生产二十碳五烯酸的微生物和二十碳五烯酸的制造方法

摘要

本发明的目的在于提供高效地生产EPA的微生物以及使用该微生物的EPA的制造方法。本发明涉及如下微生物等：其是具有生产二十二碳六烯酸(DHA)的能力的微生物，其包含由序列号2所表示的氨基酸序列中第6位、第65位、第230位、第231位和第275位的氨基酸残基中的至少一个被置换为了其他氨基酸残基的氨基酸序列构成的蛋白质(突变型OrfB)，并且能够生产二十碳五烯酸。

说明书

技术领域

本发明涉及生产二十碳五烯酸的微生物和使用该微生物制造二十碳五烯酸的方法。

背景技术

将二十二碳六烯酸(以下称为DHA)、二十碳五烯酸(以下称为EPA)、花生四烯酸(以下称为ARA)、二十二碳五烯酸(以下称为DPA)等在分子内具有多个不饱和键的长链脂肪酸称为多不饱和脂肪酸(以下称为PUFA)。已知PUFA具有动脉硬化或高脂血症的预防等各种生理功能(非专利文献1和非专利文献2)。

作为PUFA的生物合成途径，已知有需氧途径和利用多不饱和脂肪酸聚酮合酶(以下称为PUFA-PKS)的厌氧途径这两种。需氧途径是通过对利用脂肪酸合成酶合成的棕榈酸等长链脂肪酸进行基于多种去饱和酶的双键的导入或基于链延伸酶的碳链的延伸而合成PUFA的途径，是很早就已知的许多生物所具有的合成途径(非专利文献3)。

另一方面，基于PUFA-PKS的厌氧途径是由乙酰辅酶A或丙二酰辅酶A(CoA)合成PUFA的途径，已知一部分海洋细菌或网粘菌类真核生物具有该途径(非专利文献4和非专利文献5)。

PUFA-PKS是由多种蛋白质构成的复合酶(以下也称为蛋白质复合物)，在各蛋白质中存在参与PUFA的合成的多个功能结构域。

作为PUFA-PKS中存在的功能结构域，有：被认为参与丙二酰ACP与酰基ACP的缩合的β-酮脂酰-酰基载体蛋白合酶结构域(以下称为KS结构域)；被认为借助磷酸泛酰巯基乙胺基通过硫酯键与酰基结合，作为脂肪酸合成的场所发挥功能的酰基载体蛋白结构域(以下称为ACP结构域)；被认为将通过缩合而生成的羰基还原的酮还原酶结构域(以下称为KR结构域)；被认为将利用KR结构域生成的羟基脱水而形成双键的DH结构域；被认为参与碳链的延伸的链延伸因子结构域(以下称为CLF结构域)；被认为将所得到的双键还原的烯酰还原酶结构域(以下称为ER结构域)；被认为参与酰基的转移的酰基转移酶结构域(以下称为AT结构域)和丙二酰辅酶A：酰基转移酶结构域(以下称为MAT结构域)；以及被认为活化ACP结构域的磷酸泛酰巯基乙胺基转移酶结构域(以下称为PPT结构域)，认为这些多个结构域通过协同地发挥作用而使脂肪酸的碳链延伸。

已知根据PUFA-PKS的种类，所生产的PUFA的种类不同。例如，对于来源于裂殖壶菌(Schizochytrium sp.)、橙黄壶菌(Aurantiochytrium sp.)和海摩替亚氏菌(Moritellamarina)的PUFA-PKS而言，生产DHA作为主要产物；对于来源于奥奈达希瓦氏菌(Shewanellaoneidensis)和深海发光杆菌(Photobacterium profundum)的PUFA-PKS而言，生产EPA作为主要产物；对于来源于海洋金色螺旋菌(Aureispira marina)的PUFA-PKS而言，生产ARA作为主要产物，几乎不生产其他PUFA，或者即使生产其他PUFA，与主要生产物相比也是少量。

可见，PUFA-PKS具有高的生产物特异性，到目前为止已进行了很多以PUFA-PKS的功能解析为目标的研究。非专利文献4和6中，进行了由希瓦氏菌(Shewanella)属细菌或原生藻菌类真核生物克隆出PUFA-PKS基因并在异种生物中表达而生产PUFA的研究。

非专利文献7中，通过使用作为来源于生产DHA的海摩替亚氏菌(Moritellamarina)的PUFA-PKS的构成基因的pfaB基因和构成来源于生产EPA的肺鲐希瓦氏菌(Shewanella pneumatophori)的PUFA-PKS的pfaB基因的研究，公开了编码AT结构域的pfaB基因与所生产的PUFA的种类相关。

非专利文献8中，公开了在大肠杆菌中导入来源于破囊壶菌(Thraustochytrium)属的PUFA-PKS的DH结构域时，脂肪酸的生产量增加，并且不饱和脂肪酸的比例增加。

作为EPA的工业生产方法，已知有从鱼油中纯化等方法，但存在副产物多的问题(专利文献2)。

现有技术文献

专利文献

专利文献1：国际公开第2008/144473号

专利文献2：日本特开2013-055893号公报

非专利文献

非专利文献1：Annu.Nutr.Metabol.,1991,35,128-131

非专利文献2：J.Am.Clin.Nutr.,1994,13,658-664

非专利文献3：Ann.Rev.Biochem.,1983,52,537-579

非专利文献4：Science,2001,293,290-293

非专利文献5：PLoS One,2011,6,e20146

非专利文献6：Plant Physiol.Biochem.,2009,47,472-478

非专利文献7：FEMS Microbiol.Lett.,2009,295,170-176

非专利文献8：Appl.Microbiol.Biotechnol.,2018,847-856

发明内容

发明所要解决的问题

如上所述，作为EPA的工业生产方法，使用从鱼油中纯化的方法等，但存在副产物多、生产效率差的问题，因此需要高效的EPA的生产方法。

因此，本发明的目的在于提供高效地生产EPA的微生物和使用该微生物的EPA的制造方法。

用于解决问题的方法

本发明人发现，通过在具有生产DHA的能力的微生物中表达向特定的氨基酸残基中导入了突变的OrfB，能够生产以高浓度含有EPA的PUFA，从而完成了本发明。

本发明涉及下述内容。

1.一种微生物，其是具有生产DHA的能力的微生物，其包含由序列号2所表示的氨基酸序列中第6位、第65位、第230位、第231位和第275位的氨基酸残基中的至少一个被置换为了其他氨基酸残基的氨基酸序列构成的蛋白质(以下称为突变型OrfB)，并且能够生产二十碳五烯酸(以下称为EPA)。

2.一种微生物，其是具有生产DHA的能力的微生物，其包含由下述氨基酸序列构成的蛋白质(以下称为突变型OrfB同源物)，并且能够生产EPA，所述氨基酸序列是在由序列号2所表示的氨基酸序列构成的蛋白质的同源蛋白质(以下称为OrfB同源物)的氨基酸序列中，将OrfB同源物的氨基酸序列与序列号2所表示的氨基酸序列进行比对时，与序列号2的第6位、第65位、第230位、第231位和第275位的氨基酸残基中的至少一个相对应的氨基酸残基被置换为了其他氨基酸残基的氨基酸序列。

3.如上述1或2所述的微生物，其中，具有生产DHA的能力的微生物为网粘菌类微生物。

4.如上述3所述的微生物，其中，网粘菌类微生物为属于橙黄壶菌(Aurantiochytrium)属、破囊壶菌(Thraustochytrium)属、吾肯氏壶菌(Ulkenia)属、帕里蒂氏壶菌(Parietichytrium)属、网粘菌(Labyrinthula)属、不动壶菌(Aplanochytrium)属、矩圆壶菌(Oblongichytrium)属或裂殖壶菌(Schizochytrium)属的网粘菌类微生物。

5.如上述1或2所述的微生物，其中，具有生产DHA的能力的微生物是在不具有DHA代谢途径的微生物中导入了编码具有合成DHA的活性的下述(a)～(j)的各结构域的基因的微生物。

(a)KS结构域

(b)MAT结构域

(c)ACP结构域

(d)KR结构域

(e)聚酮合酶脱水酶(以下称为PS-DH)结构域

(f)CLF结构域

(g)AT结构域

(h)FabA样β-羟酰-ACP脱水酶(以下称为FabA-DH)结构域

(i)ER结构域

(j)PPT结构域

6.如上述5所述的微生物，其中，不具有DHA代谢途径的微生物为属于埃希氏菌(Escherichia)属、芽孢杆菌(Bacillus)属、棒状杆菌(Corynebacterium)属、耶氏酵母(Yarrowia)属、酵母菌(Saccharomyces)属、念珠菌(Candida)属或毕赤酵母(Pichia)属的微生物。

7.一种EPA或含有EPA的组合物的制造方法，其中，将上述1～6中任一项所述的微生物在培养基中进行培养，使EPA或含有EPA的组合物在培养物中生成、蓄积，并从该培养物中收集EPA或含有EPA的组合物。

8.一种EPA或含有EPA的组合物的制造方法，其中，使用下述(I)或(II)的能够生产EPA的微生物。

(I)具有生产DHA的能力的微生物，其包含由序列号2所表示的氨基酸序列中第6位、第65位、第230位、第231位和第275位的氨基酸残基中的至少一个被置换为了其他氨基酸残基的氨基酸序列构成的突变型OrfB，并且能够生产EPA

(II)具有生产DHA的能力的微生物，其包含由下述氨基酸序列构成的突变型OrfB同源物，并且能够生产EPA，所述氨基酸序列是在OrfB同源物的氨基酸序列中，将OrfB同源物的氨基酸序列与序列号2所表示的氨基酸序列进行比对时，序列号2的第6位、第65位、第230位、第231位和第275位的氨基酸残基中的至少一个被置换为了其他氨基酸残基的氨基酸序列

发明效果

本发明的微生物通过在具有生产DHA的能力的微生物中表达向特定的氨基酸残基中导入突变而改变了对底物的特异性的突变型OrfB，能够高效地生产EPA。本发明的EPA的制造方法通过以工业水平在能够生产DHA的微生物中表达突变型OrfB，能够低成本且高效率地生产EPA，能够应用于EPA的工业水平的生产。

附图说明

图1示出橙黄壶菌(Aurantiochytrium sp.)属的PUFA-PKS的结构的示意图。

图2示出OrfB与OrfB同源物的氨基酸序列的比对结果的一例。

具体实施方式

本发明中，“多不饱和脂肪酸(PUFA)”是指碳链长为18以上、不饱和键数为2以上的长链脂肪酸。另外，本说明书中，“结构域”是指蛋白质中的由连续的氨基酸序列构成的一部分，是在该蛋白质中具有特定的生物学活性或功能的区域。

本发明中，“PUFA-PKS”与PUFA合酶含义相同。PUFA合酶是指使用丙二酰辅酶A等作为碳源来合成特异性的长链不饱和脂肪酸的酶群，其含有KS、MAT、ACP、KR、PS-DH、CLF、AT、FabA-DH、ER、PPTase的各结构域(ACOS Lipid Library:PUFA synthase；Science,2001,293,290-293；PLoS One,2011,6,e20146等)。

KS结构域是构成具有PUFA-PKS活性的蛋白质复合物的蛋白质所具有的结构域，是指参与丙二酰ACP与酰基ACP的缩合的结构域。

MAT结构域、AT结构域是构成具有PUFA-PKS活性的蛋白质复合物的蛋白质所具有的结构域，是指参与酰基的转移的结构域。

ACP结构域是构成具有PUFA-PKS活性的蛋白质复合物的蛋白质所具有的结构域，是指借助磷酸泛酰巯基乙胺基通过硫酯键与酰基结合、作为脂肪酸合成的场所发挥功能的、PUFA-PKS活性所必需的结构域。

KR结构域是构成具有PUFA-PKS活性的蛋白质复合物的蛋白质所具有的结构域，是指参与通过缩合生成的酮基的还原的结构域。

作为DH结构域的PS-DH结构域和FabA-DH结构域是构成具有PUFA-PKS活性的蛋白质复合物的蛋白质所具有的结构域，是指参与通过将酮基还原而生成的羟基的脱水的结构域。

CLF结构域是构成具有PUFA-PKS活性的蛋白质复合物的蛋白质所具有的结构域，是指参与碳链的延伸的结构域。

ER结构域、酰基转移酶结构域、丙二酰辅酶A：ACP酰基转移酶结构域是构成具有PUFA-PKS活性的蛋白质复合物的蛋白质所具有的结构域，是指参与酰基的转移的结构域。

PPTase是构成具有PUFA-PKS活性的蛋白质复合物的酶，是指参与ACP结构域的活化的酶。

本说明书中，氨基酸序列或核苷酸序列的一致性可以使用Karlin和Altschul的算法BLAST(Pro.Natl.Acad.Sci.USA,1993,90,5873)或FASTA(Methods Enzymol.,1990,183,63)来确定。基于该算法BLAST，开发了被称为BLASTN或BLASTX的程序(J.Mol.Biol.,1990,215,403)。在基于BLAST利用BLASTN来解析核苷酸序列的情况下，参数例如设定为得分(Score)＝100、字长(wordlength)＝12。另外，在基于BLAST利用BLASTX来解析氨基酸序列的情况下，参数例如设定为得分(Score)＝50、字长(wordlength)＝3。在使用BLAST和Gapped BLAST程序的情况下，使用各程序的默认参数。这些解析方法的具体方法是公知的(参考www.ncbi.nlm.nih.gov)。

本说明书中，“外源性”是指非内源性、来源于异种，用于表示下述含义：在转化前的宿主生物不具有通过本发明应导入的基因的情况、由该基因编码的蛋白质实质上不表达的情况、以及由不同的基因编码该蛋白质的氨基酸序列但在转化后不表达内源性蛋白质的活性的情况下，将基于本发明的基因导入到宿主生物中。

[微生物]

本发明的微生物是具有生产二十二碳六烯酸(DHA)的能力的微生物，其特征在于，包含由序列号2所表示的氨基酸序列中第6位、第65位、第230位、第231位和第275位的氨基酸残基中的至少一个被置换为了其他氨基酸残基的氨基酸序列构成的蛋白质(突变型OrfB)，并且能够生产二十碳五烯酸(EPA)。

具有生产DHA的能力的微生物可以列举下述(1)和(2)。

(1)具有DHA代谢能力的微生物。

(2)通过以不具有DHA代谢能力的微生物作为宿主生物并在该宿主生物中导入了编码作为构成具有生物合成DHA的活性的PUFA-PKS的结构域的KS结构域、MAT结构域、ACP结构域、KR结构域、PS-DH结构域、CLF结构域、AT结构域、FabA-DH结构域、ER结构域和PPT结构域的基因而具有DHA生产能力的微生物。

本说明书中，“宿主生物”是指成为基因改造和转化等的对象的原始生物。在成为通过基因导入进行的转化的对象的原始生物为微生物的情况下，也称为亲本株、宿主株。

作为(1)具有DHA代谢能力的微生物，可以列举属于网粘菌类的微生物。作为属于网粘菌类的微生物，可以列举例如橙黄壶菌(Aurantiochytrium)属、破囊壶菌(Thraustochytrium)属、吾肯氏壶菌(Ulkenia)属、帕里蒂氏壶菌(Parietichytrium)属、网粘菌(Labyrinthula)属、不动壶菌(Aplanochytrium)属、矩圆壶菌(Oblongichytrium)属或裂殖壶菌(Schizochytrium)属的微生物。优选可以列举蛞蝓橙黄壶菌(Aurantiochytriumlimacinum)、金黄色破囊壶菌(Thraustochytrium aureum)等，但只要天然具有DHA代谢途径，则不限定于这些。

作为具有DHA代谢能力的微生物，具体而言，优选例如属于橙黄壶菌(Aurantiochytrium)属的微生物，可以列举例如橙黄壶菌OH4株(保藏编号FERM BP-11524)等，还可以使用作为其突变株的具有DHA生产能力的微生物。

上述橙黄壶菌OH4株保藏在位于日本茨城县筑波市东1丁目1番地中央第6(邮政编码305-8566)的独立行政法人产品评价技术基础机构(NITE)的专利微生物保藏中心。受理日(保藏日)为平成25年(公历2013年)1月11日，保藏编号为FERM BP-11524。

(2)不具有DHA代谢能力的微生物是指天然不具有DHA生产能力的微生物。作为不具有DHA代谢能力的微生物，可以列举例如细菌、微藻类、真菌、原生生物、原生动物。

作为细菌，可以列举例如属于选自由埃希氏菌(Escherichia)属、沙雷氏菌(Serratia)属、芽孢杆菌(Bacillus)属、短杆菌(Brevibacterium)属、棒状杆菌(Corynebacterium)属、微杆菌(Microbacterium)属、假单胞菌(Pseudomonas)属和金色螺旋菌(Aureispira)属组成的组中的一个属的微生物。这些之中，优选选自由大肠杆菌(Escherichia coli)XL1-Blue、大肠杆菌XL2-Blue、大肠杆菌DH1、大肠杆菌MC1000、大肠杆菌KY3276、大肠杆菌W1485、大肠杆菌JM109、大肠杆菌HB101、大肠杆菌No.49、大肠杆菌W3110、大肠杆菌NY49、大肠杆菌BL21 codon plus(Stratagene公司制造)、无花果沙雷氏菌(Serratia ficaria)、居泉沙雷氏菌(Serratia fonticola)、液化沙雷氏菌(Serratialiquefaciens)、粘质沙雷氏菌(Serratia marcescens)、枯草芽孢杆菌(Bacillussubtilis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、未成熟短杆菌(Brevibacterium immariophilum)ATCC14068、解糖短杆菌(Brevibacteriumsaccharolyticum)ATCC14066、产氨棒状杆菌(Corynebacterium ammoniagenes)、谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC13032、谷氨酸棒状杆菌ATCC14067、谷氨酸棒状杆菌ATCC13869、嗜乙酰乙酸棒状杆菌(Corynebacterium acetoacidophilum)ATCC13870、嗜氨微杆菌(Microbacterium ammoniaphilum)ATCC15354、假单胞菌(Pseudomonas sp.)D-0110和海洋金色螺旋菌(Aureispira marina)JCM23201组成的组中的一种微生物。

作为微藻类，可以列举例如裸藻纲(Euglenophyceae)[例如裸藻(Euglena)属和袋鞭藻(Peranema)属]、绿藻纲(Chrysophyceae)[例如棕鞭藻(Ochromonas)属]、锥囊藻纲(Dinobryaceae)[作为示例，有锥囊藻(Dinobryon)属、扁金藻(Platychrysis)属和金色藻(Chrysochromulina)属]、甲藻纲(Dinophyceae)[例如隐甲藻(Crypthecodinium)属、裸甲藻(Gymnodinium)属、多甲藻(Peridinium)属、角甲藻(Ceratium)属、环沟藻(Gyrodinium)属和尖尾藻(Oxyrrhis)属]、隐藻纲(Cryptophyceae)[例如隐藻(Cryptomonas)属和红胞藻(Rhodomonas)属]、黄藻纲(Xanthophyceae)[例如奥里藻(Olisthodiscus)属][并且，包括产生根黄藻类(Rhizochloridaceae)以及Aphanochaete pascheri、Bumilleriastigeoclonium和双生无隔藻(Vaucheria geminata)的孢子/配子中那样的变形虫状期的藻类的品种]、真眼点藻纲(Eustigmatophyceae)和定鞭藻纲(Prymnesiopyceae)[包括例如定鞭藻(Prymnesium)属和Diacronema属]。

这些属中的优选的种没有特别限定，可以列举微绿球藻(Nannochloropsisoculata)、寇氏隐甲藻(Crypthecodinium cohnii)、纤细裸藻(Euglena gracilis)。

作为真菌，可以列举例如：酵母菌(Saccharomyces)属[例如包括酿酒酵母(Saccharomyces cerevisiae)、卡尔斯伯酵母(Saccharomyces carlsbergensis)的酵母]、或耶氏酵母(Yarrowia)属、念珠菌(Candida)属、毕赤酵母(Pichia)属、克鲁维酵母(Kluyveromyces)属等的其他酵母；或者其他真菌、例如曲霉(Aspergillus)属、脉孢霉(Neurospora)属、青霉(Penicillium)属等的纤维状真菌等。

能够用作宿主细胞的细胞株可以是通常意义上的野生型，或者也可以是营养缺陷型突变株、抗生素抗性突变株，也可以以具有各种标记基因的方式进行了转化。可以列举例如对氯霉素、氨苄青霉素、卡那霉素、四环素等抗生素显示抗性的株。

作为用于使(2)不具有DHA代谢能力的微生物获得DHA生产能力的、编码构成具有生物合成DHA的活性的PUFA-PKS的各结构域的基因，优选编码构成上述(1)具有DHA代谢能力的微生物所具有的、具有生物合成DHA的活性的PUFA-PKS的各结构域(KS结构域、MAT结构域、ACP结构域、KR结构域、PS-DH结构域、CLF结构域、AT结构域、FabA-DH结构域、ER结构域和PPT结构域)的基因。

关于构成PUFA-PKS的各结构域，只要该结构域协同作用而生产DHA，则各结构域没有限定，可以列举例如已知的PUFA-PKS所具有的各结构域。

本说明书中，“协同作用”是指在使某种蛋白质与其他蛋白质共存时，成为一体而进行特定的反应，特别是在本说明书中，是指在使PUFA-PKS活性所需的多个结构域共存时，与其他结构域成为一体而显示出PUFA-PKS活性。

