgg atggtaatcc gattagcgat 900
ggcgtgccgg gtccgattac ccagagtatt attcgccgtt attgggaact gaatgtggaa 960
agcagcagtc tgctgacccc ggttcagtat taa 993
<210> 52
<211> 330
<212> PRT
<213> 节细菌属（Arthrobacter sp. KNK168）
<400> 52
Met Ala Phe Ser Ala Asp Thr Ser Glu Ile Val Tyr Thr His Asp Thr
1 5 10 15
Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn
20 25 30
Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro
35 40 45
Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Tyr Leu His Ser Asp
50 55 60
Val Thr Tyr Thr Val Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu
65 70 75 80
Asp Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Met Arg Ile
85 90 95
Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu
100 105 110
Val Ala Lys Thr Glu Leu Arg Glu Ala Phe Val Ser Val Ser Ile Thr
115 120 125
Arg Gly Tyr Ser Ser Thr Pro Gly Glu Arg Asp Ile Thr Lys His Arg
130 135 140
Pro Gln Val Tyr Met Tyr Ala Val Pro Tyr Gln Trp Ile Val Pro Phe
145 150 155 160
Asp Arg Ile Arg Asp Gly Val His Ala Met Val Ala Gln Ser Val Arg
165 170 175
Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp
180 185 190
Gly Asp Leu Ile Arg Ala Val Gln Glu Thr His Asp Arg Gly Phe Glu
195 200 205
Ala Pro Leu Leu Leu Asp Gly Asp Gly Leu Leu Ala Glu Gly Ser Gly
210 215 220
Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg
225 230 235 240
Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu
245 250 255
Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Leu Ala Glu Leu
260 265 270
Leu Asp Ala Asp Glu Val Leu Gly Cys Thr Thr Ala Gly Gly Val Trp
275 280 285
Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Asp Gly Val Pro Gly
290 295 300
Pro Ile Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu
305 310 315 320
Ser Ser Ser Leu Leu Thr Pro Val Gln Tyr
325 330

Claims (8)

1.一种(R)-3-氨基-1-丁醇的合成方法，包括如下步骤：
（A）以1，3-丁二醇为底物，经酶A及其辅酶催化反应生成3-酮-1-丁醇；
（B）以步骤（A）生成的3-酮-1-丁醇为底物，经酶B及其辅酶催化反应生成(R)-3-氨基-1-丁醇；
所述酶A为醇脱氢酶或所述醇脱氢酶的突变体；
所述酶B为如下（B1）-（B5）中任一转氨酶：
（B1）来源于土曲霉（Aspergillus terreus）的转氨酶，氨基酸序列为SEQ ID No.46；
（B2）来源于费希新萨托菌（Neosartorya fischeri）的转氨酶，氨基酸序列为SEQ IDNo.48；
（B3）来源于分支杆菌（Mycobacterium vanbaalenii）的转氨酶，氨基酸序列为SEQ IDNo.50；
（B4）来源于Arthrobacter sp. KNK168的转氨酶，氨基酸序列为SEQ ID No.52；
（B5）在（B1）-（B4）中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白；
所述醇脱氢酶为如下（a1）-（a20）中任一：
（a1）来源于短小乳杆菌（Lactobacillus brevis）的醇脱氢酶，氨基酸序列为SEQ IDNo.2；
（a2）来源于高温厌氧杆菌（Thermoanaerobacter brockii）的醇脱氢酶，氨基酸序列为SEQ ID No.4；
（a3）来源于Lactobacillus kefiri DSM 20587的醇脱氢酶，氨基酸序列为SEQ IDNo.6；