本说明书中，“已知的PUFA-PKS”优选列举属于选自由橙黄壶菌(Aurantiochytrium)属、破囊壶菌(Thraustochytrium)属、吾肯氏壶菌(Ulkenia)属、帕里蒂氏壶菌(Parietichytrium)属、网粘菌(Labyrinthula)属、不动壶菌(Aplanochytrium)属、矩圆壶菌(Oblongichytrium)属或裂殖壶菌(Schizochytrium)属组成的组中的属的微生物原本具有的PUFA-PKS，更优选列举选自由蛞蝓橙黄壶菌(Aurantiochytriumlimacinum)ATCC MYA-1381、裂殖壶菌(Schizochytrium sp.)ATCC20888、金黄色破囊壶菌(Thraustochytrium aureum)ATCC 34304组成的组中的微生物原本具有的PUFA-PKS。

由各结构域构成的PUFA-PKS具有DHA合成活性这一点可以如下进行确认：制造利用编码各结构域的基因进行了转化的微生物，将该微生物在培养基中进行培养，使DHA在培养物中生成、蓄积，利用气相色谱法测定该培养物中蓄积的DHA。

PUFA-PKS是由具有上述结构域的多个蛋白质构成的蛋白质复合物(复合酶)，OrfB是构成PUFA-PKS的蛋白质。图1中示出构成属于橙黄壶菌(Aurantiochytrium sp.)属的微生物中的PUFA-PKS的蛋白质复合物的结构域结构的示意图。OrfB内有一个KS结构域、CLF结构域、AT结构域、ER结构域。

作为突变型OrfB，可以列举下述(a)或(b)记载的蛋白质。

(a)由序列号2所表示的氨基酸序列中第6位、第65位、第230位、第231位和第275位的氨基酸残基中的至少一个被置换为了其他氨基酸残基的氨基酸序列构成的蛋白质

(b)由下述氨基酸序列构成的蛋白质，该氨基酸序列是在OrfB同源物的氨基酸序列中，将该氨基酸序列与序列号2所表示的氨基酸序列进行比对时，与序列号2所表示的氨基酸序列的第6位、第65位、第230位、第231位和第275位的氨基酸残基相对应的氨基酸残基中的至少一个被置换为了其他氨基酸残基的氨基酸序列。

关于上述(a)的蛋白质，优选在序列号2所表示的氨基酸序列中至少第230位的氨基酸残基被置换为其他氨基酸残基；更优选除了第230位的氨基酸残基以外，选自第6位、第65位、第231位和第275位的氨基酸残基中的至少一个进一步被置换为其他氨基酸残基；特别优选第6位和第230位的氨基酸残基、第65位和第230位的氨基酸残基、第6位、第65位和第230位的氨基酸残基或者第65位、第230位、第231位和第275位的氨基酸残基被置换为其他氨基酸残基。

另外，关于上述(b)的蛋白质，优选在OrfB同源物的氨基酸序列中将OrfB同源物的氨基酸序列与序列号2所表示的氨基酸序列进行比对时，至少与序列号2所表示的氨基酸序列的第230位的氨基酸残基相对应的氨基酸残基被置换为其他氨基酸残基；更优选除了与第230位的氨基酸残基相对应的氨基酸残基以外，选自与第6位、第65位、第231位和第275位的氨基酸残基相对应的氨基酸残基中的至少一个被置换为其他氨基酸残基；特别优选与第6位和第230位的氨基酸残基、第65位和第230位的氨基酸残基、第6位、第65位和第230位的氨基酸残基或者第65位、第230位、第231位和第275位的氨基酸残基相对应的氨基酸残基被置换为其他氨基酸残基。

OrfB同源物是指如下所述的存在于自然界中的生物所具有的蛋白质，该蛋白质由与序列号2所表示的氨基酸序列具有高同源性的氨基酸序列构成，并且结构和功能与具有序列号2所表示的氨基酸序列的OrfB类似，由此认为编码该蛋白质的基因在进化上的起源与编码原始蛋白质的基因相同。

作为OrfB同源物的具体例，可以列举序列号27所表示的来源于深海发光杆菌(Photobacterium profundum)的PhoC、序列号28所表示的来源于奥奈达希瓦氏菌(Shewanella oneidensis)的EpaC、序列号29所表示的来源于海摩替亚氏菌(Moritellamarina)的DhaC、序列号30所表示的来源于海洋金色螺旋菌(Aureispira marina)的AraC、序列号31所表示的来源于裂殖壶菌(Schizochytrium sp.)(ATCC20888)的OrfB等。图2中示出OrfB与OrfB同源物的氨基酸序列的比对结果的一例。

氨基酸序列的比对可以使用公知的比对程序ClustalW[Nucelic Acids Research22,4673,(1994)]来制作。ClustalW可从http://www.ebi.ac.uk/clustalw/(EuropeanBioinformatics Institute)来利用。使用ClustalW制作比对时的参数例如使用默认值。

作为突变型OrfB，更优选列举在上述(a)或(b)记载的蛋白质的氨基酸序列中进行了至少一种下述氨基酸残基的置换的蛋白质。

(i)序列号2的氨基酸序列的第6位的氨基酸残基或OrfB同源物的氨基酸序列的与该氨基酸残基相对应的氨基酸残基被置换为丝氨酸；

(ii)序列号2的氨基酸序列的第65位的氨基酸残基或OrfB同源物的氨基酸序列的与该氨基酸残基相对应的氨基酸残基被置换为亮氨酸；

(iii)序列号2的氨基酸序列的第230位的氨基酸残基或OrfB同源物的氨基酸序列的与该氨基酸残基相对应的氨基酸残基被置换为亮氨酸、L-色氨酸、L-天冬酰胺、甘氨酸、L-天冬氨酸或L-丙氨酸；

(iv)序列号2的氨基酸序列的第231位的氨基酸残基或OrfB同源物的氨基酸序列的与该氨基酸残基相对应的氨基酸残基被置换为苏氨酸；

(v)序列号2的氨基酸序列的第275位的氨基酸残基或OrfB同源物的氨基酸序列的与该氨基酸残基相对应的氨基酸残基被置换为甘氨酸。

上述置换后的氨基酸残基可以是能够相互置换的氨基酸。以下示出能够相互置换的氨基酸的示例。同一组中包含的氨基酸能够相互置换。

A组：亮氨酸、异亮氨酸、正亮氨酸、缬氨酸、正缬氨酸、丙氨酸、2-氨基丁酸、甲硫氨酸、邻甲基丝氨酸、叔丁基甘氨酸、叔丁基丙氨酸、环己基丙氨酸

B组：天冬氨酸、谷氨酸、异天冬氨酸、异谷氨酸、2-氨基己二酸、2-氨基辛二酸

C组：天冬酰胺、谷氨酰胺

D组：赖氨酸、精氨酸、鸟氨酸、2,4-二氨基丁酸、2,3-二氨基丙酸

E组：脯氨酸、3-羟基脯氨酸、4-羟基脯氨酸

F组：丝氨酸、苏氨酸、高丝氨酸

G组：苯丙氨酸、酪氨酸

上述被置换的氨基酸可以为天然型或非天然型。作为天然型氨基酸，可以列举L-丙氨酸、L-天冬酰胺、L-天冬氨酸、L-谷氨酰胺、L-谷氨酸、甘氨酸、L-组氨酸、L-异亮氨酸、L-亮氨酸、L-赖氨酸、L-精氨酸、L-甲硫氨酸、L-苯丙氨酸、L-脯氨酸、L-丝氨酸、L-苏氨酸、L-色氨酸、L-酪氨酸、L-缬氨酸、L-半胱氨酸等。

[微生物的制作方法]

作为使具有生产DHA的能力的微生物表达突变型OrfB或突变型OrfB同源物的方法，可以列举例如下述的(I)和(II)。

(I)向具有生产DHA的能力的微生物中导入编码突变型OrfB或突变型OrfB同源物的外源性基因。

(II)向具有生产DHA的能力的微生物中的编码内源性的OrfB或OrfB同源物的基因中导入突变。

关于上述(I)，作为编码突变型OrfB或突变型OrfB同源物的外源性基因的导入，包括：以能够自主复制的质粒的形式存在于该宿主生物的细胞中的情况、将该细胞中的置换对象的基因置换为对应的外源性基因的情况、将编码突变型OrfB或突变型OrfB同源物的外源性基因整合到该细胞中的染色体DNA中的与编码OrfB的基因不同的区域的情况。需要说明的是，导入外源性基因时，优选参考作为宿主的微生物的密码子使用频率来进行序列的优化。

关于上述(II)，例如可以通过使用Molecular Cloning,A Laboratory Manual,Third Edition,Cold Spring Harbor Laboratory Press(2001)(以下简称为分子克隆第3版)、Current Protocols in Molecular Biology,John Wiley&Sons(1987-1997)(以下简称为分子生物学实验室指南)、Nucleic Acids Research,10,6487(1982)、Proc.Natl.Acad.Sci.USA,79,6409(1982)、Gene,34,315(1985)、Nucleic AcidsResearch,13,4431(1985)、Proc.Natl.Acad.Sci.USA,82,488(1985)等中记载的定点诱变法导入定点突变而在编码内源性的OrfB或OrfB同源物的基因中导入突变。

本说明书中，“基因”是指除了蛋白质的编码区以外还可以包含转录调节区、启动子区和终止子区等的DNA。在宿主生物使用细菌等原核生物作为亲本株的情况下，作为该DNA，优选使用将作为核糖体结合区的夏因-达尔加诺(Shine-Dalgarno)序列与起始密码子之间调节为适当距离(例如6～18个碱基)的质粒。该DNA中，转录终止因子对于该DNA的表达不是必需的，但优选紧挨结构基因的下游配置转录终止序列。

作为导入到宿主生物中的基因，例如可以通过制成插入到适当表达载体的启动子的下游的重组基因而导入到宿主细胞中。表达载体可以还包含启动子、转录终止信号、用于选择转化体的选择标记基因(例如卡那霉素抗性基因、链霉素抗性基因、萎锈灵抗性基因、博来霉素抗性基因、潮霉素抗性基因等药剂抗性基因、与亮氨酸、组氨酸、甲硫氨酸、精氨酸、色氨酸、赖氨酸等氨基酸缺陷型突变互补的基因等、与尿嘧啶、腺嘌呤等核酸碱基缺陷型突变互补的基因等)。在尿嘧啶缺陷型株的情况下，作为标记基因，可以列举例如乳清酸核苷-5’-磷酸脱羧酶基因(ura3基因)或乳清酸焦磷酸化酶基因(ura5基因)。

作为启动子，无论是结构性启动子还是调节启动子，均定义为使RNA聚合酶与DNA结合而开始RNA合成的DNA的碱基序列。强启动子是指以高频率开始mRNA合成的启动子，优选使用。可以根据其宿主细胞的性质等使用lac系统、trp系统、TAC或TRC系统、λ噬菌体的主要操纵子和启动子区、fd包被蛋白的调控区、针对糖酵解酶(例如3-磷酸甘油酸激酶、甘油醛-3-磷酸脱氢酶)、谷氨酸脱羧酶A、丝氨酸羟甲基转移酶的启动子等。

除了启动子和终止子序列以外，作为其他调节元件，可以列举例如选择标记、扩增信号、复制起点等。作为优选的调节序列，可以列举例如“Gene Expression Technology:Methods in Enzymology 185”、Academic Press(1990)中记载的序列。

作为载体，只要能够使目的基因进行表达，则没有特别限定。对用于构建载体的试剂类、例如限制酶或连接酶等的种类也没有特别限定，可以适当使用市售品。

作为使用网粘菌类微生物作为宿主生物的情况下的启动子，只要是在网粘菌类微生物的细胞中发挥功能的启动子，则没有特别限定，可以列举例如肌动蛋白启动子、微管蛋白启动子、延伸因子Tu启动子、糖酵解系统基因的表达启动子。

在亲本株使用属于埃希氏菌属的微生物的情况下，作为表达载体，可以列举例如pColdI(宝生物公司制造)、pET21a、pCOLADuet-1、pACYCDuet-1、pCDF-1b、pRSF-1b(均为Novagen公司制造)、pMAL-c2x(New England Biolabs公司制造)、pGEX-4T-1(GEHealthcare Biosciences公司制造)、pTrcHis(Invitrogen公司制造)、pSE280(Invitrogen公司制造)、pGEMEX-1(Promega公司制造)、pQE-30(Qiagen公司制造)、pET-3(Novagen公司制造)、pTrc99A(GE Healthcare Biosciences公司制造)、pKYP10(日本特开昭58-110600号公报)、pKYP200[Agric.Biol.Chem.,48,669(1984)]、pLSA1[Agric.Biol.Chem.,53,277(1989)]、pGEL1[Proc.Natl.Acad.Sci.,USA,82,4306(1985)]、pBluescriptII SK(+)、pBluescriptII KS(-)(Stratagene公司制造)、pTrS30[由大肠杆菌JM109/pTrS30(FermBP-5407)制备]、pTrS32[由大肠杆菌JM109/pTrS32(Ferm BP-5408)制备]、pTK31[APPLIEDAND ENVIRONMENTAL MICROBIOLOGY,2007,Vol.73,No.20,p6378-6385]、pPAC31(国际公开第98/12343号)、pUC19[Gene,33,103(1985)]、pSTV28(宝生物公司制造)、pUC118(宝生物公司制造)、pPA1(日本特开昭63-233798号公报)、pHSG298(宝生物公司制造)、pUC18(宝生物公司制造)。

作为使用上述表达载体的情况下的启动子，只要是在属于埃希氏菌属的微生物的细胞中发挥功能的启动子，则没有特别限定，可以列举例如trp启动子(Ptrp)、lac启动子(Plac)、PL启动子、PR启动子、PSE启动子、T7启动子等来源于大肠杆菌或噬菌体等的启动子。另外，可以列举例如将2个Ptrp串联而成的启动子、tac启动子、trc启动子、lacT7启动子、letI启动子等人为设计改造的启动子。

在亲本株使用棒状细菌的情况下，作为表达载体，可以列举例如pCG1(日本特开昭57-134500号公报)、pCG2(日本特开昭58-35197号公报)、pCG4(日本特开昭57-183799号公报)、pCG11(日本特开昭57-134500号公报)、pCG116、pCE54、pCB101(均为日本特开昭58-105999号公报)、pCE51、pCE52、pCE53[均为Molecular and General Genetics,196,175(1984)]等。

作为使用上述表达载体的情况下的启动子，只要是在棒状细菌的细胞中发挥功能的启动子，则没有特别限定，可以列举例如P54-6启动子[Appl.Microbiol.Biotechnol.,53,674-679(2000)]。

在亲本株使用酵母菌株的情况下，作为表达载体，可以列举例如YEp13(ATCC37115)、YEp24(ATCC37051)、YCp51(ATCC37419)、pHS19、pHS15等。

作为使用上述表达载体的情况下的启动子，只要是在酵母菌株的细胞中发挥功能的启动子，则没有特别限定，可以列举例如PH05启动子、PGK启动子、GAP启动子、ADH启动子、gal 1启动子、gal 10启动子、热休克多肽启动子、MFα1启动子、CUP1启动子等启动子。

作为将重组基因整合到宿主生物的染色体中的方法，可以使用同源重组法。作为同源重组法，可以列举例如利用同源重组系统将重组基因导入的方法，所述利用同源重组系统可以通过与具有在希望导入的亲本株内无法自主复制的药剂抗性基因的质粒DNA连接来制作。作为利用在大肠杆菌中频繁使用的同源重组的方法，可以列举利用λ噬菌体的同源重组系统将重组基因导入的方法[Proc.Natl.Acad.Sci.USA,97,6641-6645(2000)]。

此外，可以使用利用了通过与重组基因一起整合到染色体上的枯草杆菌果聚糖蔗糖酶而使大肠杆菌成为蔗糖敏感性这一点的选择法、利用了通过在具有链霉素抗性的突变rpsL基因的大肠杆菌中整合野生型rpsL基因而使大肠杆菌成为链霉素敏感性这一点的选择法[Mol.Microbiol.,55,137(2005)、Biosci.Biotechnol.Biochem.,71,2905(2007)]等，获得将亲本株的染色体DNA上的目标区域置换为重组体DNA的微生物。

另外，作为同源重组法，可以列举例如借助农杆菌的ATMT法[Appl.Environ.Microbiol.,(2009),vol.75,p.5529-5535]。此外，只要能够获得稳定保持目标性状的转化体，也可包括ATMT法的改良法等，并不限定于这些。

作为将待导入的基因以能够在宿主生物中自主复制的质粒的形式导入的方法，可以列举例如使用钙离子的方法[Proc.Natl.Acad.Sci.,USA,69,2110(1972)]、原生质体法(日本特开昭63-248394号公报)、电穿孔法[Nucleic Acids Res.,16,6127(1988)]等方法。

通过上述方法获得的微生物为目标微生物这一点可以通过对该微生物进行培养并利用气相色谱法对其培养物中蓄积的EPA进行检测来确认。

本发明的微生物的例如在20℃下培养48小时时产生的最终产物(PUFA)中的EPA/DHA比在利用实施例中后述的气相色谱质谱分析法测定时优选为0.1以上，更优选为0.2以上，进一步优选为0.5以上。

[EPA或含有EPA的组合物的制造方法]

本发明包括EPA或含有EPA的组合物的制造方法(以下称为本发明的制造方法)，其特征在于，将上述制作的微生物在培养基中进行培养，使EPA或含有EPA的组合物在培养物中生成、蓄积，并从该培养物中收集EPA或含有EPA的组合物。

含有EPA的组合物可以列举例如含有EPA的油脂或含有EPA的磷脂，优选列举含有EPA的油脂。该微生物的培养物通过将该微生物接种到适当的培养基中并按照常规方法进行培养而得到。

作为培养基，包含碳源、氮源和无机盐等的公知的培养基均可以使用。例如，作为碳源，除了葡萄糖、果糖、半乳糖等碳水化合物以外，还可以例示油酸、大豆油等油脂类、甘油、乙酸钠等。这些碳源例如可以以每1升培养基中为20～300g的浓度使用。根据特别优选的方式，可以通过在初始碳源被消耗后供给碳源来继续培养。通过在这样的条件下进行培养，能够增大所消耗的碳源的量，能够提高含有EPA的组合物的生产量。

另外，作为氮源，可以列举例如酵母提取物、玉米浆、多聚蛋白胨、谷氨酸钠、尿素等有机氮、或乙酸铵、硫酸铵、氯化铵、硝酸钠、硝酸铵、氨等无机氮。作为无机盐，可以适当组合使用磷酸钾等。

含有上述各成分的培养基优选通过加入适当的酸或碱将pH调节至4.0～9.5的范围内后，利用高压釜进行杀菌后使用。培养温度通常为10～45℃，优选为20～37℃。培养温度优选控制为能够生产含有EPA的组合物的培养温度。培养时的pH通常为3.5～9.5，优选为4.5～9.5。特别优选的pH根据目的而不同，为了大量生产油脂，pH为5.0～8.0。

培养时间例如可以设定为2～7天，可以通过通气搅拌培养等进行培养。从培养物中分离培养液和微生物的方法可以通过本领域技术人员公知的常规方法进行，例如可以通过离心分离法或过滤等进行。将从上述培养物中分离出的微生物利用例如超声波或戴诺磨等破碎后，利用例如氯仿、己烷、丁醇等进行溶剂提取，由此得到含有EPA的组合物。

对于通过上述制造方法制造的含有EPA的组合物，可以通过例如低温溶剂分提法[高桥是太郎,油化学,40:931-941(1991)]、或利用脂肪酶等水解酶将短链的脂肪酸游离除去的方法[高桥是太郎,油化学,40:931-941(1991)]等方法将含有EPA的组合物浓缩，得到EPA含量高的含有EPA的组合物。

通过从含有EPA的组合物中分离收集EPA，能够制造EPA。例如，通过水解法从含有EPA的组合物中制备含有EPA的混合脂肪酸，然后，通过例如尿素添加法、冷却分离法、高效液相色谱法或超临界色谱法等分离收集EPA，由此能够制造EPA。

另外，可以通过从含有EPA的组合物中分离收集EPA烷基酯来制造EPA烷基酯。EPA烷基酯只要是EPA烷基酯则没有特别限定，优选列举EPA乙酯。

为了从含有EPA的组合物中分离收集EPA烷基酯，例如可以通过利用醇解法从含有EPA的组合物中制备含有EPA烷基酯的混合脂肪酸烷基酯，然后，通过例如尿素添加法、冷却分离法、高效液相色谱法或超临界色谱法等分离收集EPA烷基酯来进行。

实施例

以下示出实施例，但本发明并不限定于下述实施例。

[实施例1]

使用生产突变型OrfB的大肠杆菌的EPA的制造-1

(1)各表达质粒的制作

[OrfA蛋白表达质粒的制作]

通过与林等人(Sci.Rep.,2016,6,35441)同样的方法，得到具有编码来源于裂殖壶菌(Schizochytrium sp.)(ATCC20888)株的OrfA蛋白的DNA(由序列号4所表示的碱基序列构成的DNA)的表达质粒pET21-orfA。

[OrfC蛋白表达质粒的制作]

以利用常规方法提取出的橙黄壶菌(Auranctiochytrium sp.)OH4株的基因组DNA作为模板，使用序列号7和8所表示的引物进行PCR，得到包含编码OrfC蛋白的DNA(由序列号3所表示的碱基序列构成的DNA)的DNA片段。将所得到的DNA和大肠杆菌载体pCOLADuet-1(Merck Millipore公司制造)分别用限制酶NdeI和MfeI进行处理，将所得到的限制酶处理片段连接，由此得到来源于橙黄壶菌(Auranctiochytrium sp.)OH4株的OrfC蛋白的表达质粒pCOLA-OH4_orfC。

[HetI蛋白表达质粒的制作]

通过与林等人(Sci.Rep.,2016,6,35441)同样的方法，得到具有编码来源于念珠藻(Nostoc sp.)PCC7120(ATCC27893)株的HetI蛋白的DNA(由序列号5所表示的碱基序列构成的DNA)的表达质粒pSTV-hetI。

(2)编码突变型OrfB的DNA文库的构建

[野生型OrfB表达质粒的制作]

将来源于裂殖壶菌(Schizochytrium sp.)(ATCC20888)株的OrfB表达质粒pCDF-orfB1(Sci.Rep.,2016,6,35441)用AgeI处理而获得AgeI处理片段，使用Blunting high试剂盒(东洋纺公司制造)将该AgeI处理片段的末端平滑化后进行自连接。由此，得到pCDF-orfB1的T7终止子下游的AgeI识别序列被删除的pCDF-orfB1’。

接着，以利用常规方法提取出的橙黄壶菌(Aurantiochytrium sp.)OH4株的基因组DNA作为模板，使用序列号9、10、11和12所表示的引物进行重叠延伸PCR，扩增出包含编码OrfB的DNA(由序列号1所表示的碱基序列构成的DNA)的DNA片段。该扩增出的DNA片段中，编码区的第4713位的碱基从腺嘌呤被置换为胸腺嘧啶，NdeI识别序列(第4712～4717位的碱基序列)被删除。将所得到的DNA片段和pCDF-orfB1’分别用限制酶NdeI和EcoRI进行处理，将所得到的限制酶处理片段连接，由此得到pCDF-OH4_orfB。

接着，以pCDF-OH4_orfB作为模板，使用序列号9、12、13和14所表示的引物进行重叠延伸PCR，扩增出包含编码OrfB的DNA的DNA片段。该DNA片段中，编码区的第2625位的碱基从鸟嘌呤被置换为腺嘌呤，并在第2623～2628位的碱基序列中导入了SphI识别序列。将所得到的DNA片段和pCDF-orfB1’分别用限制酶NdeI和EcoRI进行处理，将所得到的限制酶处理片段连接，由此得到表达来源于橙黄壶菌(Auranctiochytrium sp.)OH4株的野生型OrfB的质粒pCDF-OH4_orfBs。

[编码突变型OrfB的DNA文库的构建]

接着，以pCDF-OH4_orfBs作为模板，使用序列号15和16所表示的引物，使用TaKaRaTaq Hot Start Version(宝生物公司制造)进行易错PCR。易错PCR中，为了诱发突变，将PCR反应液中的MgCl的浓度设定为5mM。

将通过易错PCR得到的DNA片段纯化后，用限制酶NdeI和AgeI进行处理，与进行了相同的限制酶处理的pCDF-OH4_orfBs连接。由此，构建编码突变型OrfB的DNA文库。

(3)EPA的生产率评价

通过与林等人(Sci.Rep.,2016,6,35441)同样的方法，制作编码酰基辅酶A脱氢酶FadE(由序列号6所表示的氨基酸序列构成的蛋白质)的基因发生了缺失的大肠杆菌BLR(DE3)ΔfadE株。

利用编码pET21-orfA、pCOLA-OH4_orfC和pSTV-hetI、以及pCDF-OH4_orfBs或突变型OrfB的DNA文库对大肠杆菌BLR(DE3)ΔfadE株进行转化。

将所得到的大肠杆菌接种到含有氨苄青霉素100mg/L、卡那霉素20mg/L、氯霉素30mg/L、链霉素20mg/L的极品肉汤(Terrific Broth)培养基(Becton,Dickinson andCompany公司制造)2mL中，在30℃下进行16小时振荡培养。

将所得到的培养液1mL接种到装有新制备的含有氨苄青霉素100mg/L、卡那霉素20mg/L、氯霉素30mg/L、链霉素20mg/L、1mM IPTG的极品肉汤培养基(Becton,Dickinsonand Company公司制造)20mL的200mL带桨叶的烧瓶中，在230rpm、20℃下培养48小时。

培养后，收集培养液，利用Bligh-Dyer法[Bligh,e.G.and Dyer,W.J.(1959)Can.J.Biochem.Physiol.37,911-917]进行脂质提取后，利用三氟化硼-甲醇溶液将脂肪酸甲基化，利用气相色谱质谱分析法进行分析。利用气相色谱质谱分析法由与DHA甲酯、EPA甲酯相对应的峰的面积算出培养液中的DHA和EPA的丰度，进一步还计算出EPA与DHA的丰度比。

其结果，生产野生型OrfB的大肠杆菌不生产EPA，与此相对，在利用编码突变型OrfB的DNA文库进行了转化的大肠杆菌中，确认到生产EPA的菌株。

对编码该生产EPA的大肠杆菌所生产的突变型OrfB的DNA的碱基序列进行确定，结果，OrfB的氨基酸序列中第230位的L-苯丙氨酸被置换为L-亮氨酸。

(4)进一步的突变型OrfB的获得

进一步，以编码由第230位的L-苯丙氨酸被置换为L-亮氨酸的氨基酸序列构成的突变型OrfB的DNA作为模板，利用与上述同样的方法进行易错PCR，并利用与上述同样的方法导入到大肠杆菌BLR(DE3)ΔfadE株中，确认EPA的生产率。

其结果，确认到与上述获得的生产由第230位的L-苯丙氨酸被置换为L-亮氨酸的氨基酸序列构成的突变型OrfB的大肠杆菌相比进一步提高了EPA的生产率的菌株。

对编码提高了EPA的生产率的大肠杆菌所表达的突变型OrfB的DNA的碱基序列进行确定，结果，在OrfB的氨基酸序列中第230位的L-苯丙氨酸被置换为L-亮氨酸的基础上，第6位的L-天冬酰胺被置换为L-丝氨酸、第65位的L-苯丙氨酸被置换为L-亮氨酸。

将对上述培养液中的EPA、DHA和DPA进行测定的结果总结示于表1。

[表1]

如表1所示可知，通过使用生产将OrfB的氨基酸序列中第230位的氨基酸残基置换为L-亮氨酸的突变型OrfB、或者将第6位的氨基酸残基置换为L-丝氨酸、将第65位的氨基酸残基置换为L-亮氨酸并且将第230位的氨基酸残基置换为L-亮氨酸的突变型OrfB的大肠杆菌，与使用生产野生型OrfB的大肠杆菌的情况相比，能够高效地制造EPA。

[实施例2]

使用生产突变型OrfB的大肠杆菌的EPA的制造-2

(1)各表达质粒的制作

以实施例1(2)中获得的pCDF-OH4_orfB作为模板，使用序列号9和17所表示的引物进行PCR，扩增出包含编码OrfB的KS结构域的N末端区的DNA的DNA片段。

另外，以pCDF-OH4_orfB作为模板，使用序列号16所表示的引物和序列号18、19、20、21或22所表示的引物进行PCR，扩增出包含编码OrfB的氨基酸序列中第230位的氨基酸残基被置换为L-色氨酸、L-天冬酰胺、甘氨酸、L-天冬氨酸或L-丙氨酸的突变型OrfB的KS结构域的C末端区的DNA的DNA片段。

使用所获得的、编码KS结构域的N末端区或C末端区的DNA片段以及序列号9和16所表示的引物进行重叠延伸PCR，获得包含编码OrfB的氨基酸序列中第230位的氨基酸残基被置换为L-色氨酸、L-天冬酰胺、甘氨酸、L-天冬氨酸或L-丙氨酸的突变型OrfB的KS结构域的全长的DNA的DNA片段。

将该DNA片段和pCDF-OH4_orfBs分别用限制酶NdeI和AgeI进行处理，将所得到的限制酶处理片段连接，由此得到pCDF-OH4_orfB-F230W、pCDF-OH4_orfB-F230N、pCDF-OH4_orfB-F230G、pCDF-OH4_orfB-F230D和pCDF-OH4_orfB-F230A。

另外，由实施例1(3)中获得的大肠杆菌获得具有编码OrfB的氨基酸序列中第230位的氨基酸残基被置换为L-亮氨酸的突变型OrfB的DNA的质粒pCDF-OH4_orfB-F230L。

(2)EPA的制造

利用pET21-orfA、pCOLA-OH4_orfC和pSTV-hetI、以及野生型OrfB或6种突变型OrfB的表达质粒(pCDF-OH4_orfBs、pCDF-OH4_orfB-F230L、pCDF-OH4_orfB-F230W、pCDF-OH4_orfB-F230N、pCDF-OH4_orfB-F230G、pCDF-OH4_orfB-F230D或pCDF-OH4_orfB-F230A)对大肠杆菌BLR(DE3)ΔfadE株进行转化。

将所得到的大肠杆菌接种到含有氨苄青霉素100mg/L、卡那霉素20mg/L、氯霉素30mg/L、链霉素20mg/L的极品肉汤培养基(Becton,Dickinson and Company公司制造)2mL中，在30℃下进行16小时振荡培养。

将所得到的培养液1mL接种到装有新制备的含有氨苄青霉素100mg/L、卡那霉素20mg/L、氯霉素30mg/L、链霉素20mg/L、1mM IPTG的极品肉汤培养基(Becton,Dickinsonand Company公司制造)20mL的200mL带桨叶的烧瓶中，在230rpm、20℃下培养48小时。

培养后，收集培养液，利用Bligh-Dyer法进行脂质提取后，利用三氟化硼-甲醇溶液将脂肪酸甲基化，利用气相色谱质谱分析法进行分析。

将对培养液中的EPA、DHA和DPA进行测定的结果示于表2。

[表2]

如表2所示可知，与使用将OrfB的氨基酸序列中第230位的氨基酸残基置换为L-亮氨酸的突变型OrfB时同样，使用生产将OrfB的氨基酸序列中第230位的氨基酸残基置换为L-色氨酸、L-天冬酰胺、甘氨酸、L-天冬氨酸或L-丙氨酸的突变型OrfB的大肠杆菌时，与使用生产野生型OrfB的大肠杆菌的情况相比，也能够高效地制造EPA。

[实施例3]

使用生产突变型OrfB的大肠杆菌的EPA的制造-3

(1)各表达质粒的制作

[pCDF-OH4_orfB-N6S-F230L的制作]

以pCDF-OH4_orfB-F230L作为模板，使用序列号23和16所表示的引物进行PCR，得到包含编码OrfB的KS结构域的DNA的DNA片段。将所得到的DNA片段和pCDF-OH4_orfB-F230L分别用限制酶NdeI和AgeI进行处理，将所得到的限制酶处理片段连接，由此得到pCDF-OH4_orfB-N6S-F230L。

pCDF-OH4_orfB-N6S-F230L具有编码来源于橙黄壶菌(Auranctiochytrium sp.)OH4株的OrfB的氨基酸序列中第6位的氨基酸残基被置换为L-丝氨酸、第230位的氨基酸残基被置换为L-亮氨酸的氨基酸序列的DNA。

[pCDF-OH4_orfB-F65L-F230L的制作]

以pCDF-OH4_orfB-F230L作为模板，使用序列号24、25、26和16所表示的引物进行重叠延伸PCR，得到包含编码OrfB的KS结构域的DNA的DNA片段。将所得到的DNA片段和pCDF-OH4_orfB-F230L分别用限制酶NdeI和AgeI进行处理，将所得到的限制酶处理片段连接，由此得到pCDF-OH4_orfB-F65L-F230L。

pCDF-OH4_orfB-F65L-F230L具有编码来源于橙黄壶菌(Auranctiochytrium sp.)OH4株的OrfB的氨基酸序列中第65位的氨基酸残基被置换为L-亮氨酸、第230位的氨基酸残基被置换为L-亮氨酸的氨基酸序列的DNA。

[pCDF-OH4_orfB-N6S-F65L-F230L的制作]

从实施例1(4)中获得的、生产第230位的L-苯丙氨酸被置换为L-亮氨酸、第6位的L-天冬酰胺被置换为L-丝氨酸、第65位的L-苯丙氨酸被置换为L-亮氨酸的OrfB的大肠杆菌中提取质粒，得到pCDF-OF4_orfB-N6S-F65L-F230L。

pCDF-OF4_orfB-N6S-F65L-F230L具有编码来源于橙黄壶菌(Auranctiochytriumsp.)OH4株的OrfB的氨基酸序列中第6位的氨基酸残基被置换为L-丝氨酸、第65位的氨基酸残基被置换为L-亮氨酸、并且第230位的氨基酸残基被置换为L-亮氨酸的氨基酸序列的DNA。

(2)EPA的制造

利用pET21-orfA、pCOLA-OH4_orfC和pSTV-hetI、以及野生型OrfB或4种突变型OrfB的表达质粒(pCDF-OH4_orfBs、pCDF-OH4_orfB-F230L、pCDF-OH4_orfB-N6S-F230L、pCDF-OH4_orfB-F65L-F230L或pCDF-OH4_orfB-N6S-F65L-F230L)对大肠杆菌BLR(DE3)ΔfadE株进行转化。

将所得到的大肠杆菌接种到含有氨苄青霉素100mg/L、卡那霉素20mg/L、氯霉素30mg/L、链霉素20mg/L的极品肉汤培养基(Becton,Dickinson and Company公司制造)2mL中，在30℃下进行16小时振荡培养。

将所得到的培养液1mL接种到装有新制备的含有氨苄青霉素100mg/L、卡那霉素20mg/L、氯霉素30mg/L、链霉素20mg/L、1mM IPTG的极品肉汤培养基(Becton,Dickinsonand Company公司制造)20mL的200mL带桨叶的烧瓶中，在230rpm、20℃下培养48小时。

培养后，收集培养液，利用Bligh-Dyer法进行脂质提取后，利用三氟化硼-甲醇溶液将脂肪酸甲基化，利用气相色谱质谱分析法进行分析。

将对培养液中的EPA、DHA和DPA进行测定的结果示于表3。

[表3]

如表3所示可知，使用生产在OrfB的氨基酸序列中第230位的氨基酸残基的置换的基础上将第6位的氨基酸残基和/或第65位的氨基酸残基分别置换为L-丝氨酸、L-亮氨酸的突变型OrfB的大肠杆菌时，与使用生产OrfB的氨基酸序列中第230位的氨基酸残基被置换为L-亮氨酸的突变型OrfB的大肠杆菌的情况相比，能够更高效地制造EPA。

[实施例4]

使用生产突变型OrfB的大肠杆菌的EPA的制造-4

以实施例3中获得的、编码由第230位的L-苯丙氨酸被置换为L-亮氨酸、第65位的L-苯丙氨酸被置换为L-亮氨酸的氨基酸序列构成的突变型OrfB的DNA作为模板，利用与实施例1(2)同样的方法进行易错PCR，利用与实施例1(3)同样的方法导入到大肠杆菌BLR(DE3)ΔfadE株中，确认EPA的生产率。

其结果，确认到与生产由第230位的L-苯丙氨酸被置换为L-亮氨酸、第65位的L-苯丙氨酸被置换为L-亮氨酸的氨基酸序列构成的突变型OrfB的大肠杆菌相比进一步提高了EPA的生产率的菌株。

对编码提高了EPA的生产率的大肠杆菌所表达的突变型OrfB的DNA的碱基序列进行确定，结果，在OrfB的氨基酸序列中第230位的L-苯丙氨酸被置换为L-亮氨酸、第65位的L-苯丙氨酸被置换为L-亮氨酸的基础上，第231位的L-异亮氨酸被置换为L-苏氨酸、第275位的L-天冬氨酸被置换为L-甘氨酸。

将对上述培养液中的EPA、DHA和DPA进行测定的结果总结示于表4。

[表4]

如表4所示可知，使用生产在OrfB的氨基酸序列中第230位的氨基酸残基和第65位的氨基酸残基的置换的基础上将第231位的氨基酸残基置换为L-苏氨酸、将第275位的氨基酸残基置换为甘氨酸的突变型OrfB的大肠杆菌时，与使用生产OrfB的氨基酸序列中第230位的氨基酸残基和第65位的氨基酸残基分别被置换为L-亮氨酸的突变型OrfB的大肠杆菌的情况相比，能够更高效地制造EPA。

参考特定的方式详细地对本发明进行了说明，但对于本领域技术人员显而易见的是，可以在不脱离本发明的精神和范围的情况下进行各种变更和修正。需要说明的是，本申请基于2018年8月10日提交的日本专利申请(日本特愿2018-151234)，通过引用将其全部内容进行援引。另外，在此引用的所有参考作为整体并入本说明书中。

序列表

<110> 协和发酵生化株式会社

<120> 生产二十碳五烯酸的微生物和二十碳五烯酸的制造方法

<130> W527738

<140> JP2018-151234

<141> 2018-08-10

<160> 31

<170> PatentIn version 3.5

<210> 1

<211> 6105

<212> DNA

<213> 橙黄壶菌OH4（Aurantiochytrium sp. OH4）

<220>

<221> CDS

<222> (1)..(6105)

<400> 1

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Met Ala Ser Arg Lys Asn Val Ser Ala Ala His Glu Met His Asp Glu

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Lys Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys

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Asp Lys Glu Glu Phe Trp Lys Val Val Met Gly Gly Glu Ala Ala Trp

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Phe Lys Ala Glu Arg Ser Lys Phe Ala Asp Thr Phe Cys Asn Glu Asn

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Tyr Gly Cys Val Asp Asp Ser Val Asp Asn Glu His Glu Leu Leu Leu

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Lys Leu Ser Lys Lys Ala Leu Ser Glu Thr Ser Val Ser Asp Ser Thr

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Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu

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Gln Gly Glu Leu Leu Asn Val Tyr Gln Asn His Val Glu Lys Lys Leu

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Gly Ala Arg Val Phe Lys Asp Ala Ser Lys Trp Ser Glu Arg Glu Gln

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Ser Gln Asn Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala

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Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Pro Leu His Tyr Ser Val

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Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp

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His Leu Val Ser Gly Ala Ala Asp Val Met Leu Ala Gly Ala Thr Cys

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Phe Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala

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Met Pro Val Ser Gly Asp Gly Ile Ser Tyr Pro Leu His Lys Asp Ser

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Gln Gly Leu Thr Pro Gly Glu Gly Gly Ala Ile Met Val Leu Lys Arg

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Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu Leu

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Gly Ala Thr Ile Ser Asn Ala Gly Cys Gly Leu Pro Leu Lys Pro His

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Leu Pro Ser Glu Lys Ser Cys Leu Ile Asp Thr Tyr Lys Arg Val Asn

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Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly Thr

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Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe Glu

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Gly Lys Val Pro Arg Phe Gly Ser Ser Lys Gly Asn Phe Gly His Thr

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Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ala Met

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Lys His Gly Val Ile Pro Pro Thr Pro Gly Val Asp Gly Ser Ser Gln

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Met Asp Pro Leu Val Val Ser Glu Pro Ile Pro Trp Pro Asp Thr Glu

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Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly Gly Thr

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Asn Ala His Ala Val Phe Glu Glu Phe Asp Arg Ser Lys Ala Ala Cys

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Ala Thr His Asp Ser Ile Ser Ser Leu Ser Ser Arg Cys Gly Gly Glu

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Gly Asn Met Arg Ile Ala Ile Thr Gly Met Asp Ala Thr Phe Gly Ser

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Leu Lys Gly Leu Asp Ala Phe Glu Arg Ala Ile Tyr Asn Gly Gln His

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Gly Ala Val Pro Leu Pro Glu Lys Arg Trp Arg Phe Leu Gly Lys Asp

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Lys Asp Phe Leu Asp Leu Cys Gly Val Lys Glu Val Pro His Gly Cys

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Ile Asp Arg Ala Ile Leu Asn Ser Gly Leu Lys Lys Gly Gly Lys Val

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Ala Val Phe Val Gly Leu Gly Thr Asp Leu Glu Leu Tyr Arg His Arg

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Ala Arg Val Ala Leu Lys Glu Arg Ala Arg Pro Glu Ala Ala Ala Ala

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Leu Asn Asp Met Met Ser Tyr Ile Asn Asp Cys Gly Thr Ala Thr Ser

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Tyr Thr Ser Tyr Ile Gly Asn Leu Val Ala Thr Arg Val Ser Ser Gln

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Trp Gly Phe Glu Gly Pro Ser Phe Thr Ile Thr Glu Gly Asn Asn Ser

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Val Glu Ala Val Val Ile Ala Gly Val Asp Leu Cys Ala Ser Ala Glu

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Asn Leu Tyr Val Lys Ser Arg Arg Phe Lys Val Ser Glu Gln Glu Ser

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Pro Arg Ala Ser Phe Asp Ser Gly Ala Asp Gly Tyr Phe Val Gly Glu