（a4）来源于Thermoanaerobacter wiegelii Rt8.B1的醇脱氢酶，氨基酸序列为SEQ IDNo.8；
（a5）来源于酒球菌（Oenococcus alcoholitolerans）的醇脱氢酶，氨基酸序列为SEQID No.10；
（a6）来源于致黑脱硫肠状菌（Desulfotomaculum nigrificans）的醇脱氢酶，氨基酸序列为SEQ ID No.12；
（a7）来源于一氧化碳嗜热窦菌（Thermosinus carboxydivorans）的醇脱氢酶，氨基酸序列为SEQ ID No.14；
（a8）来源于烷嗜热厌氧杆菌（Thermoanaerobacter mathranii）的醇脱氢酶，氨基酸序列为SEQ ID No.16；
（a9）来源于Firmicutes bacterium CAG:137的醇脱氢酶，氨基酸序列为SEQ IDNo.18；
（a10）来源于甲烷八叠球菌（Methanosarcina thermophila）的醇脱氢酶，氨基酸序列为SEQ ID No.20；
（a11）来源于拜氏梭菌（Clostridium beijerinckii）的醇脱氢酶，氨基酸序列为SEQID No.22；
（a12）来源于未培养梭菌（uncultured Clostridium sp.）的醇脱氢酶，氨基酸序列为SEQ ID No.24；
（a13）来源于丝状孢子梭菌（Clostridium taeniosporum）的醇脱氢酶，氨基酸序列为SEQ ID No.26；
（a14）来源于脱硫肠状菌（Desulfotomaculum putei）的醇脱氢酶，氨基酸序列为SEQID No.28；
（a15）来源于二醇梭菌（Clostridium diolis）的醇脱氢酶，氨基酸序列为SEQ IDNo.30；
（a16）来源于匙形梭菌（Clostridium cochlearium）的醇脱氢酶，氨基酸序列为SEQ IDNo.32；
（a17）来源于笃斯梭菌（Clostridium uliginosum）的醇脱氢酶，氨基酸序列为SEQ IDNo.34；
（a18）来源于Clostridiales bacterium SK-Y3的醇脱氢酶，氨基酸序列为SEQ IDNo.36；
（a19）来源于近平滑假丝酵母（Candida parapsilosis）的醇脱氢酶，氨基酸序列为SEQID No.38；
（a20）在（a1）-（a19）中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白；
所述醇脱氢酶的突变体为如下（c1）-（c5）：
（c1）与SEQ ID No.2所示来源于短小乳杆菌的醇脱氢酶相比，仅存在如下突变中的至少一种：I11V、G37D；
（c2）与SEQ ID No.4所示来源于高温厌氧杆菌的醇脱氢酶相比，仅存在如下突变：A85G/I86A或者A85G/I86A/ W110A或者A85G/I86A/ G198D；
（c3）与SEQ ID No.6所示来源于高加索酸奶乳杆菌的醇脱氢酶相比，仅存在如下突变：G37D；
（c4）与SEQ ID No.8所示来源于威吉利热厌氧杆菌的醇脱氢酶相比，仅存在如下突变：G198D；
（c5）在（c1）-（c4）中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
2.如权利要求1所述的方法，其特征在于：所述方法中，酶A和酶B均是以粗酶液、粗酶液冻干粉、纯酶或全细胞的形式发生催化作用的。
3.如权利要求2所述的方法，其特征在于：所述粗酶液、粗酶液冻干粉或纯酶按照包括如下步骤的方法制备得到：在宿主细胞中表达所述酶A和/或所述酶B，得到重组细胞；裂解所述重组细胞获得所述粗酶液、粗酶液冻干粉或纯酶；
所述全细胞按照包括如下步骤的方法制备得到：在宿主细胞中表达所述酶A和/或所述酶B，得到的重组细胞即为所述全细胞。
4.如权利要求3所述的方法，其特征在于：所述重组细胞是按照包括如下步骤的方法制备获得的：向所述宿主细胞到导入能够表达所述酶A和/或所述酶B的核酸分子，经诱导培养后获得表达所述酶A和/或所述酶B的所述重组细胞。
5.如权利要求4所述的方法，其特征在于：所述能够表达所述酶A和/或所述酶B的核酸分子是通过重组载体的形式导入到所述宿主细胞中的；所述重组载体为携带有所述酶A和/或所述酶B的编码基因的细菌质粒、噬菌体、酵母质粒或逆转录病毒包装质粒；
所述宿主细胞为原核细胞或低等真核细胞；
所述原核细胞为细菌；所述低等真核细胞为酵母细胞；
所述细菌为大肠杆菌。
6.根据权利要求1所述的方法，其特征在于：
所述来源于短小乳杆菌的醇脱氢酶的编码基因的序列为SEQ ID No.1或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于高温厌氧杆菌的醇脱氢酶的编码基因的序列为SEQ ID No.3或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于Lactobacillus kefiri DSM 20587的醇脱氢酶的编码基因的序列为SEQID No.5或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于Thermoanaerobacter wiegelii Rt8.B1的醇脱氢酶的编码基因的序列为SEQ ID No.7或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于酒球菌的醇脱氢酶的编码基因的序列为SEQ ID No.9或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于致黑脱硫肠状菌的醇脱氢酶的编码基因的序列为SEQ ID