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Gly Cys Gly Ala Leu Val Leu Lys Arg Glu Ser Asp Cys Thr Lys Asp

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Glu Arg Ile Tyr Ala Cys Met Asp Ala Ile Val Pro Gly Asn Met Pro

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Ala Ala Cys Met Glu Glu Ala Leu Ala Gln Ala Arg Val Asn Pro Lys

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Asp Val Glu Met Leu Glu Leu Ser Ala Asp Ser Ala Arg His Leu Lys

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Asn Pro Ser Val Leu Pro Lys Glu Leu Thr Ala Glu Glu Glu Ile Arg

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Gly Ile Glu Ala Ile Leu Ser Gln Arg Ser Ser Asn Glu Ala Val Glu

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Pro His Asn Val Ala Val Ser Ser Val Lys Ser Thr Val Gly Asp Thr

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Gly Tyr Ala Ser Gly Ala Ala Ser Leu Ile Lys Thr Ala Leu Cys Leu

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Tyr Asn Arg Tyr Leu Pro Ser Asn Gly Ala Ser Trp Glu Glu Pro Ala

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Pro Glu Thr Gln Trp Gly Lys Ser Leu Tyr Ala Cys Gln Ser Ser Arg

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Ala Trp Leu Lys Asn Pro Gly Ala Arg Arg His Ala Ala Val Ser Gly

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Val Ser Glu Thr Arg Ser Cys Tyr Thr Val Leu Leu Ser Asp Val Glu

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Gly His His Glu Thr Lys Ser Arg Ile Ser Leu Asp Asp Asp Ala Val

915 920 925

aaa ctc ctc gta atc cgc gga gac tcc cac gac gct atc acg cag cgt 2832

Lys Leu Leu Val Ile Arg Gly Asp Ser His Asp Ala Ile Thr Gln Arg

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Val Asp Lys Leu Arg Glu Arg Leu Ala Gln Pro Ser Ala Asn Val Arg

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Leu Ala Phe Met Glu Leu Leu Gly Glu Ser Ile Ala Gln Glu Thr Lys

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Thr Pro Leu Pro Ala Phe Ala Leu Cys Leu Val Thr Ser Pro Ser Lys

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Leu Gln Lys Glu Leu Glu Leu Ala Ser Lys Gly Ile Pro Arg Ser Leu

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Lys Met Gly Arg Asp Trp Thr Ser Pro Ser Gly Ser His Phe Ala

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Pro Lys Pro Leu Ser Ser Asp Arg Val Ala Phe Met Tyr Gly Glu

1025 1030 1035

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Gly Arg Ser Pro Tyr Tyr Gly Ile Gly Leu Asp Ile His Arg Ile

1040 1045 1050

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Trp Pro Glu Leu His Glu Phe Val Asn Ala Lys Thr Asn Lys Leu

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Trp Asp Gln Gly Asp Arg Trp Leu Ile Pro Arg Ala Ser Thr Lys

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Glu Glu Leu Lys Ala Gln Glu Asp Glu Phe Ser Arg Asn Gln Val

1085 1090 1095

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Glu Met Phe Arg Leu Gly Ile Leu Met Ser Met Cys Phe Thr His

1100 1105 1110

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Ile Ala Arg Asp Val Leu Gly Ile Gln Pro Lys Ala Ala Phe Gly

1115 1120 1125

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Leu Ser Leu Gly Glu Ile Ser Met Val Phe Ala Phe Ser Glu Lys

1130 1135 1140

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Asn Gly Leu Val Ser Glu Glu Leu Thr Thr Lys Leu Arg Asn Ser

1145 1150 1155

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Glu Val Trp Arg Lys Ala Leu Ala Val Glu Phe Asp Ala Leu Arg

1160 1165 1170

aag gcc tgg aat att ccc caa gat acc cct gtc agc gag ttc tgg 3564

Lys Ala Trp Asn Ile Pro Gln Asp Thr Pro Val Ser Glu Phe Trp

1175 1180 1185

caa gga tac gtg gta cgt gga acc cgc gag gcc gtt gaa gcg gcc 3609

Gln Gly Tyr Val Val Arg Gly Thr Arg Glu Ala Val Glu Ala Ala

1190 1195 1200

atc ggc ccc aac aat aag tac gtg cac ttg acc att gtc aac gat 3654

Ile Gly Pro Asn Asn Lys Tyr Val His Leu Thr Ile Val Asn Asp

1205 1210 1215

gcc aac agt gct ctc atc agt ggc aag cct gaa gat tgc aag gct 3699

Ala Asn Ser Ala Leu Ile Ser Gly Lys Pro Glu Asp Cys Lys Ala

1220 1225 1230

gcc att gct cgc ctg agc agc aac ctc cct gct ttg ccc gtg gac 3744

Ala Ile Ala Arg Leu Ser Ser Asn Leu Pro Ala Leu Pro Val Asp

1235 1240 1245

ctt ggt atg tgt ggc cac tgc ccc gtg gtc gag ccg tac ggc aag 3789

Leu Gly Met Cys Gly His Cys Pro Val Val Glu Pro Tyr Gly Lys

1250 1255 1260

cag atc gct gag atc cat agc gtc ctc gag att ccc gag gtt gcc 3834

Gln Ile Ala Glu Ile His Ser Val Leu Glu Ile Pro Glu Val Ala

1265 1270 1275

ggc ctt gac ctg tac acg agc gtc aac cag aag aag ctt gtt aac 3879

Gly Leu Asp Leu Tyr Thr Ser Val Asn Gln Lys Lys Leu Val Asn

1280 1285 1290

aag tcc act gga gcc agc gac gag tac gca ccc agc ttt ggt gaa 3924

Lys Ser Thr Gly Ala Ser Asp Glu Tyr Ala Pro Ser Phe Gly Glu

1295 1300 1305

tac gca gca cag ctg tac act gtt cag gca gac ttt cct aag atc 3969

Tyr Ala Ala Gln Leu Tyr Thr Val Gln Ala Asp Phe Pro Lys Ile

1310 1315 1320

gcc aag acc gtt agc gac aag aac ttt gac gtc ttt gtt gag act 4014

Ala Lys Thr Val Ser Asp Lys Asn Phe Asp Val Phe Val Glu Thr

1325 1330 1335

ggt ccc aac gct cac cgt agc gcc gca att cgc gcc acc ctt gga 4059

Gly Pro Asn Ala His Arg Ser Ala Ala Ile Arg Ala Thr Leu Gly

1340 1345 1350

aat agc aag cct ttt gtc acc gga tcc atg gac cgc cag aac gag 4104

Asn Ser Lys Pro Phe Val Thr Gly Ser Met Asp Arg Gln Asn Glu

1355 1360 1365

aat gct tgg aca acc atg gtc aag ctg gtt gcc tct ctc caa gcc 4149

Asn Ala Trp Thr Thr Met Val Lys Leu Val Ala Ser Leu Gln Ala

1370 1375 1380

cac cgc gtg cct ggc gtg aag gtc tcc cct ctg tac cac ccc gag 4194

His Arg Val Pro Gly Val Lys Val Ser Pro Leu Tyr His Pro Glu

1385 1390 1395

act gtt gag gag gct acg cag agt tac aac gat atg gtg gct ggc 4239

Thr Val Glu Glu Ala Thr Gln Ser Tyr Asn Asp Met Val Ala Gly

1400 1405 1410

aag aag cct act aag aac aag ttc ttg cgt aag att gtg gtc aat 4284

Lys Lys Pro Thr Lys Asn Lys Phe Leu Arg Lys Ile Val Val Asn

1415 1420 1425

ggt cgc tat gac ccc aaa aag cag ctc gtg ccg ccc cag gtg cta 4329

Gly Arg Tyr Asp Pro Lys Lys Gln Leu Val Pro Pro Gln Val Leu

1430 1435 1440

gct aag ctt cct cct gcg gac ccc aag atc gag gct ctt atc cag 4374

Ala Lys Leu Pro Pro Ala Asp Pro Lys Ile Glu Ala Leu Ile Gln

1445 1450 1455

gct cgc aag atg cag cct att gcc ccc aag ttc atg gag cgt ctc 4419

Ala Arg Lys Met Gln Pro Ile Ala Pro Lys Phe Met Glu Arg Leu

1460 1465 1470

gac att cag gag caa gac gcc aca cgc gac cct att ctc aac aag 4464

Asp Ile Gln Glu Gln Asp Ala Thr Arg Asp Pro Ile Leu Asn Lys

1475 1480 1485

gat aac aaa cct tcc gct gct cct gcc ctt gtc cct gct gct ccg 4509

Asp Asn Lys Pro Ser Ala Ala Pro Ala Leu Val Pro Ala Ala Pro

1490 1495 1500

gcc cct gct ccg gcc cgc agc gcc tcc gga gct gtt gtg gct tcc 4554

Ala Pro Ala Pro Ala Arg Ser Ala Ser Gly Ala Val Val Ala Ser

1505 1510 1515

tct gag gct ctc cgt gcc aaa ctt ttg gag ctc aac agc act ttg 4599

Ser Glu Ala Leu Arg Ala Lys Leu Leu Glu Leu Asn Ser Thr Leu

1520 1525 1530

atg ctt ggt gtc aac gcc aac ggt gat ctc gtt gaa gca agc cca 4644

Met Leu Gly Val Asn Ala Asn Gly Asp Leu Val Glu Ala Ser Pro

1535 1540 1545

agt gaa gca tct att gtt gtg ccc aag tgc gat atc aag gat ctt 4689

Ser Glu Ala Ser Ile Val Val Pro Lys Cys Asp Ile Lys Asp Leu

1550 1555 1560

ggc agc cgt gcc ttc atg gag aca tat ggt gta tcc gcc ccc atg 4734

Gly Ser Arg Ala Phe Met Glu Thr Tyr Gly Val Ser Ala Pro Met

1565 1570 1575

tac acc ggc gcc atg gca aag ggc att gca tcc gct gag atg gtt 4779

Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala Ser Ala Glu Met Val

1580 1585 1590

atc gct gcc gga aag cgc ggc atc ctt ggt tct ctc ggt gct ggt 4824

Ile Ala Ala Gly Lys Arg Gly Ile Leu Gly Ser Leu Gly Ala Gly

1595 1600 1605

ggt ctt cct atc gcc acc gta cgc aag gct ctc gaa gct atc cag 4869

Gly Leu Pro Ile Ala Thr Val Arg Lys Ala Leu Glu Ala Ile Gln

1610 1615 1620

gct gaa ctg ccc aag ggc cct tac gct gtc aac ctc atc cac tct 4914

Ala Glu Leu Pro Lys Gly Pro Tyr Ala Val Asn Leu Ile His Ser

1625 1630 1635

ccc ttc gac agc aac ctc gag aag ggt aac gtc gac ctc ttc ctc 4959

Pro Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu Phe Leu

1640 1645 1650

gag aag ggc gtc act gtc gtt gaa gcc tcc gcc ttt atg acc ttg 5004

Glu Lys Gly Val Thr Val Val Glu Ala Ser Ala Phe Met Thr Leu

1655 1660 1665

acc ccg cag ctc gtg cgc tac cgt gct gca ggt ctc tct cgc gct 5049

Thr Pro Gln Leu Val Arg Tyr Arg Ala Ala Gly Leu Ser Arg Ala

1670 1675 1680

gct gat ggc tcc acg gtt att aag aac cgc gtc atc ggt aag gtt 5094

Ala Asp Gly Ser Thr Val Ile Lys Asn Arg Val Ile Gly Lys Val

1685 1690 1695

tct cgc aca gag ctt gcc gca atg ttt atc cgt ccc gcg ccc gag 5139

Ser Arg Thr Glu Leu Ala Ala Met Phe Ile Arg Pro Ala Pro Glu

1700 1705 1710

aat ctc ctc gag aag ctg ctg aag tcc ggc gag atc acc caa gag 5184

Asn Leu Leu Glu Lys Leu Leu Lys Ser Gly Glu Ile Thr Gln Glu

1715 1720 1725

cag gct gct ctc gca cgc aca gtg cct gtg gca gac gac att gcc 5229

Gln Ala Ala Leu Ala Arg Thr Val Pro Val Ala Asp Asp Ile Ala

1730 1735 1740

gtt gag gcg gac tcc ggt ggc cac acc gat aac cgc ccc atc cac 5274

Val Glu Ala Asp Ser Gly Gly His Thr Asp Asn Arg Pro Ile His

1745 1750 1755

gtc atc ctc cct ctc att gtc aac ctc cgt gat cgt ctg cac aag 5319

Val Ile Leu Pro Leu Ile Val Asn Leu Arg Asp Arg Leu His Lys

1760 1765 1770

gag tgc ggc tac cct gcc cac ctt cgc gtt cgc gtt ggt gct ggt 5364

Glu Cys Gly Tyr Pro Ala His Leu Arg Val Arg Val Gly Ala Gly

1775 1780 1785

ggt ggc att gga tgc cct cag gcc gcc att gcc acc ttc aac atg 5409

Gly Gly Ile Gly Cys Pro Gln Ala Ala Ile Ala Thr Phe Asn Met

1790 1795 1800

ggc gcg gcc ttc atc gtc act ggt acc gta aac cag atg agt aag 5454

Gly Ala Ala Phe Ile Val Thr Gly Thr Val Asn Gln Met Ser Lys

1805 1810 1815

caa gct gga acc tgt gac acc gtt cgc aag cag ctc tca caa gcc 5499

Gln Ala Gly Thr Cys Asp Thr Val Arg Lys Gln Leu Ser Gln Ala

1820 1825 1830

acc tac tcc gac atc tgc atg gcc cca gca gct gac atg ttt gag 5544

Thr Tyr Ser Asp Ile Cys Met Ala Pro Ala Ala Asp Met Phe Glu

1835 1840 1845

gaa ggt gtc aag ctc cag gtg ctc aag aag gga act atg ttc ccc 5589

Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr Met Phe Pro

1850 1855 1860

tcg cgt gcc aac aag ctc tat gag ctc ttc gtc aag tat gac tcc 5634

Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Val Lys Tyr Asp Ser

1865 1870 1875

ttt gag tcc atg gct cct gga gag ctg gaa cgt gtg gag aag cgc 5679

Phe Glu Ser Met Ala Pro Gly Glu Leu Glu Arg Val Glu Lys Arg

1880 1885 1890

att ttc aag aag tct ctg tca gag gtt tgg gaa gag acc aag gac 5724

Ile Phe Lys Lys Ser Leu Ser Glu Val Trp Glu Glu Thr Lys Asp

1895 1900 1905

ttc tac atc aac agg ttg cag aac ccg gag aag att gag cgc gcg 5769

Phe Tyr Ile Asn Arg Leu Gln Asn Pro Glu Lys Ile Glu Arg Ala

1910 1915 1920

gag cgt gac ccc aag ctt aag atg tcc ttg tgc ttc cgc tgg tac 5814

Glu Arg Asp Pro Lys Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr

1925 1930 1935

ctt ggt ttg gcg agc ttc tgg gca aac gct ggc atc ccg gac cgt 5859

Leu Gly Leu Ala Ser Phe Trp Ala Asn Ala Gly Ile Pro Asp Arg

1940 1945 1950

gcc atg gac tac cag gtt tgg tgt ggc cca gcg att gga tct ttc 5904

Ala Met Asp Tyr Gln Val Trp Cys Gly Pro Ala Ile Gly Ser Phe

1955 1960 1965

aac gac ttc atc aag ggt acc tac ctt gac ccc gcc gtt gcc aac 5949

Asn Asp Phe Ile Lys Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn

1970 1975 1980

gag tac ccc gat gtt gtg caa atc aac ttg cag atc ctc cgt ggt 5994

Glu Tyr Pro Asp Val Val Gln Ile Asn Leu Gln Ile Leu Arg Gly

1985 1990 1995

gcc tgc ttc ttg cgc cgc ctc gaa gct gtc cgt aat gcc ccg ctg 6039

Ala Cys Phe Leu Arg Arg Leu Glu Ala Val Arg Asn Ala Pro Leu

2000 2005 2010

aag gct aac gcc aag cag gtt gct gcc gag att gat gac atc tac 6084

Lys Ala Asn Ala Lys Gln Val Ala Ala Glu Ile Asp Asp Ile Tyr

2015 2020 2025

gtg ccc act gag cgc ctg taa 6105

Val Pro Thr Glu Arg Leu

2030

<210> 2

<211> 2034

<212> PRT

<213> 橙黄壶菌OH4（Aurantiochytrium sp. OH4）

<400> 2

Met Ala Ser Arg Lys Asn Val Ser Ala Ala His Glu Met His Asp Glu

1 5 10 15

Lys Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys

20 25 30

Asp Lys Glu Glu Phe Trp Lys Val Val Met Gly Gly Glu Ala Ala Trp

35 40 45

Thr Lys Ile Ser Asp Lys Arg Leu Gly Ser Asn Lys Arg Ala Glu His

50 55 60

Phe Lys Ala Glu Arg Ser Lys Phe Ala Asp Thr Phe Cys Asn Glu Asn

65 70 75 80

Tyr Gly Cys Val Asp Asp Ser Val Asp Asn Glu His Glu Leu Leu Leu

85 90 95

Lys Leu Ser Lys Lys Ala Leu Ser Glu Thr Ser Val Ser Asp Ser Thr

100 105 110

Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu

115 120 125

Gln Gly Glu Leu Leu Asn Val Tyr Gln Asn His Val Glu Lys Lys Leu

130 135 140

Gly Ala Arg Val Phe Lys Asp Ala Ser Lys Trp Ser Glu Arg Glu Gln

145 150 155 160

Ser Gln Asn Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala

165 170 175

Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Pro Leu His Tyr Ser Val

180 185 190

Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp

195 200 205

His Leu Val Ser Gly Ala Ala Asp Val Met Leu Ala Gly Ala Thr Cys

210 215 220

Phe Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala

225 230 235 240

Met Pro Val Ser Gly Asp Gly Ile Ser Tyr Pro Leu His Lys Asp Ser

245 250 255

Gln Gly Leu Thr Pro Gly Glu Gly Gly Ala Ile Met Val Leu Lys Arg

260 265 270

Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu Leu

275 280 285

Gly Ala Thr Ile Ser Asn Ala Gly Cys Gly Leu Pro Leu Lys Pro His

290 295 300

Leu Pro Ser Glu Lys Ser Cys Leu Ile Asp Thr Tyr Lys Arg Val Asn

305 310 315 320

Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly Thr

325 330 335

Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe Glu

340 345 350

Gly Lys Val Pro Arg Phe Gly Ser Ser Lys Gly Asn Phe Gly His Thr

355 360 365

Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ala Met

370 375 380

Lys His Gly Val Ile Pro Pro Thr Pro Gly Val Asp Gly Ser Ser Gln

385 390 395 400

Met Asp Pro Leu Val Val Ser Glu Pro Ile Pro Trp Pro Asp Thr Glu

405 410 415

Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly Gly Thr

420 425 430

Asn Ala His Ala Val Phe Glu Glu Phe Asp Arg Ser Lys Ala Ala Cys

435 440 445

Ala Thr His Asp Ser Ile Ser Ser Leu Ser Ser Arg Cys Gly Gly Glu

450 455 460

Gly Asn Met Arg Ile Ala Ile Thr Gly Met Asp Ala Thr Phe Gly Ser

465 470 475 480

Leu Lys Gly Leu Asp Ala Phe Glu Arg Ala Ile Tyr Asn Gly Gln His

485 490 495

Gly Ala Val Pro Leu Pro Glu Lys Arg Trp Arg Phe Leu Gly Lys Asp

500 505 510

Lys Asp Phe Leu Asp Leu Cys Gly Val Lys Glu Val Pro His Gly Cys

515 520 525

Tyr Ile Glu Asp Val Glu Val Asp Phe Ser Arg Leu Arg Thr Pro Met

530 535 540

Thr Pro Asp Asp Met Leu Arg Pro Met Gln Leu Leu Ala Val Thr Thr

545 550 555 560

Ile Asp Arg Ala Ile Leu Asn Ser Gly Leu Lys Lys Gly Gly Lys Val

565 570 575

Ala Val Phe Val Gly Leu Gly Thr Asp Leu Glu Leu Tyr Arg His Arg

580 585 590

Ala Arg Val Ala Leu Lys Glu Arg Ala Arg Pro Glu Ala Ala Ala Ala

595 600 605

Leu Asn Asp Met Met Ser Tyr Ile Asn Asp Cys Gly Thr Ala Thr Ser

610 615 620

Tyr Thr Ser Tyr Ile Gly Asn Leu Val Ala Thr Arg Val Ser Ser Gln

625 630 635 640

Trp Gly Phe Glu Gly Pro Ser Phe Thr Ile Thr Glu Gly Asn Asn Ser

645 650 655

Val Tyr Arg Cys Ala Glu Leu Gly Lys Tyr Leu Leu Glu Thr Gly Glu

660 665 670

Val Glu Ala Val Val Ile Ala Gly Val Asp Leu Cys Ala Ser Ala Glu

675 680 685

Asn Leu Tyr Val Lys Ser Arg Arg Phe Lys Val Ser Glu Gln Glu Ser

690 695 700

Pro Arg Ala Ser Phe Asp Ser Gly Ala Asp Gly Tyr Phe Val Gly Glu

705 710 715 720

Gly Cys Gly Ala Leu Val Leu Lys Arg Glu Ser Asp Cys Thr Lys Asp

725 730 735

Glu Arg Ile Tyr Ala Cys Met Asp Ala Ile Val Pro Gly Asn Met Pro

740 745 750

Ala Ala Cys Met Glu Glu Ala Leu Ala Gln Ala Arg Val Asn Pro Lys

755 760 765

Asp Val Glu Met Leu Glu Leu Ser Ala Asp Ser Ala Arg His Leu Lys

770 775 780

Asn Pro Ser Val Leu Pro Lys Glu Leu Thr Ala Glu Glu Glu Ile Arg

785 790 795 800

Gly Ile Glu Ala Ile Leu Ser Gln Arg Ser Ser Asn Glu Ala Val Glu

805 810 815

Pro His Asn Val Ala Val Ser Ser Val Lys Ser Thr Val Gly Asp Thr

820 825 830

Gly Tyr Ala Ser Gly Ala Ala Ser Leu Ile Lys Thr Ala Leu Cys Leu

835 840 845

Tyr Asn Arg Tyr Leu Pro Ser Asn Gly Ala Ser Trp Glu Glu Pro Ala

850 855 860

Pro Glu Thr Gln Trp Gly Lys Ser Leu Tyr Ala Cys Gln Ser Ser Arg

865 870 875 880

Ala Trp Leu Lys Asn Pro Gly Ala Arg Arg His Ala Ala Val Ser Gly

885 890 895

Val Ser Glu Thr Arg Ser Cys Tyr Thr Val Leu Leu Ser Asp Val Glu

900 905 910

Gly His His Glu Thr Lys Ser Arg Ile Ser Leu Asp Asp Asp Ala Val

915 920 925

Lys Leu Leu Val Ile Arg Gly Asp Ser His Asp Ala Ile Thr Gln Arg

930 935 940

Val Asp Lys Leu Arg Glu Arg Leu Ala Gln Pro Ser Ala Asn Val Arg

945 950 955 960

Leu Ala Phe Met Glu Leu Leu Gly Glu Ser Ile Ala Gln Glu Thr Lys

965 970 975

Thr Pro Leu Pro Ala Phe Ala Leu Cys Leu Val Thr Ser Pro Ser Lys

980 985 990

Leu Gln Lys Glu Leu Glu Leu Ala Ser Lys Gly Ile Pro Arg Ser Leu

995 1000 1005

Lys Met Gly Arg Asp Trp Thr Ser Pro Ser Gly Ser His Phe Ala

1010 1015 1020

Pro Lys Pro Leu Ser Ser Asp Arg Val Ala Phe Met Tyr Gly Glu

1025 1030 1035

Gly Arg Ser Pro Tyr Tyr Gly Ile Gly Leu Asp Ile His Arg Ile

1040 1045 1050

Trp Pro Glu Leu His Glu Phe Val Asn Ala Lys Thr Asn Lys Leu

1055 1060 1065

Trp Asp Gln Gly Asp Arg Trp Leu Ile Pro Arg Ala Ser Thr Lys

1070 1075 1080

Glu Glu Leu Lys Ala Gln Glu Asp Glu Phe Ser Arg Asn Gln Val

1085 1090 1095

Glu Met Phe Arg Leu Gly Ile Leu Met Ser Met Cys Phe Thr His

1100 1105 1110

Ile Ala Arg Asp Val Leu Gly Ile Gln Pro Lys Ala Ala Phe Gly

1115 1120 1125

Leu Ser Leu Gly Glu Ile Ser Met Val Phe Ala Phe Ser Glu Lys

1130 1135 1140

Asn Gly Leu Val Ser Glu Glu Leu Thr Thr Lys Leu Arg Asn Ser

1145 1150 1155

Glu Val Trp Arg Lys Ala Leu Ala Val Glu Phe Asp Ala Leu Arg

1160 1165 1170

Lys Ala Trp Asn Ile Pro Gln Asp Thr Pro Val Ser Glu Phe Trp

1175 1180 1185

Gln Gly Tyr Val Val Arg Gly Thr Arg Glu Ala Val Glu Ala Ala

1190 1195 1200

Ile Gly Pro Asn Asn Lys Tyr Val His Leu Thr Ile Val Asn Asp

1205 1210 1215

Ala Asn Ser Ala Leu Ile Ser Gly Lys Pro Glu Asp Cys Lys Ala

1220 1225 1230

Ala Ile Ala Arg Leu Ser Ser Asn Leu Pro Ala Leu Pro Val Asp

1235 1240 1245

Leu Gly Met Cys Gly His Cys Pro Val Val Glu Pro Tyr Gly Lys

1250 1255 1260

Gln Ile Ala Glu Ile His Ser Val Leu Glu Ile Pro Glu Val Ala

1265 1270 1275

Gly Leu Asp Leu Tyr Thr Ser Val Asn Gln Lys Lys Leu Val Asn

1280 1285 1290

Lys Ser Thr Gly Ala Ser Asp Glu Tyr Ala Pro Ser Phe Gly Glu

1295 1300 1305

Tyr Ala Ala Gln Leu Tyr Thr Val Gln Ala Asp Phe Pro Lys Ile

1310 1315 1320

Ala Lys Thr Val Ser Asp Lys Asn Phe Asp Val Phe Val Glu Thr

1325 1330 1335

Gly Pro Asn Ala His Arg Ser Ala Ala Ile Arg Ala Thr Leu Gly

1340 1345 1350

Asn Ser Lys Pro Phe Val Thr Gly Ser Met Asp Arg Gln Asn Glu

1355 1360 1365

Asn Ala Trp Thr Thr Met Val Lys Leu Val Ala Ser Leu Gln Ala

1370 1375 1380

His Arg Val Pro Gly Val Lys Val Ser Pro Leu Tyr His Pro Glu

1385 1390 1395

Thr Val Glu Glu Ala Thr Gln Ser Tyr Asn Asp Met Val Ala Gly

1400 1405 1410

Lys Lys Pro Thr Lys Asn Lys Phe Leu Arg Lys Ile Val Val Asn

1415 1420 1425

Gly Arg Tyr Asp Pro Lys Lys Gln Leu Val Pro Pro Gln Val Leu

1430 1435 1440

Ala Lys Leu Pro Pro Ala Asp Pro Lys Ile Glu Ala Leu Ile Gln

1445 1450 1455

Ala Arg Lys Met Gln Pro Ile Ala Pro Lys Phe Met Glu Arg Leu

1460 1465 1470

Asp Ile Gln Glu Gln Asp Ala Thr Arg Asp Pro Ile Leu Asn Lys

1475 1480 1485

Asp Asn Lys Pro Ser Ala Ala Pro Ala Leu Val Pro Ala Ala Pro

1490 1495 1500

Ala Pro Ala Pro Ala Arg Ser Ala Ser Gly Ala Val Val Ala Ser

1505 1510 1515

Ser Glu Ala Leu Arg Ala Lys Leu Leu Glu Leu Asn Ser Thr Leu

1520 1525 1530

Met Leu Gly Val Asn Ala Asn Gly Asp Leu Val Glu Ala Ser Pro

1535 1540 1545

Ser Glu Ala Ser Ile Val Val Pro Lys Cys Asp Ile Lys Asp Leu

1550 1555 1560

Gly Ser Arg Ala Phe Met Glu Thr Tyr Gly Val Ser Ala Pro Met

1565 1570 1575

Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala Ser Ala Glu Met Val

1580 1585 1590

Ile Ala Ala Gly Lys Arg Gly Ile Leu Gly Ser Leu Gly Ala Gly

1595 1600 1605

Gly Leu Pro Ile Ala Thr Val Arg Lys Ala Leu Glu Ala Ile Gln

1610 1615 1620

Ala Glu Leu Pro Lys Gly Pro Tyr Ala Val Asn Leu Ile His Ser

1625 1630 1635

Pro Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu Phe Leu

1640 1645 1650

Glu Lys Gly Val Thr Val Val Glu Ala Ser Ala Phe Met Thr Leu

1655 1660 1665

Thr Pro Gln Leu Val Arg Tyr Arg Ala Ala Gly Leu Ser Arg Ala

1670 1675 1680

Ala Asp Gly Ser Thr Val Ile Lys Asn Arg Val Ile Gly Lys Val

1685 1690 1695

Ser Arg Thr Glu Leu Ala Ala Met Phe Ile Arg Pro Ala Pro Glu

1700 1705 1710

Asn Leu Leu Glu Lys Leu Leu Lys Ser Gly Glu Ile Thr Gln Glu

1715 1720 1725

Gln Ala Ala Leu Ala Arg Thr Val Pro Val Ala Asp Asp Ile Ala

1730 1735 1740

Val Glu Ala Asp Ser Gly Gly His Thr Asp Asn Arg Pro Ile His

1745 1750 1755

Val Ile Leu Pro Leu Ile Val Asn Leu Arg Asp Arg Leu His Lys

1760 1765 1770

Glu Cys Gly Tyr Pro Ala His Leu Arg Val Arg Val Gly Ala Gly

1775 1780 1785

Gly Gly Ile Gly Cys Pro Gln Ala Ala Ile Ala Thr Phe Asn Met

1790 1795 1800

Gly Ala Ala Phe Ile Val Thr Gly Thr Val Asn Gln Met Ser Lys

1805 1810 1815

Gln Ala Gly Thr Cys Asp Thr Val Arg Lys Gln Leu Ser Gln Ala

1820 1825 1830

Thr Tyr Ser Asp Ile Cys Met Ala Pro Ala Ala Asp Met Phe Glu

1835 1840 1845

Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr Met Phe Pro

1850 1855 1860

Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Val Lys Tyr Asp Ser

1865 1870 1875

Phe Glu Ser Met Ala Pro Gly Glu Leu Glu Arg Val Glu Lys Arg

1880 1885 1890

Ile Phe Lys Lys Ser Leu Ser Glu Val Trp Glu Glu Thr Lys Asp

1895 1900 1905

Phe Tyr Ile Asn Arg Leu Gln Asn Pro Glu Lys Ile Glu Arg Ala

1910 1915 1920

Glu Arg Asp Pro Lys Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr

1925 1930 1935

Leu Gly Leu Ala Ser Phe Trp Ala Asn Ala Gly Ile Pro Asp Arg

1940 1945 1950

Ala Met Asp Tyr Gln Val Trp Cys Gly Pro Ala Ile Gly Ser Phe

1955 1960 1965

Asn Asp Phe Ile Lys Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn

1970 1975 1980

Glu Tyr Pro Asp Val Val Gln Ile Asn Leu Gln Ile Leu Arg Gly

1985 1990 1995

Ala Cys Phe Leu Arg Arg Leu Glu Ala Val Arg Asn Ala Pro Leu

2000 2005 2010

Lys Ala Asn Ala Lys Gln Val Ala Ala Glu Ile Asp Asp Ile Tyr

2015 2020 2025

Val Pro Thr Glu Arg Leu

2030

<210> 3

<211> 4302

<212> DNA

<213> 橙黄壶菌OH4（Aurantiochytrium sp. OH4）

<220>

<221> CDS

<222> (1)..(4302)