No.11或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于一氧化碳嗜热窦菌的醇脱氢酶的编码基因的序列为SEQ ID No.13或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于烷嗜热厌氧杆菌的醇脱氢酶的编码基因的序列为SEQ ID No.15或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于Firmicutes bacterium CAG:137的醇脱氢酶的编码基因的序列为SEQ IDNo.17或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于甲烷八叠球菌的醇脱氢酶的编码基因的序列为SEQ ID No.19或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于拜氏梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.21或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于未培养梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.23或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于丝状孢子梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.25或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于脱硫肠状菌的醇脱氢酶的编码基因的序列为SEQ ID No.27或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于二醇梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.29或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于匙形梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.31或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于笃斯梭菌的醇脱氢酶的编码基因的序列为SEQ ID No.33或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于Clostridiales bacterium SK-Y3的醇脱氢酶的编码基因的序列为SEQ IDNo.35或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于近平滑假丝酵母的醇脱氢酶的编码基因的序列为SEQ ID No.37或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于土曲霉的转氨酶的编码基因的序列为SEQ ID No.45或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于费希新萨托菌的转氨酶的编码基因的序列为SEQ ID No.47或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于分支杆菌的转氨酶的编码基因的序列为SEQ ID No.49或在其5’端和/或3’端连接标签编码序列后得到的融合序列；
所述来源于Arthrobacter sp. KNK168的转氨酶的编码基因的序列为SEQ ID No.51或在其5’端和/或3’端连接标签编码序列后得到的融合序列。
7.如权利要求1所述的方法，其特征在于：步骤（A）和步骤（B）中，所述催化反应的温度均为25～37℃；所述催化反应的时间均为4～48h；
和/或当所述酶A和所述酶B是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，步骤（A）和步骤（B）中，所述酶A和所述酶B在各自反应体系中的浓度均为0.1g/L～10 g/L；当所述酶A和所述酶B以共表达所述酶A和所述酶B的全细胞的形式发生催化作用时，每升反应体系中含有所述全细胞的湿重为100g；
和/或当所述酶A和所述酶B是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，步骤（A）中，所述催化反应在如下（h1）所示缓冲液中进行；步骤（B）中，所述催化反应在如下（h2）所示缓冲液中进行；当所述酶A和所述酶B以共表达所述酶A和所述酶B的全细胞的形式发生催化作用时，步骤（A）和步骤（B）的所述催化反应均是在如下（h1）所示缓冲液中进行；（h1）浓度为50～100mM，pH值为6.5～8.0的磷酸盐缓冲液；（h2）浓度为50～100mM，pH值为7.5～8.5的磷酸盐缓冲液。
8.如权利要求2所述的方法，其特征在于：当酶A和酶B以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，步骤（A）中，所述催化反应的反应体系中除了含有1，3-丁二醇和所述酶A及其辅酶外，还含有丙酮；
和/或当酶A和酶B是以粗酶液、粗酶液冻干粉或纯酶的形式发生催化作用时，步骤（B）中，所述催化反应的反应体系中除了含有3-酮-1-丁醇、所述酶B及其辅酶磷酸吡哆醛外，还含有异丙胺或丙氨酸。
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