<400> 3

atg gcc act cgc gtg aag acc aac aag aaa cca tgc tgg gag atg acc 48

Met Ala Thr Arg Val Lys Thr Asn Lys Lys Pro Cys Trp Glu Met Thr

1 5 10 15

aag gag gag ctc acc agc ggc aag aac gtc gtt ttc gac tat gac gag 96

Lys Glu Glu Leu Thr Ser Gly Lys Asn Val Val Phe Asp Tyr Asp Glu

20 25 30

ctc ctt gag ttc gcc gag ggt gac atc agc aag gtc ttc ggc ccc gaa 144

Leu Leu Glu Phe Ala Glu Gly Asp Ile Ser Lys Val Phe Gly Pro Glu

35 40 45

ttc agc cag atc gac cag tac aag cgt cgc gtt cgt ctc ccc gcc cgc 192

Phe Ser Gln Ile Asp Gln Tyr Lys Arg Arg Val Arg Leu Pro Ala Arg

50 55 60

gag tac ctc ctc gtc acc cgc gtc acc ctc atg gac gcc gag gtc aac 240

Glu Tyr Leu Leu Val Thr Arg Val Thr Leu Met Asp Ala Glu Val Asn

65 70 75 80

aac tac cgc gtc ggt gcc cgc atg gtc act gag tac gac ctc ccc gtc 288

Asn Tyr Arg Val Gly Ala Arg Met Val Thr Glu Tyr Asp Leu Pro Val

85 90 95

aac ggt gag ctc tct gag ggt ggt gac tgc ccc tgg gcc gtg ctc gtc 336

Asn Gly Glu Leu Ser Glu Gly Gly Asp Cys Pro Trp Ala Val Leu Val

100 105 110

gag agt ggc cag tgt gat ctc atg ctc atc tcc tac atg ggt att gac 384

Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr Met Gly Ile Asp

115 120 125

ttc cag aac aag agc gac cgc gtc tac cgt ctg ctc aac acc acc ctc 432

Phe Gln Asn Lys Ser Asp Arg Val Tyr Arg Leu Leu Asn Thr Thr Leu

130 135 140

acc ttc tac ggt gtt gcc cag gag ggc gag acc ctg gag tac gat atc 480

Thr Phe Tyr Gly Val Ala Gln Glu Gly Glu Thr Leu Glu Tyr Asp Ile

145 150 155 160

cgc gtg acc ggc ttc gcc aag cgt ctc gac ggt gac atc tcc atg ttc 528

Arg Val Thr Gly Phe Ala Lys Arg Leu Asp Gly Asp Ile Ser Met Phe

165 170 175

ttc ttc gag tac gac tgc tac gtc aac ggc cgt ctc ctc atc gag atg 576

Phe Phe Glu Tyr Asp Cys Tyr Val Asn Gly Arg Leu Leu Ile Glu Met

180 185 190

cgc gac ggc tgt gcc ggt ttc ttc acc aac gag gag ctc gcc gcc ggc 624

Arg Asp Gly Cys Ala Gly Phe Phe Thr Asn Glu Glu Leu Ala Ala Gly

195 200 205

aag ggt gtc gtc ttt acc cgc gct gat ctc ctc gcc cgc gag aag acc 672

Lys Gly Val Val Phe Thr Arg Ala Asp Leu Leu Ala Arg Glu Lys Thr

210 215 220

aag aag cag gac atc acc ccg tac gcc att gcc ccg cgt ctt aac aag 720

Lys Lys Gln Asp Ile Thr Pro Tyr Ala Ile Ala Pro Arg Leu Asn Lys

225 230 235 240

acc gtt ctc aac gag act gag atg cag tcc ctc gtg gac aag aac tgg 768

Thr Val Leu Asn Glu Thr Glu Met Gln Ser Leu Val Asp Lys Asn Trp

245 250 255

acc aag gtt ttc ggc ccc gag aac ggc atg gac cag atc aac tac aaa 816

Thr Lys Val Phe Gly Pro Glu Asn Gly Met Asp Gln Ile Asn Tyr Lys

260 265 270

ctc tgc gcc cgt aag atg ctc atg att gac cgc gtc acc aag att gac 864

Leu Cys Ala Arg Lys Met Leu Met Ile Asp Arg Val Thr Lys Ile Asp

275 280 285

tac acc ggt ggc ccc tac ggc ctt ggt ctc ctc gtt ggt gag aag atc 912

Tyr Thr Gly Gly Pro Tyr Gly Leu Gly Leu Leu Val Gly Glu Lys Ile

290 295 300

ctc gag cgc gac cac tgg tac ttc ccg tgc cac ttc gtc gga gac cag 960

Leu Glu Arg Asp His Trp Tyr Phe Pro Cys His Phe Val Gly Asp Gln

305 310 315 320

gtc atg gct gga tcc ctc gtg tct gac ggc tgc agc cag ctc ctc aag 1008

Val Met Ala Gly Ser Leu Val Ser Asp Gly Cys Ser Gln Leu Leu Lys

325 330 335

atg tac atg ctc tgg ctc ggc ctc cac ctt aag acc ggt ccc ttc gac 1056

Met Tyr Met Leu Trp Leu Gly Leu His Leu Lys Thr Gly Pro Phe Asp

340 345 350

ttc cgc ccc gtc aac ggc cac ccc aac aag gtc cgc tgc cgt ggc cag 1104

Phe Arg Pro Val Asn Gly His Pro Asn Lys Val Arg Cys Arg Gly Gln

355 360 365

atc tcc ccg cac aag ggt aag ctc gtc tac gtc atg gag atc aag gag 1152

Ile Ser Pro His Lys Gly Lys Leu Val Tyr Val Met Glu Ile Lys Glu

370 375 380

atg gga tac gac gag gct ggt gac ccg tac gcc att gcc gat gtc aac 1200

Met Gly Tyr Asp Glu Ala Gly Asp Pro Tyr Ala Ile Ala Asp Val Asn

385 390 395 400

att ctc gac att gac ttc gag aag ggc cag act ttc gac ctt gcc aac 1248

Ile Leu Asp Ile Asp Phe Glu Lys Gly Gln Thr Phe Asp Leu Ala Asn

405 410 415

ctc cac gag tac ggc aag ggc gac ctc aac aag aag atc gtc gtc gac 1296

Leu His Glu Tyr Gly Lys Gly Asp Leu Asn Lys Lys Ile Val Val Asp

420 425 430

ttc aag ggt att gcc ctc aag ctc cag aag cgc tct ggc cct gcc gtt 1344

Phe Lys Gly Ile Ala Leu Lys Leu Gln Lys Arg Ser Gly Pro Ala Val

435 440 445

gtc gct ccc gag aag ccc ctc gct ctc aac aag gac ctt tgc gcc ccg 1392

Val Ala Pro Glu Lys Pro Leu Ala Leu Asn Lys Asp Leu Cys Ala Pro

450 455 460

gct gtt gag gcc atc cct gag cac atc ctc aag ggc gat gct ctt gcc 1440

Ala Val Glu Ala Ile Pro Glu His Ile Leu Lys Gly Asp Ala Leu Ala

465 470 475 480

cct aac cag atg acc tgg cac ccg atg tcc aag atc gct ggc aac ccc 1488

Pro Asn Gln Met Thr Trp His Pro Met Ser Lys Ile Ala Gly Asn Pro

485 490 495

acg ccc tcg ttc tct ccc tcg gcc tac cct ccc cgt ccc atc acc ttc 1536

Thr Pro Ser Phe Ser Pro Ser Ala Tyr Pro Pro Arg Pro Ile Thr Phe

500 505 510

acc ccg ttc ccc ggc aac aag aac gac aac aac cac gtg ccc ggc gag 1584

Thr Pro Phe Pro Gly Asn Lys Asn Asp Asn Asn His Val Pro Gly Glu

515 520 525

atg ccg ctc tcg tgg tac aac atg gct gag ttc atg gcc ggc aag gtc 1632

Met Pro Leu Ser Trp Tyr Asn Met Ala Glu Phe Met Ala Gly Lys Val

530 535 540

agc ctc tgc ctc ggc cct gag ttc gcc aag ttc gat gac tcc aac acc 1680

Ser Leu Cys Leu Gly Pro Glu Phe Ala Lys Phe Asp Asp Ser Asn Thr

545 550 555 560

agc cgc agc cct gca tgg gat ctt gct ctt gtg act cgt gtg gtc tcc 1728

Ser Arg Ser Pro Ala Trp Asp Leu Ala Leu Val Thr Arg Val Val Ser

565 570 575

gtt tct gac atg gag tgg gtc cag tgg aag aac gtg gac tgc aac ccg 1776

Val Ser Asp Met Glu Trp Val Gln Trp Lys Asn Val Asp Cys Asn Pro

580 585 590

tcc aag gga acc atg gtt ggc gag ttc gac tgc ccc atc gac gcc tgg 1824

Ser Lys Gly Thr Met Val Gly Glu Phe Asp Cys Pro Ile Asp Ala Trp

595 600 605

ttc ttc cag gga tct tgt aac gac ggc cac atg ccg tac tcc atc ctc 1872

Phe Phe Gln Gly Ser Cys Asn Asp Gly His Met Pro Tyr Ser Ile Leu

610 615 620

atg gag atc gcc ctc cag acc tct ggt gtc ctc acc tct gtg ctc aag 1920

Met Glu Ile Ala Leu Gln Thr Ser Gly Val Leu Thr Ser Val Leu Lys

625 630 635 640

gcc ccg ctc acc atg gag aag aag gac att ctc ttc cgc aac ctt gac 1968

Ala Pro Leu Thr Met Glu Lys Lys Asp Ile Leu Phe Arg Asn Leu Asp

645 650 655

gcc aac gcc gag atg gtt cgc tct gat att gac ctc cgc ggc aag acc 2016

Ala Asn Ala Glu Met Val Arg Ser Asp Ile Asp Leu Arg Gly Lys Thr

660 665 670

atc cac aac ctc acc aag tgt acc ggc tac agc atg ctc gga gac atg 2064

Ile His Asn Leu Thr Lys Cys Thr Gly Tyr Ser Met Leu Gly Asp Met

675 680 685

ggt gtc cac cgc ttc agc ttc gag ctc tct gtt gat ggt gta gtc ttc 2112

Gly Val His Arg Phe Ser Phe Glu Leu Ser Val Asp Gly Val Val Phe

690 695 700

tac aag ggt acc acc tcc ttc ggc tgg ttc gtc cct gag gtc ttc atc 2160

Tyr Lys Gly Thr Thr Ser Phe Gly Trp Phe Val Pro Glu Val Phe Ile

705 710 715 720

tcc cag act ggt ctc gac aac ggt cgc cgc acc cag ccc tgg cac att 2208

Ser Gln Thr Gly Leu Asp Asn Gly Arg Arg Thr Gln Pro Trp His Ile

725 730 735

gag tcc aag gtg cct tcc gcc cag gtc ctc acc tac gac gtt acc ccc 2256

Glu Ser Lys Val Pro Ser Ala Gln Val Leu Thr Tyr Asp Val Thr Pro

740 745 750

aac ggt gcc ggt cgc acc cag ctc tac gcc aac gct ccc aag ggt gct 2304

Asn Gly Ala Gly Arg Thr Gln Leu Tyr Ala Asn Ala Pro Lys Gly Ala

755 760 765

cag ctc agt cgc cgc tgg aac cag tgc cag tac ctt gac acc atc gac 2352

Gln Leu Ser Arg Arg Trp Asn Gln Cys Gln Tyr Leu Asp Thr Ile Asp

770 775 780

ctt gtg gtc gcc ggt ggc tcc gcc ggt ctt ggc tac ggt cat ggc cgc 2400

Leu Val Val Ala Gly Gly Ser Ala Gly Leu Gly Tyr Gly His Gly Arg

785 790 795 800

aag cag gtg aac ccc aag gac tgg ttc ttc tcg tgc cac ttc tgg ttc 2448

Lys Gln Val Asn Pro Lys Asp Trp Phe Phe Ser Cys His Phe Trp Phe

805 810 815

gac tcc gtc atg ccc ggc tcg ctc ggt gtg gag tct atg ttc cag ctc 2496

Asp Ser Val Met Pro Gly Ser Leu Gly Val Glu Ser Met Phe Gln Leu

820 825 830

gtc gag tcc atc gct gtc aag cag gac ctc gcc ggc aag tac ggc atc 2544

Val Glu Ser Ile Ala Val Lys Gln Asp Leu Ala Gly Lys Tyr Gly Ile

835 840 845

acc aac ccg acc ttc gct cat gct ccg ggc aag atc tcc tgg aag tac 2592

Thr Asn Pro Thr Phe Ala His Ala Pro Gly Lys Ile Ser Trp Lys Tyr

850 855 860

cgt ggt cag ctc acc ccc acc tcc aag ttc atg gac tcc gag gcc cac 2640

Arg Gly Gln Leu Thr Pro Thr Ser Lys Phe Met Asp Ser Glu Ala His

865 870 875 880

att gtc tcc atc gag gcc cac gac ggc gtc gtc gac atc gtt gcc aat 2688

Ile Val Ser Ile Glu Ala His Asp Gly Val Val Asp Ile Val Ala Asn

885 890 895

ggt aac ctc tgg gct gat ggc ctc cgc gtc tac aac gtc agc aac atc 2736

Gly Asn Leu Trp Ala Asp Gly Leu Arg Val Tyr Asn Val Ser Asn Ile

900 905 910

cgt gtt cgc att acc atc acc ctc aag cag ctc aag gct gag ctt ctt 2784

Arg Val Arg Ile Thr Ile Thr Leu Lys Gln Leu Lys Ala Glu Leu Leu

915 920 925

gac gtt gag aag cct ctc tac atc tcc tcc agc aac ggc cag gtc aag 2832

Asp Val Glu Lys Pro Leu Tyr Ile Ser Ser Ser Asn Gly Gln Val Lys

930 935 940

aag cac gcc gat gtg gct ggt ggc cag gcc acc att gtg cag gct tgc 2880

Lys His Ala Asp Val Ala Gly Gly Gln Ala Thr Ile Val Gln Ala Cys

945 950 955 960

agc ctc agt gac ctc ggt gat gaa ggc ttc atg aag acc tac ggt gtt 2928

Ser Leu Ser Asp Leu Gly Asp Glu Gly Phe Met Lys Thr Tyr Gly Val

965 970 975

gtg gct cct ctc tac acc ggt gcc atg gcc aag ggt att gcc tct gct 2976

Val Ala Pro Leu Tyr Thr Gly Ala Met Ala Lys Gly Ile Ala Ser Ala

980 985 990

gac ctt gtg att gcc act ggt aag cgt aag atc ctc ggt tcc ttc ggt 3024

Asp Leu Val Ile Ala Thr Gly Lys Arg Lys Ile Leu Gly Ser Phe Gly

995 1000 1005

gct ggt ggt ctc ccc atg cac att gtc cgt gcc gct gtt gag aag 3069

Ala Gly Gly Leu Pro Met His Ile Val Arg Ala Ala Val Glu Lys

1010 1015 1020

atc cag gct gag ctc ccg aac ggc ccc ttc gcc gtc aac ctc atc 3114

Ile Gln Ala Glu Leu Pro Asn Gly Pro Phe Ala Val Asn Leu Ile

1025 1030 1035

cac tcc ccc ttc gat agc aac ctt gag aag ggc aac gtt gac ctc 3159

His Ser Pro Phe Asp Ser Asn Leu Glu Lys Gly Asn Val Asp Leu

1040 1045 1050

ttc ctc gag aag ggc gtc act gtc gtc gag gcc tcc gcc ttc atg 3204

Phe Leu Glu Lys Gly Val Thr Val Val Glu Ala Ser Ala Phe Met

1055 1060 1065

acc ttg acc ccg caa gtc gtc cgc tac cgt gct gct ggt ctt tcc 3249

Thr Leu Thr Pro Gln Val Val Arg Tyr Arg Ala Ala Gly Leu Ser

1070 1075 1080

cgt aac gct gat ggc tcc att aac atc aag aac cgc atc atc ggt 3294

Arg Asn Ala Asp Gly Ser Ile Asn Ile Lys Asn Arg Ile Ile Gly

1085 1090 1095

aag gtc tcc cgt acc gag ctc gct gag atg ttc atc cgc cct gcc 3339

Lys Val Ser Arg Thr Glu Leu Ala Glu Met Phe Ile Arg Pro Ala

1100 1105 1110

ccg cag aac ctc ctc gac aag ctc atc cag tct ggt gag att acc 3384

Pro Gln Asn Leu Leu Asp Lys Leu Ile Gln Ser Gly Glu Ile Thr

1115 1120 1125

aag gag cag gct gag ctt gcc aag ctc gtc ccc gtc gcc gac gat 3429

Lys Glu Gln Ala Glu Leu Ala Lys Leu Val Pro Val Ala Asp Asp

1130 1135 1140

atc gcc gtc gag gcc gac tct ggt ggc cac acc gac aac cgc ccc 3474

Ile Ala Val Glu Ala Asp Ser Gly Gly His Thr Asp Asn Arg Pro

1145 1150 1155

atc cac gtc atc ctc ccc ctt atc atc aac ctc cgc aac cgc ctc 3519

Ile His Val Ile Leu Pro Leu Ile Ile Asn Leu Arg Asn Arg Leu

1160 1165 1170

cac aag gag tgc ggc tac ccc gct cac ctc cgc gtg cgc gtt gga 3564

His Lys Glu Cys Gly Tyr Pro Ala His Leu Arg Val Arg Val Gly

1175 1180 1185

gct ggt ggt ggt gtt gga tgc ccc cag gcc gct gcc gct gct ctc 3609

Ala Gly Gly Gly Val Gly Cys Pro Gln Ala Ala Ala Ala Ala Leu

1190 1195 1200

gct atg ggt gct gcc ttc ctt gtt acc ggc act gtc aac cag gtc 3654

Ala Met Gly Ala Ala Phe Leu Val Thr Gly Thr Val Asn Gln Val

1205 1210 1215

gcc aag cag tcc ggc acc tgc gac aat gtc cgc aag cag ctc tgc 3699

Ala Lys Gln Ser Gly Thr Cys Asp Asn Val Arg Lys Gln Leu Cys

1220 1225 1230

atg gcc acc tac tct gac gtc tgc atg gct ccc gct gct gac atg 3744

Met Ala Thr Tyr Ser Asp Val Cys Met Ala Pro Ala Ala Asp Met

1235 1240 1245

ttc gag gag ggc gtc aag ctc cag gtc ctc aag aag gga acc atg 3789

Phe Glu Glu Gly Val Lys Leu Gln Val Leu Lys Lys Gly Thr Met

1250 1255 1260

ttc ccg tcc agg gct aac aag ctc tac gag ctc ttc tgc aag tac 3834

Phe Pro Ser Arg Ala Asn Lys Leu Tyr Glu Leu Phe Cys Lys Tyr

1265 1270 1275

gac tcc ttc gag tcc atg cct gcc gca gag ctc gag cgt gtt gag 3879

Asp Ser Phe Glu Ser Met Pro Ala Ala Glu Leu Glu Arg Val Glu

1280 1285 1290

aag cgc atc ttc cag tgc cct ctt gct gat gtc tgg gct gag acc 3924

Lys Arg Ile Phe Gln Cys Pro Leu Ala Asp Val Trp Ala Glu Thr

1295 1300 1305

tcc gac ttc tac atc aac cgc ctc cac aac ccg gag aag atc acc 3969

Ser Asp Phe Tyr Ile Asn Arg Leu His Asn Pro Glu Lys Ile Thr

1310 1315 1320

cgt gcc gag cgt gac ccc aag ctc aag atg tct ctc tgc ttc cgc 4014

Arg Ala Glu Arg Asp Pro Lys Leu Lys Met Ser Leu Cys Phe Arg

1325 1330 1335

tgg tac ctt ggt ctt gcc tct cgc tgg gcc aac acc ggt gag gct 4059

Trp Tyr Leu Gly Leu Ala Ser Arg Trp Ala Asn Thr Gly Glu Ala

1340 1345 1350

gga cgc gtc atg gac tac cag gtc tgg tgt ggc cct gcc att gga 4104

Gly Arg Val Met Asp Tyr Gln Val Trp Cys Gly Pro Ala Ile Gly

1355 1360 1365

gcc ttc aac gac ttc atc aag ggc tcc tac ctt gac ccg gcc gtc 4149

Ala Phe Asn Asp Phe Ile Lys Gly Ser Tyr Leu Asp Pro Ala Val

1370 1375 1380

tct ggt gag tac ccg gac gtc gtg cag atc aac ttg cag atc ctt 4194

Ser Gly Glu Tyr Pro Asp Val Val Gln Ile Asn Leu Gln Ile Leu

1385 1390 1395

cgc ggt gcc tgc tac ctc cgc cgt ctc aat gcc atc cgc aac gac 4239

Arg Gly Ala Cys Tyr Leu Arg Arg Leu Asn Ala Ile Arg Asn Asp

1400 1405 1410

ccg cgt gtc agc att gag gtc gag gat gct gag ttc gtc tac gag 4284

Pro Arg Val Ser Ile Glu Val Glu Asp Ala Glu Phe Val Tyr Glu

1415 1420 1425

ccc acc aac gcc ctc taa 4302

Pro Thr Asn Ala Leu

1430

<210> 4

<211> 8733

<212> DNA

<213> 裂殖壶菌（Shizochytrium sp.）

<220>

<221> CDS

<222> (1)..(8733)

<400> 4

atg gcg gcc cgt ctg cag gag caa aag gga ggc gag atg gat acc cgc 48

Met Ala Ala Arg Leu Gln Glu Gln Lys Gly Gly Glu Met Asp Thr Arg

1 5 10 15

att gcc atc atc ggc atg tcg gcc atc ctc ccc tgc ggc acg acc gtg 96

Ile Ala Ile Ile Gly Met Ser Ala Ile Leu Pro Cys Gly Thr Thr Val

20 25 30

cgc gag tcg tgg gag acc atc cgc gcc ggc atc gac tgc ctg tcg gat 144

Arg Glu Ser Trp Glu Thr Ile Arg Ala Gly Ile Asp Cys Leu Ser Asp

35 40 45

ctc ccc gag gac cgc gtc gac gtg acg gcg tac ttt gac ccc gtc aag 192

Leu Pro Glu Asp Arg Val Asp Val Thr Ala Tyr Phe Asp Pro Val Lys

50 55 60

acc acc aag gac aag atc tac tgc aag cgc ggt ggc ttc att ccc gag 240

Thr Thr Lys Asp Lys Ile Tyr Cys Lys Arg Gly Gly Phe Ile Pro Glu

65 70 75 80

tac gac ttt gac gcc cgc gag ttc gga ctc aac atg ttc cag atg gag 288

Tyr Asp Phe Asp Ala Arg Glu Phe Gly Leu Asn Met Phe Gln Met Glu

85 90 95

gac tcg gac gca aac cag acc atc tcg ctt ctc aag gtc aag gag gcc 336

Asp Ser Asp Ala Asn Gln Thr Ile Ser Leu Leu Lys Val Lys Glu Ala

100 105 110

ctc cag gac gcc ggc atc gac gcc ctc ggc aag gaa aag aag aac atc 384

Leu Gln Asp Ala Gly Ile Asp Ala Leu Gly Lys Glu Lys Lys Asn Ile

115 120 125

ggc tgc gtg ctc ggc att ggc ggc ggc caa aag tcc agc cac gag ttc 432

Gly Cys Val Leu Gly Ile Gly Gly Gly Gln Lys Ser Ser His Glu Phe

130 135 140

tac tcg cgc ctt aat tat gtt gtc gtg gag aag gtc ctc cgc aag atg 480

Tyr Ser Arg Leu Asn Tyr Val Val Val Glu Lys Val Leu Arg Lys Met

145 150 155 160

ggc atg ccc gag gag gac gtc aag gtc gcc gtc gaa aag tac aag gcc 528

Gly Met Pro Glu Glu Asp Val Lys Val Ala Val Glu Lys Tyr Lys Ala

165 170 175

aac ttc ccc gag tgg cgc ctc gac tcc ttc cct ggc ttc ctc ggc aac 576

Asn Phe Pro Glu Trp Arg Leu Asp Ser Phe Pro Gly Phe Leu Gly Asn

180 185 190

gtc acc gcc ggt cgc tgc acc aac acc ttc aac ctc gac ggc atg aac 624

Val Thr Ala Gly Arg Cys Thr Asn Thr Phe Asn Leu Asp Gly Met Asn

195 200 205

tgc gtt gtc gac gcc gca tgc gcc tcg tcc ctc atc gcc gtc aag gtc 672

Cys Val Val Asp Ala Ala Cys Ala Ser Ser Leu Ile Ala Val Lys Val

210 215 220

gcc atc gac gag ctg ctc tac ggt gac tgc gac atg atg gtc acc ggt 720

Ala Ile Asp Glu Leu Leu Tyr Gly Asp Cys Asp Met Met Val Thr Gly

225 230 235 240

gcc acc tgc acg gat aac tcc atc ggc atg tac atg gcc ttc tcc aag 768

Ala Thr Cys Thr Asp Asn Ser Ile Gly Met Tyr Met Ala Phe Ser Lys

245 250 255

acc ccc gtg ttc tcc acg gac ccc agc gtg cgc gcc tac gac gaa aag 816

Thr Pro Val Phe Ser Thr Asp Pro Ser Val Arg Ala Tyr Asp Glu Lys

260 265 270

aca aag ggc atg ctc atc ggc gag ggc tcc gcc atg ctc gtc ctc aag 864

Thr Lys Gly Met Leu Ile Gly Glu Gly Ser Ala Met Leu Val Leu Lys

275 280 285

cgc tac gcc gac gcc gtc cgc gac ggc gat gag atc cac gct gtt att 912

Arg Tyr Ala Asp Ala Val Arg Asp Gly Asp Glu Ile His Ala Val Ile

290 295 300

cgc ggc tgc gcc tcc tcc agt gat ggc aag gcc gcc ggc atc tac acg 960

Arg Gly Cys Ala Ser Ser Ser Asp Gly Lys Ala Ala Gly Ile Tyr Thr

305 310 315 320

ccc acc att tcg ggc cag gag gag gcc ctc cgc cgc gcc tac aac cgc 1008

Pro Thr Ile Ser Gly Gln Glu Glu Ala Leu Arg Arg Ala Tyr Asn Arg

325 330 335

gcc tgt gtc gac ccg gcc acc gtc act ctc gtc gag ggt cac ggc acc 1056

Ala Cys Val Asp Pro Ala Thr Val Thr Leu Val Glu Gly His Gly Thr

340 345 350

ggt act ccc gtt ggc gac cgc atc gag ctc acc gcc ttg cgc aac ctc 1104

Gly Thr Pro Val Gly Asp Arg Ile Glu Leu Thr Ala Leu Arg Asn Leu

355 360 365

ttt gac aag gcc tac ggc gag ggc aac acc gaa aag gtc gct gtg ggc 1152

Phe Asp Lys Ala Tyr Gly Glu Gly Asn Thr Glu Lys Val Ala Val Gly

370 375 380

agc atc aag tcc agc atc ggc cat ctc aag gcc gtc gcc ggt ctc gcc 1200

Ser Ile Lys Ser Ser Ile Gly His Leu Lys Ala Val Ala Gly Leu Ala

385 390 395 400

ggt atg atc aag gtc atc atg gcg ctc aag cac aag act ctc ccg ggc 1248

Gly Met Ile Lys Val Ile Met Ala Leu Lys His Lys Thr Leu Pro Gly

405 410 415

acc atc aac gtc gac aac cca ccc aac ctc tac gac aac acg ccc atc 1296

Thr Ile Asn Val Asp Asn Pro Pro Asn Leu Tyr Asp Asn Thr Pro Ile

420 425 430

aac gag tcc tcg ctc tac att aac acc atg aac cgc ccc tgg ttc ccg 1344

Asn Glu Ser Ser Leu Tyr Ile Asn Thr Met Asn Arg Pro Trp Phe Pro

435 440 445

ccc cct ggt gtg ccc cgc cgc gcc ggc att tcg agc ttt ggc ttt ggt 1392

Pro Pro Gly Val Pro Arg Arg Ala Gly Ile Ser Ser Phe Gly Phe Gly

450 455 460

ggc gcc aac tac cac gcc gtc ctc gag gag gcc gag ccc gag cac acg 1440

Gly Ala Asn Tyr His Ala Val Leu Glu Glu Ala Glu Pro Glu His Thr

465 470 475 480

acc gcg tac cgc ctc aac aag cgc ccg cag ccc gtg ctc atg atg gcc 1488

Thr Ala Tyr Arg Leu Asn Lys Arg Pro Gln Pro Val Leu Met Met Ala

485 490 495

gcc acg ccc gcg gcc ctc cag tcg ctc tgc gag gcc cag ctc aag gag 1536

Ala Thr Pro Ala Ala Leu Gln Ser Leu Cys Glu Ala Gln Leu Lys Glu

500 505 510

ttc gag gcc gcc atc aag gag aac gag acc gtc aag aac acc gcc tac 1584

Phe Glu Ala Ala Ile Lys Glu Asn Glu Thr Val Lys Asn Thr Ala Tyr

515 520 525

atc aag tgc gtc aag ttc ggc gag cag ttc aaa ttc cct ggc tcc atc 1632

Ile Lys Cys Val Lys Phe Gly Glu Gln Phe Lys Phe Pro Gly Ser Ile

530 535 540

ccg gcc aca aac gcg cgc ctc ggc ttc ctc gtc aag gat gct gag gat 1680

Pro Ala Thr Asn Ala Arg Leu Gly Phe Leu Val Lys Asp Ala Glu Asp

545 550 555 560

gcc tgc tcc acc ctc cgt gcc atc tgc gcc caa ttc gcc aag gat gtc 1728

Ala Cys Ser Thr Leu Arg Ala Ile Cys Ala Gln Phe Ala Lys Asp Val

565 570 575

acc aag gag gcc tgg cgc ctc ccc cgc gag ggc gtc agc ttc cgc gcc 1776

Thr Lys Glu Ala Trp Arg Leu Pro Arg Glu Gly Val Ser Phe Arg Ala

580 585 590

aag ggc atc gcc acc aac ggc gct gtc gcc gcg ctc ttc tcc ggc cag 1824

Lys Gly Ile Ala Thr Asn Gly Ala Val Ala Ala Leu Phe Ser Gly Gln

595 600 605

ggc gcg cag tac acg cac atg ttt agc gag gtg gcc atg aac tgg ccc 1872

Gly Ala Gln Tyr Thr His Met Phe Ser Glu Val Ala Met Asn Trp Pro

610 615 620

cag ttc cgc cag agc att gcc gcc atg gac gcc gcc cag tcc aag gtc 1920

Gln Phe Arg Gln Ser Ile Ala Ala Met Asp Ala Ala Gln Ser Lys Val

625 630 635 640

gct gga agc gac aag gac ttt gag cgc gtc tcc cag gtc ctc tac ccg 1968

Ala Gly Ser Asp Lys Asp Phe Glu Arg Val Ser Gln Val Leu Tyr Pro

645 650 655

cgc aag ccg tac gag cgt gag ccc gag cag gac cac aag aag atc tcc 2016

Arg Lys Pro Tyr Glu Arg Glu Pro Glu Gln Asp His Lys Lys Ile Ser

660 665 670

ctc acc gcc tac tcg cag ccc tcg acc ctg gcc tgc gct ctc ggt gcc 2064

Leu Thr Ala Tyr Ser Gln Pro Ser Thr Leu Ala Cys Ala Leu Gly Ala

675 680 685

ttt gag atc ttc aag gag gcc ggc ttc acc ccg gac ttt gcc gcc ggc 2112

Phe Glu Ile Phe Lys Glu Ala Gly Phe Thr Pro Asp Phe Ala Ala Gly

690 695 700

cat tcg ctc ggt gag ttc gcc gcc ctc tac gcc gcg ggc tgc gtc gac 2160

His Ser Leu Gly Glu Phe Ala Ala Leu Tyr Ala Ala Gly Cys Val Asp

705 710 715 720

cgc gac gag ctc ttt gag ctt gtc tgc cgc cgc gcc cgc atc atg ggc 2208

Arg Asp Glu Leu Phe Glu Leu Val Cys Arg Arg Ala Arg Ile Met Gly

725 730 735

ggc aag gac gca ccg gcc acc ccc aag ggc tgc atg gcc gcc gtc att 2256

Gly Lys Asp Ala Pro Ala Thr Pro Lys Gly Cys Met Ala Ala Val Ile

740 745 750

ggc ccc aac gcc gag aac atc aag gtc cag gcc gcc aac gtc tgg ctc 2304

Gly Pro Asn Ala Glu Asn Ile Lys Val Gln Ala Ala Asn Val Trp Leu

755 760 765

ggc aac tcc aac tcg cct tcg cag acc gtc atc acc ggc tcc gtc gaa 2352

Gly Asn Ser Asn Ser Pro Ser Gln Thr Val Ile Thr Gly Ser Val Glu

770 775 780

ggt atc cag gcc gag agc gcc cgc ctc cag aag gag ggc ttc cgc gtc 2400

Gly Ile Gln Ala Glu Ser Ala Arg Leu Gln Lys Glu Gly Phe Arg Val

785 790 795 800

gtg cct ctt gcc tgc gag agc gcc ttc cac tcg ccc cag atg gag aac 2448

Val Pro Leu Ala Cys Glu Ser Ala Phe His Ser Pro Gln Met Glu Asn

805 810 815

gcc tcg tcg gcc ttc aag gac gtc atc tcc aag gtc tcc ttc cgc acc 2496

Ala Ser Ser Ala Phe Lys Asp Val Ile Ser Lys Val Ser Phe Arg Thr

820 825 830

ccc aag gcc gag acc aag ctc ttc agc aac gtc tct ggc gag acc tac 2544

Pro Lys Ala Glu Thr Lys Leu Phe Ser Asn Val Ser Gly Glu Thr Tyr

835 840 845

ccc acg gac gcc cgc gag atg ctt acg cag cac atg acc agc agc gtc 2592

Pro Thr Asp Ala Arg Glu Met Leu Thr Gln His Met Thr Ser Ser Val

850 855 860

aag ttc ctc acc cag gtc cgc aac atg cac cag gcc ggt gcg cgc atc 2640

Lys Phe Leu Thr Gln Val Arg Asn Met His Gln Ala Gly Ala Arg Ile

865 870 875 880

ttt gtc gag ttc gga ccc aag cag gtg ctc tcc aag ctt gtc tcc gag 2688

Phe Val Glu Phe Gly Pro Lys Gln Val Leu Ser Lys Leu Val Ser Glu

885 890 895

acc ctc aag gat gac ccc tcg gtt gtc acc gtc tct gtc aac ccg gcc 2736

Thr Leu Lys Asp Asp Pro Ser Val Val Thr Val Ser Val Asn Pro Ala

900 905 910

tcg ggc acg gat tcg gac atc cag ctc cgc gac gcg gcc gtc cag ctc 2784

Ser Gly Thr Asp Ser Asp Ile Gln Leu Arg Asp Ala Ala Val Gln Leu

915 920 925

gtt gtc gct ggc gtc aac ctt cag ggc ttt gac aag tgg gac gcc ccc 2832

Val Val Ala Gly Val Asn Leu Gln Gly Phe Asp Lys Trp Asp Ala Pro

930 935 940

gat gcc acc cgc atg cag gcc atc aag aag aag cgc act acc ctc cgc 2880

Asp Ala Thr Arg Met Gln Ala Ile Lys Lys Lys Arg Thr Thr Leu Arg

945 950 955 960

ctt tcg gcc gcc acc tac gtc tcg gac aag acc aag aag gtc cgc gac 2928

Leu Ser Ala Ala Thr Tyr Val Ser Asp Lys Thr Lys Lys Val Arg Asp

965 970 975

gcc gcc atg aac gat ggc cgc tgc gtc acc tac ctc aag ggc gcc gca 2976

Ala Ala Met Asn Asp Gly Arg Cys Val Thr Tyr Leu Lys Gly Ala Ala

980 985 990

ccg ctc atc aag gcc ccg gag ccc gtt gtc gac gag gcc gcc aag cgc 3024

Pro Leu Ile Lys Ala Pro Glu Pro Val Val Asp Glu Ala Ala Lys Arg

995 1000 1005

gag gcc gag cgt ctc cag aag gag ctt cag gat gcc cag cgc cag 3069

Glu Ala Glu Arg Leu Gln Lys Glu Leu Gln Asp Ala Gln Arg Gln

1010 1015 1020

ctc gac gac gcc aag cgc gcc gcc gcc gag gcc aac tcc aag ctc 3114

Leu Asp Asp Ala Lys Arg Ala Ala Ala Glu Ala Asn Ser Lys Leu

1025 1030 1035

gcc gct gcc aag gag gag gcc aag acc gcc gct gct tcg gcc aag 3159

Ala Ala Ala Lys Glu Glu Ala Lys Thr Ala Ala Ala Ser Ala Lys

1040 1045 1050

ccc gca gtt gac act gct gtt gtc gaa aag cat cgt gcc atc ctc 3204

Pro Ala Val Asp Thr Ala Val Val Glu Lys His Arg Ala Ile Leu

1055 1060 1065

aag tcc atg ctc gcg gag ctc gat ggc tac gga tcg gtc gac gct 3249

Lys Ser Met Leu Ala Glu Leu Asp Gly Tyr Gly Ser Val Asp Ala

1070 1075 1080

tct tcc ctc cag cag cag cag cag cag cag acg gcc ccc gcc ccg 3294

Ser Ser Leu Gln Gln Gln Gln Gln Gln Gln Thr Ala Pro Ala Pro

1085 1090 1095

gtc aag gct gct gcg cct gcc gcc ccc gtt gcc tcg gcc cct gcc 3339

Val Lys Ala Ala Ala Pro Ala Ala Pro Val Ala Ser Ala Pro Ala

1100 1105 1110

ccg gct gtc tcg aac gag ctt ctt gag aag gcc gag act gtc gtc 3384

Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr Val Val

1115 1120 1125

atg gag gtc ctc gcc gcc aag acc ggc tac gag acc gac atg atc 3429

Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile

1130 1135 1140

gag gct gac atg gag ctc gag acc gag ctc ggc att gac tcc atc 3474

Glu Ala Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile

1145 1150 1155

aag cgt gtc gag atc ctc tcc gag gtc cag gcc atg ctc aat gtc 3519

Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val

1160 1165 1170

gag gcc aag gat gtc gat gcc ctc agc cgc act cgc act gtt ggt 3564

Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val Gly

1175 1180 1185

gag gtt gtc aac gcc atg aag gcc gag atc gct ggc agc tct gcc 3609

Glu Val Val Asn Ala Met Lys Ala Glu Ile Ala Gly Ser Ser Ala

1190 1195 1200

ccg gcg cct gct gcc gct gct ccg gct ccg gcc aag gct gcc cct 3654

Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Lys Ala Ala Pro

1205 1210 1215

gcc gcc gct gcg cct gct gtc tcg aac gag ctt ctc gag aag gcc 3699

Ala Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala

1220 1225 1230

gag acc gtc gtc atg gag gtc ctc gcc gcc aag act ggc tac gag 3744

Glu Thr Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu

1235 1240 1245

act gac atg atc gag tcc gac atg gag ctc gag act gag ctc ggc 3789

Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly

1250 1255 1260

att gac tcc atc aag cgt gtc gag atc ctc tcc gag gtt cag gcc 3834

Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala

1265 1270 1275

atg ctc aac gtc gag gcc aag gac gtc gac gct ctc agc cgc act 3879

Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr

1280 1285 1290

cgc act gtg ggt gag gtc gtc aac gcc atg aag gct gag atc gct 3924

Arg Thr Val Gly Glu Val Val Asn Ala Met Lys Ala Glu Ile Ala

1295 1300 1305

ggt ggc tct gcc ccg gcg cct gcc gcc gct gcc cca ggt ccg gct 3969

Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Gly Pro Ala

1310 1315 1320

gct gcc gcc cct gcg cct gcc gcc gcc gcc cct gct gtc tcg aac 4014

Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Asn

1325 1330 1335

gag ctt ctt gag aag gcc gag acc gtc gtc atg gag gtc ctc gcc 4059

Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala

1340 1345 1350

gcc aag act ggc tac gag act gac atg atc gag tcc gac atg gag 4104

Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu

1355 1360 1365

ctc gag acc gag ctc ggc att gac tcc atc aag cgt gtc gag att 4149

Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile

1370 1375 1380

ctc tcc gag gtc cag gcc atg ctc aac gtc gag gcc aag gac gtc 4194

Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val

1385 1390 1395

gac gct ctc agc cgc acc cgc act gtt ggc gag gtc gtc gat gcc 4239

Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala

1400 1405 1410

atg aag gcc gag atc gct ggt ggc tct gcc ccg gcg cct gcc gcc 4284

Met Lys Ala Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala

1415 1420 1425

gct gct cct gct ccg gct gct gcc gcc cct gcg cct gcc gcc cct 4329

Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Pro

1430 1435 1440

gcg cct gct gtc tcg agc gag ctt ctc gag aag gcc gag act gtc 4374

Ala Pro Ala Val Ser Ser Glu Leu Leu Glu Lys Ala Glu Thr Val

1445 1450 1455

gtc atg gag gtc ctc gcc gcc aag act ggc tac gag act gac atg 4419

Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met

1460 1465 1470

atc gag tcc gac atg gag ctc gag acc gag ctc ggc att gac tcc 4464

Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser

1475 1480 1485

atc aag cgt gtc gag att ctc tcc gag gtc cag gcc atg ctc aac 4509

Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn

1490 1495 1500

gtc gag gcc aag gac gtc gac gct ctc agc cgc acc cgc act gtt 4554

Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val

1505 1510 1515

ggc gag gtc gtc gat gcc atg aag gcc gag atc gct ggt ggc tct 4599

Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly Gly Ser

1520 1525 1530

gcc ccg gcg cct gcc gcc gct gct cct gct ccg gct gct gcc gcc 4644

Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala

1535 1540 1545

cct gcg cct gcc gcc cct gcg cct gcc gcc cct gcg cct gct gtc 4689

Pro Ala Pro Ala Ala Pro Ala Pro Ala Ala Pro Ala Pro Ala Val

1550 1555 1560

tcg agc gag ctt ctc gag aag gcc gag act gtc gtc atg gag gtc 4734

Ser Ser Glu Leu Leu Glu Lys Ala Glu Thr Val Val Met Glu Val

1565 1570 1575

ctc gcc gcc aag act ggc tac gag act gac atg att gag tcc gac 4779

Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp

1580 1585 1590

atg gag ctc gag acc gag ctc ggc att gac tcc atc aag cgt gtc 4824

Met Glu Leu Glu Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val

1595 1600 1605

gag att ctc tcc gag gtt cag gcc atg ctc aac gtc gag gcc aag 4869

Glu Ile Leu Ser Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys

1610 1615 1620

gac gtc gac gct ctc agc cgc act cgc act gtt ggt gag gtc gtc 4914

Asp Val Asp Ala Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val

1625 1630 1635

gat gcc atg aag gct gag atc gct ggc agc tcc gcc tcg gcg cct 4959

Asp Ala Met Lys Ala Glu Ile Ala Gly Ser Ser Ala Ser Ala Pro

1640 1645 1650

gcc gcc gct gct cct gct ccg gct gct gcc gct cct gcg ccc gct 5004

Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala

1655 1660 1665

gcc gcc gcc cct gct gtc tcg aac gag ctt ctc gag aaa gcc gag 5049

Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu

1670 1675 1680

act gtc gtc atg gag gtc ctc gcc gcc aag act ggc tac gag act 5094

Thr Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr

1685 1690 1695

gac atg atc gag tcc gac atg gag ctc gag act gag ctc ggc att 5139

Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile

1700 1705 1710

gac tcc atc aag cgt gtc gag atc ctc tcc gag gtt cag gcc atg 5184

Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met

1715 1720 1725

ctc aac gtc gag gcc aag gac gtc gat gcc ctc agc cgc acc cgc 5229

Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg

1730 1735 1740

act gtt ggc gag gtt gtc gat gcc atg aag gcc gag atc gct ggt 5274

Thr Val Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly

1745 1750 1755

ggc tct gcc ccg gcg cct gcc gcc gct gcc cct gct ccg gct gcc 5319

Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala

1760 1765 1770

gcc gcc cct gct gtc tcg aac gag ctt ctc gag aag gcc gag act 5364

Ala Ala Pro Ala Val Ser Asn Glu Leu Leu Glu Lys Ala Glu Thr

1775 1780 1785

gtc gtc atg gag gtc ctc gcc gcc aag act ggc tac gag acc gac 5409

Val Val Met Glu Val Leu Ala Ala Lys Thr Gly Tyr Glu Thr Asp

1790 1795 1800

atg atc gag tcc gac atg gag ctc gag acc gag ctc ggc att gac 5454

Met Ile Glu Ser Asp Met Glu Leu Glu Thr Glu Leu Gly Ile Asp

1805 1810 1815

tcc atc aag cgt gtc gag att ctc tcc gag gtt cag gcc atg ctc 5499

Ser Ile Lys Arg Val Glu Ile Leu Ser Glu Val Gln Ala Met Leu

1820 1825 1830

aac gtc gag gcc aag gac gtc gat gct ctc agc cgc act cgc act 5544

Asn Val Glu Ala Lys Asp Val Asp Ala Leu Ser Arg Thr Arg Thr

1835 1840 1845

gtt ggc gag gtc gtc gat gcc atg aag gct gag atc gcc ggc agc 5589

Val Gly Glu Val Val Asp Ala Met Lys Ala Glu Ile Ala Gly Ser

1850 1855 1860

tcc gcc ccg gcg cct gcc gcc gct gct cct gct ccg gct gct gcc 5634

Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Pro Ala Ala Ala

1865 1870 1875

gct cct gcg ccc gct gcc gct gcc cct gct gtc tcg agc gag ctt 5679

Ala Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Ser Glu Leu

1880 1885 1890

ctc gag aag gcc gag acc gtc gtc atg gag gtc ctc gcc gcc aag 5724

Leu Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala Ala Lys

1895 1900 1905

act ggc tac gag act gac atg att gag tcc gac atg gag ctc gag 5769

Thr Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu

1910 1915 1920

act gag ctc ggc att gac tcc atc aag cgt gtc gag atc ctc tcc 5814

Thr Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser

1925 1930 1935

gag gtt cag gcc atg ctc aac gtc gag gcc aag gac gtc gat gcc 5859

Glu Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala

1940 1945 1950

ctc agc cgc acc cgc act gtt ggc gag gtt gtc gat gcc atg aag 5904

Leu Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala Met Lys

1955 1960 1965

gcc gag atc gct ggt ggc tct gcc ccg gcg cct gcc gcc gct gcc 5949

Ala Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala

1970 1975 1980

cct gct ccg gct gcc gcc gcc cct gct gtc tcg aac gag ctt ctt 5994

Pro Ala Pro Ala Ala Ala Ala Pro Ala Val Ser Asn Glu Leu Leu

1985 1990 1995

gag aag gcc gag acc gtc gtc atg gag gtc ctc gcc gcc aag act 6039

Glu Lys Ala Glu Thr Val Val Met Glu Val Leu Ala Ala Lys Thr

2000 2005 2010

ggc tac gag acc gac atg atc gag tcc gac atg gag ctc gag acc 6084

Gly Tyr Glu Thr Asp Met Ile Glu Ser Asp Met Glu Leu Glu Thr

2015 2020 2025

gag ctc ggc att gac tcc atc aag cgt gtc gag att ctc tcc gag 6129

Glu Leu Gly Ile Asp Ser Ile Lys Arg Val Glu Ile Leu Ser Glu

2030 2035 2040

gtt cag gcc atg ctc aac gtc gag gcc aag gac gtc gac gct ctc 6174

Val Gln Ala Met Leu Asn Val Glu Ala Lys Asp Val Asp Ala Leu

2045 2050 2055

agc cgc act cgc act gtt ggc gag gtc gtc gat gcc atg aag gct 6219

Ser Arg Thr Arg Thr Val Gly Glu Val Val Asp Ala Met Lys Ala

2060 2065 2070

gag atc gct ggt ggc tct gcc ccg gcg cct gcc gcc gct gct cct 6264

Glu Ile Ala Gly Gly Ser Ala Pro Ala Pro Ala Ala Ala Ala Pro

2075 2080 2085

gcc tcg gct ggc gcc gcg cct gcg gtc aag att gac tcg gtc cac 6309

Ala Ser Ala Gly Ala Ala Pro Ala Val Lys Ile Asp Ser Val His

2090 2095 2100

ggc gct gac tgt gat gat ctt tcc ctg atg cac gcc aag gtg gtt 6354

Gly Ala Asp Cys Asp Asp Leu Ser Leu Met His Ala Lys Val Val

2105 2110 2115

gac atc cgc cgc ccg gac gag ctc atc ctg gag cgc ccc gag aac 6399

Asp Ile Arg Arg Pro Asp Glu Leu Ile Leu Glu Arg Pro Glu Asn

2120 2125 2130

cgc ccc gtt ctc gtt gtc gat gac ggc agc gag ctc acc ctc gcc 6444

Arg Pro Val Leu Val Val Asp Asp Gly Ser Glu Leu Thr Leu Ala

2135 2140 2145

ctg gtc cgc gtc ctc ggc gcc tgc gcc gtt gtc ctg acc ttt gag 6489

Leu Val Arg Val Leu Gly Ala Cys Ala Val Val Leu Thr Phe Glu

2150 2155 2160

ggt ctc cag ctc gct cag cgc gct ggt gcc gct gcc atc cgc cac 6534

Gly Leu Gln Leu Ala Gln Arg Ala Gly Ala Ala Ala Ile Arg His

2165 2170 2175

gtg ctc gcc aag gat ctt tcc gcg gag agc gcc gag aag gcc atc 6579

Val Leu Ala Lys Asp Leu Ser Ala Glu Ser Ala Glu Lys Ala Ile

2180 2185 2190

aag gag gcc gag cag cgc ttt ggc gct ctc ggc ggc ttc atc tcg 6624

Lys Glu Ala Glu Gln Arg Phe Gly Ala Leu Gly Gly Phe Ile Ser

2195 2200 2205

cag cag gcg gag cgc ttc gag ccc gcc gaa atc ctc ggc ttc acg 6669

Gln Gln Ala Glu Arg Phe Glu Pro Ala Glu Ile Leu Gly Phe Thr

2210 2215 2220

ctc atg tgc gcc aag ttc gcc aag gct tcc ctc tgc acg gct gtg 6714

Leu Met Cys Ala Lys Phe Ala Lys Ala Ser Leu Cys Thr Ala Val

2225 2230 2235

gct ggc ggc cgc ccg gcc ttt atc ggt gtg gcg cgc ctt gac ggc 6759

Ala Gly Gly Arg Pro Ala Phe Ile Gly Val Ala Arg Leu Asp Gly

2240 2245 2250

cgc ctc gga ttc act tcg cag ggc act tct gac gcg ctc aag cgt 6804

Arg Leu Gly Phe Thr Ser Gln Gly Thr Ser Asp Ala Leu Lys Arg

2255 2260 2265

gcc cag cgt ggt gcc atc ttt ggc ctc tgc aag acc atc ggc ctc 6849

Ala Gln Arg Gly Ala Ile Phe Gly Leu Cys Lys Thr Ile Gly Leu

2270 2275 2280

gag tgg tcc gag tct gac gtc ttt tcc cgc ggc gtg gac att gct 6894

Glu Trp Ser Glu Ser Asp Val Phe Ser Arg Gly Val Asp Ile Ala

2285 2290 2295

cag ggc atg cac ccc gag gat gcc gcc gtg gcg att gtg cgc gag 6939

Gln Gly Met His Pro Glu Asp Ala Ala Val Ala Ile Val Arg Glu

2300 2305 2310

atg gcg tgc gct gac att cgc att cgc gag gtc ggc att ggc gca 6984

Met Ala Cys Ala Asp Ile Arg Ile Arg Glu Val Gly Ile Gly Ala

2315 2320 2325

aac cag cag cgc tgc acg atc cgt gcc gcc aag ctc gag acc ggc 7029

Asn Gln Gln Arg Cys Thr Ile Arg Ala Ala Lys Leu Glu Thr Gly

2330 2335 2340

aac ccg cag cgc cag atc gcc aag gac gac gtg ctg ctc gtt tct 7074

Asn Pro Gln Arg Gln Ile Ala Lys Asp Asp Val Leu Leu Val Ser

2345 2350 2355

ggc ggc gct cgc ggc atc acg cct ctt tgc atc cgg gag atc acg 7119

Gly Gly Ala Arg Gly Ile Thr Pro Leu Cys Ile Arg Glu Ile Thr

2360 2365 2370

cgc cag atc gcg ggc ggc aag tac att ctg ctt ggc cgc agc aag 7164

Arg Gln Ile Ala Gly Gly Lys Tyr Ile Leu Leu Gly Arg Ser Lys

2375 2380 2385

gtc tct gcg agc gaa ccg gca tgg tgc gct ggc atc act gac gag 7209

Val Ser Ala Ser Glu Pro Ala Trp Cys Ala Gly Ile Thr Asp Glu

2390 2395 2400

aag gct gtg caa aag gct gct acc cag gag ctc aag cgc gcc ttt 7254

Lys Ala Val Gln Lys Ala Ala Thr Gln Glu Leu Lys Arg Ala Phe

2405 2410 2415

agc gct ggc gag ggc ccc aag ccc acg ccc cgc gct gtc act aag 7299

Ser Ala Gly Glu Gly Pro Lys Pro Thr Pro Arg Ala Val Thr Lys

2420 2425 2430

ctt gtg ggc tct gtt ctt ggc gct cgc gag gtg cgc agc tct att 7344

Leu Val Gly Ser Val Leu Gly Ala Arg Glu Val Arg Ser Ser Ile

2435 2440 2445

gct gcg att gaa gcg ctc ggc ggc aag gcc atc tac tcg tcg tgc 7389

Ala Ala Ile Glu Ala Leu Gly Gly Lys Ala Ile Tyr Ser Ser Cys

2450 2455 2460

gac gtg aac tct gcc gcc gac gtg gcc aag gcc gtg cgc gat gcc 7434

Asp Val Asn Ser Ala Ala Asp Val Ala Lys Ala Val Arg Asp Ala

2465 2470 2475

gag tcc cag ctc ggt gcc cgc gtc tcg ggc atc gtt cat gcc tcg 7479

Glu Ser Gln Leu Gly Ala Arg Val Ser Gly Ile Val His Ala Ser

2480 2485 2490

ggc gtg ctc cgc gac cgt ctc atc gag aag aag ctc ccc gac gag 7524

Gly Val Leu Arg Asp Arg Leu Ile Glu Lys Lys Leu Pro Asp Glu

2495 2500 2505

ttc gac gcc gtc ttt ggc acc aag gtc acc ggt ctc gag aac ctc 7569

Phe Asp Ala Val Phe Gly Thr Lys Val Thr Gly Leu Glu Asn Leu

2510 2515 2520

ctc gcc gcc gtc gac cgc gcc aac ctc aag cac atg gtc ctc ttc 7614

Leu Ala Ala Val Asp Arg Ala Asn Leu Lys His Met Val Leu Phe

2525 2530 2535

agc tcg ctc gcc ggc ttc cac ggc aac gtc ggc cag tct gac tac 7659

Ser Ser Leu Ala Gly Phe His Gly Asn Val Gly Gln Ser Asp Tyr

2540 2545 2550

gcc atg gcc aac gag gcc ctt aac aag atg ggc ctc gag ctc gcc 7704

Ala Met Ala Asn Glu Ala Leu Asn Lys Met Gly Leu Glu Leu Ala

2555 2560 2565

aag gac gtc tcg gtc aag tcg atc tgc ttc ggt ccc tgg gac ggt 7749

Lys Asp Val Ser Val Lys Ser Ile Cys Phe Gly Pro Trp Asp Gly

2570 2575 2580

ggc atg gtg acg ccg cag ctc aag aag cag ttc cag gag atg ggc 7794

Gly Met Val Thr Pro Gln Leu Lys Lys Gln Phe Gln Glu Met Gly

2585 2590 2595

gtg cag atc atc ccc cgc gag ggc ggc gct gat acc gtg gcg cgc 7839

Val Gln Ile Ile Pro Arg Glu Gly Gly Ala Asp Thr Val Ala Arg

2600 2605 2610

atc gtg ctc ggc tcc tcg ccg gct gag atc ctt gtc ggc aac tgg 7884

Ile Val Leu Gly Ser Ser Pro Ala Glu Ile Leu Val Gly Asn Trp

2615 2620 2625

cgc acc ccg tcc aag aag gtc ggc tcg gac acc atc acc ctg cac 7929

Arg Thr Pro Ser Lys Lys Val Gly Ser Asp Thr Ile Thr Leu His

2630 2635 2640

cgc aag att tcc gcc aag tcc aac ccc ttc ctc gag gac cac gtc 7974

Arg Lys Ile Ser Ala Lys Ser Asn Pro Phe Leu Glu Asp His Val

2645 2650 2655

atc cag ggc cgc cgc gtg ctg ccc atg acg ctg gcc att ggc tcg 8019

Ile Gln Gly Arg Arg Val Leu Pro Met Thr Leu Ala Ile Gly Ser

2660 2665 2670

ctc gcg gag acc tgc ctc ggc ctc ttc ccc ggc tac tcg ctc tgg 8064

Leu Ala Glu Thr Cys Leu Gly Leu Phe Pro Gly Tyr Ser Leu Trp

2675 2680 2685

gcc att gac gac gcc cag ctc ttc aag ggt gtc act gtc gac ggc 8109

Ala Ile Asp Asp Ala Gln Leu Phe Lys Gly Val Thr Val Asp Gly

2690 2695 2700

gac gtc aac tgc gag gtg acc ctc acc ccg tcg acg gcg ccc tcg 8154

Asp Val Asn Cys Glu Val Thr Leu Thr Pro Ser Thr Ala Pro Ser

2705 2710 2715

ggc cgc gtc aac gtc cag gcc acg ctc aag acc ttt tcc agc ggc 8199

Gly Arg Val Asn Val Gln Ala Thr Leu Lys Thr Phe Ser Ser Gly

2720 2725 2730

aag ctg gtc ccg gcc tac cgc gcc gtc atc gtg ctc tcc aac cag 8244

Lys Leu Val Pro Ala Tyr Arg Ala Val Ile Val Leu Ser Asn Gln

2735 2740 2745

ggc gcg ccc ccg gcc aac gcc acc atg cag ccg ccc tcg ctc gat 8289

Gly Ala Pro Pro Ala Asn Ala Thr Met Gln Pro Pro Ser Leu Asp

2750 2755 2760

gcc gat ccg gcg ctc cag ggc tcc gtc tac gac ggc aag acc ctc 8334

Ala Asp Pro Ala Leu Gln Gly Ser Val Tyr Asp Gly Lys Thr Leu

2765 2770 2775

ttc cac ggc ccg gcc ttc cgc ggc atc gat gac gtg ctc tcg tgc 8379

Phe His Gly Pro Ala Phe Arg Gly Ile Asp Asp Val Leu Ser Cys

2780 2785 2790

acc aag agc cag ctt gtg gcc aag tgc agc gct gtc ccc ggc tcc 8424

Thr Lys Ser Gln Leu Val Ala Lys Cys Ser Ala Val Pro Gly Ser

2795 2800 2805

gac gcc gct cgc ggc gag ttt gcc acg gac act gac gcc cat gac 8469

Asp Ala Ala Arg Gly Glu Phe Ala Thr Asp Thr Asp Ala His Asp

2810 2815 2820

ccc ttc gtg aac gac ctg gcc ttt cag gcc atg ctc gtc tgg gtg 8514

Pro Phe Val Asn Asp Leu Ala Phe Gln Ala Met Leu Val Trp Val

2825 2830 2835

cgc cgc acg ctc ggc cag gct gcg ctc ccc aac tcg atc cag cgc 8559

Arg Arg Thr Leu Gly Gln Ala Ala Leu Pro Asn Ser Ile Gln Arg

2840 2845 2850

atc gtc cag cac cgc ccg gtc ccg cag gac aag ccc ttc tac att 8604

Ile Val Gln His Arg Pro Val Pro Gln Asp Lys Pro Phe Tyr Ile

2855 2860 2865

acc ctc cgc tcc aac cag tcg ggc ggt cac tcc cag cac aag cac 8649

Thr Leu Arg Ser Asn Gln Ser Gly Gly His Ser Gln His Lys His

2870 2875 2880

gcc ctt cag ttc cac aac gag cag ggc gat ctc ttc att gat gtc 8694

Ala Leu Gln Phe His Asn Glu Gln Gly Asp Leu Phe Ile Asp Val

2885 2890 2895

cag gct tcg gtc atc gcc acg gac agc ctt gcc ttc taa 8733

Gln Ala Ser Val Ile Ala Thr Asp Ser Leu Ala Phe

2900 2905 2910

<210> 5

<211> 714

<212> DNA

<213> 念珠藻PCC7120（Nostoc sp. PCC7120）

<220>

<221> CDS

<222> (1)..(714)

<400> 5

atg ctg caa cat acc tgg ctg ccg aaa ccg cct aat ctg acc ctg ctg 48

Met Leu Gln His Thr Trp Leu Pro Lys Pro Pro Asn Leu Thr Leu Leu

1 5 10 15

agt gat gaa gtt cat ctg tgg cgt att ccg ctg gat cag ccg gaa agc 96

Ser Asp Glu Val His Leu Trp Arg Ile Pro Leu Asp Gln Pro Glu Ser

20 25 30

cag ctg caa gac ctg gca gca acc ctg agc agt gat gaa ctg gca cgt 144

Gln Leu Gln Asp Leu Ala Ala Thr Leu Ser Ser Asp Glu Leu Ala Arg

35 40 45

gca aat cgt ttc tat ttt ccg gaa cat cgt cgt cgt ttt acc gca ggt 192

Ala Asn Arg Phe Tyr Phe Pro Glu His Arg Arg Arg Phe Thr Ala Gly

50 55 60

cgt ggt att ctg cgt agc att ctg ggt ggt tat ctg ggt gtt gaa ccg 240

Arg Gly Ile Leu Arg Ser Ile Leu Gly Gly Tyr Leu Gly Val Glu Pro

65 70 75 80

ggt cag gtt aaa ttt gat tat gaa agc cgt ggt aaa ccg att ctg ggc 288

Gly Gln Val Lys Phe Asp Tyr Glu Ser Arg Gly Lys Pro Ile Leu Gly

85 90 95

gat cgt ttt gca gaa agc ggt ctg ctg ttt aat ctg agc cat agc cag 336

Asp Arg Phe Ala Glu Ser Gly Leu Leu Phe Asn Leu Ser His Ser Gln

100 105 110

aat ctg gca ctg tgt gca gtg aat tat acc cgt cag att ggt atc gat 384

Asn Leu Ala Leu Cys Ala Val Asn Tyr Thr Arg Gln Ile Gly Ile Asp

115 120 125

ctg gaa tat ctg cgt ccg acc agt gat ctg gaa agc ctg gcc aaa cgt 432

Leu Glu Tyr Leu Arg Pro Thr Ser Asp Leu Glu Ser Leu Ala Lys Arg

130 135 140

ttt ttt ctg cct cgt gaa tat gaa ctg ctg cgt agc ctg ccg gat gaa 480

Phe Phe Leu Pro Arg Glu Tyr Glu Leu Leu Arg Ser Leu Pro Asp Glu

145 150 155 160

cag aaa cag aaa atc ttt ttt cgt tat tgg acc tgc aaa gaa gcc tat 528

Gln Lys Gln Lys Ile Phe Phe Arg Tyr Trp Thr Cys Lys Glu Ala Tyr

165 170 175

ctg aaa gca acc ggt gat ggt att gca aaa ctg gaa gaa att gaa att 576

Leu Lys Ala Thr Gly Asp Gly Ile Ala Lys Leu Glu Glu Ile Glu Ile

180 185 190

gca ctg acc ccg acc gaa ccg gca aaa ctg caa acc gca ccg gca tgg 624

Ala Leu Thr Pro Thr Glu Pro Ala Lys Leu Gln Thr Ala Pro Ala Trp

195 200 205

tca ctg ctg gaa ctg gtt ccg gat gat aat tgt gtt gca gcc gtt gca 672

Ser Leu Leu Glu Leu Val Pro Asp Asp Asn Cys Val Ala Ala Val Ala

210 215 220

gtt gca ggt ttt ggt tgg cag ccg aaa ttt tgg cat tat taa 714

Val Ala Gly Phe Gly Trp Gln Pro Lys Phe Trp His Tyr

225 230 235

<210> 6

<211> 814

<212> PRT

<213> 大肠杆菌（Escherichia coli）

<400> 6

Met Met Ile Leu Ser Ile Leu Ala Thr Val Val Leu Leu Gly Ala Leu

1 5 10 15

Phe Tyr His Arg Val Ser Leu Phe Ile Ser Ser Leu Ile Leu Leu Ala

20 25 30

Trp Thr Ala Ala Leu Gly Val Ala Gly Leu Trp Ser Ala Trp Val Leu

35 40 45

Val Pro Leu Ala Ile Ile Leu Val Pro Phe Asn Phe Ala Pro Met Arg

50 55 60

Lys Ser Met Ile Ser Ala Pro Val Phe Arg Gly Phe Arg Lys Val Met

65 70 75 80

Pro Pro Met Ser Arg Thr Glu Lys Glu Ala Ile Asn Ala Gly Thr Thr

85 90 95

Trp Trp Glu Gly Asp Leu Phe Gln Gly Lys Pro Asp Trp Lys Lys Leu

100 105 110

His Asn Tyr Pro Gln Pro Arg Leu Thr Ala Glu Glu Gln Ala Phe Leu

115 120 125

Asp Gly Pro Val Glu Glu Ala Cys Arg Met Ala Asn Asp Phe Gln Ile

130 135 140

Thr His Glu Leu Ala Asp Leu Pro Pro Glu Leu Trp Ala Tyr Leu Lys

145 150 155 160

Glu His Arg Phe Phe Ala Met Ile Ile Lys Lys Glu Tyr Gly Gly Leu

165 170 175

Glu Phe Ser Ala Tyr Ala Gln Ser Arg Val Leu Gln Lys Leu Ser Gly

180 185 190

Val Ser Gly Ile Leu Ala Ile Thr Val Gly Val Pro Asn Ser Leu Gly

195 200 205

Pro Gly Glu Leu Leu Gln His Tyr Gly Thr Asp Glu Gln Lys Asp His

210 215 220

Tyr Leu Pro Arg Leu Ala Arg Gly Gln Glu Ile Pro Cys Phe Ala Leu

225 230 235 240

Thr Ser Pro Glu Ala Gly Ser Asp Ala Gly Ala Ile Pro Asp Thr Gly

245 250 255

Ile Val Cys Met Gly Glu Trp Gln Gly Gln Gln Val Leu Gly Met Arg

260 265 270

Leu Thr Trp Asn Lys Arg Tyr Ile Thr Leu Ala Pro Ile Ala Thr Val

275 280 285

Leu Gly Leu Ala Phe Lys Leu Ser Asp Pro Glu Lys Leu Leu Gly Gly

290 295 300

Ala Glu Asp Leu Gly Ile Thr Cys Ala Leu Ile Pro Thr Thr Thr Pro

305 310 315 320

Gly Val Glu Ile Gly Arg Arg His Phe Pro Leu Asn Val Pro Phe Gln

325 330 335

Asn Gly Pro Thr Arg Gly Lys Asp Val Phe Val Pro Ile Asp Tyr Ile

340 345 350

Ile Gly Gly Pro Lys Met Ala Gly Gln Gly Trp Arg Met Leu Val Glu

355 360 365

Cys Leu Ser Val Gly Arg Gly Ile Thr Leu Pro Ser Asn Ser Thr Gly

370 375 380

Gly Val Lys Ser Val Ala Leu Ala Thr Gly Ala Tyr Ala His Ile Arg

385 390 395 400

Arg Gln Phe Lys Ile Ser Ile Gly Lys Met Glu Gly Ile Glu Glu Pro

405 410 415

Leu Ala Arg Ile Ala Gly Asn Ala Tyr Val Met Asp Ala Ala Ala Ser

420 425 430

Leu Ile Thr Tyr Gly Ile Met Leu Gly Glu Lys Pro Ala Val Leu Ser

435 440 445

Ala Ile Val Lys Tyr His Cys Thr His Arg Gly Gln Gln Ser Ile Ile

450 455 460

Asp Ala Met Asp Ile Thr Gly Gly Lys Gly Ile Met Leu Gly Gln Ser

465 470 475 480

Asn Phe Leu Ala Arg Ala Tyr Gln Gly Ala Pro Ile Ala Ile Thr Val

485 490 495

Glu Gly Ala Asn Ile Leu Thr Arg Ser Met Met Ile Phe Gly Gln Gly

500 505 510

Ala Ile Arg Cys His Pro Tyr Val Leu Glu Glu Met Glu Ala Ala Lys

515 520 525

Asn Asn Asp Val Asn Ala Phe Asp Lys Leu Leu Phe Lys His Ile Gly

530 535 540

His Val Gly Ser Asn Lys Val Arg Ser Phe Trp Leu Gly Leu Thr Arg

545 550 555 560

Gly Leu Thr Ser Ser Thr Pro Thr Gly Asp Ala Thr Lys Arg Tyr Tyr

565 570 575

Gln His Leu Asn Arg Leu Ser Ala Asn Leu Ala Leu Leu Ser Asp Val

580 585 590

Ser Met Ala Val Leu Gly Gly Ser Leu Lys Arg Arg Glu Arg Ile Ser

595 600 605

Ala Arg Leu Gly Asp Ile Leu Ser Gln Leu Tyr Leu Ala Ser Ala Val

610 615 620

Leu Lys Arg Tyr Asp Asp Glu Gly Arg Asn Glu Ala Asp Leu Pro Leu

625 630 635 640

Val His Trp Gly Val Gln Asp Ala Leu Tyr Gln Ala Glu Gln Ala Met

645 650 655

Asp Asp Leu Leu Gln Asn Phe Pro Asn Arg Val Val Ala Gly Leu Leu

660 665 670

Asn Val Val Ile Phe Pro Thr Gly Arg His Tyr Leu Ala Pro Ser Asp

675 680 685

Lys Leu Asp His Lys Val Ala Lys Ile Leu Gln Val Pro Asn Ala Thr

690 695 700

Arg Ser Arg Ile Gly Arg Gly Gln Tyr Leu Thr Pro Ser Glu His Asn

705 710 715 720

Pro Val Gly Leu Leu Glu Glu Ala Leu Val Asp Val Ile Ala Ala Asp

725 730 735

Pro Ile His Gln Arg Ile Cys Lys Glu Leu Gly Lys Asn Leu Pro Phe

740 745 750

Thr Arg Leu Asp Glu Leu Ala His Asn Ala Leu Val Lys Gly Leu Ile

755 760 765

Asp Lys Asp Glu Ala Ala Ile Leu Val Lys Ala Glu Glu Ser Arg Leu

770 775 780

Arg Ser Ile Asn Val Asp Asp Phe Asp Pro Glu Glu Leu Ala Thr Lys

785 790 795 800

Pro Val Lys Leu Pro Glu Lys Val Arg Lys Val Glu Ala Ala

805 810

<210> 7

<211> 40

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 7

aaaaaacata tggccactcg cgtgaagacc aacaagaaac 40

<210> 8

<211> 44

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 8

aaacaattgt tagagggcgt tggtgggctc gtagacgaac tcag 44

<210> 9

<211> 38

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 9

aaaaaacata tggcctctcg caagaatgtg agcgctgc 38

<210> 10

<211> 39

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 10

aaagaattct tacaggcgct cagtgggcac gtagatgtc 39

<210> 11

<211> 41

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 11

gccttcatgg agacttatgg tgtatccgcc cccatgtaca c 41

<210> 12

<211> 28

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 12

acaccataag tctccatgaa ggcacggc 28

<210> 13

<211> 37

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 13

gtctctgtac gcatgccagt cctcgcgggc ctggttg 37

<210> 14

<211> 39

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 14

gaggactggc atgcgtacag agacttgccc cactgtgtc 39

<210> 15

<211> 29

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 15

ctctcttccg ggcgctatca tgccatacc 29

<210> 16

<211> 29

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 16

gcacagcacc atgttggcca ttgtagatg 29

<210> 17

<211> 24

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 17

aaagggctcc gggaagcaag ttgc 24

<210> 18

<211> 51

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 18

gcaacttgct tcccggagcc cttttggatt ctctccggat tctccacttt c 51

<210> 19

<211> 51

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 19

gcaacttgct tcccggagcc ctttaatatt ctctccggat tctccacttt c 51

<210> 20

<211> 51

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 20

gcaacttgct tcccggagcc ctttgggatt ctctccggat tctccacttt c 51

<210> 21

<211> 51

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 21

gcaacttgct tcccggagcc ctttgacatt ctctccggat tctccacttt c 51

<210> 22

<211> 51

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 22

gcaacttgct tcccggagcc ctttgctatt ctctccggat tctccacttt c 51

<210> 23

<211> 48

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 23

gatatacata tggcctctcg caagagtgtg agcgctgctc acgaaatg 48

<210> 24

<211> 29

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 24

ctctcttccg ggcgctatca tgccatacc 29

<210> 25

<211> 24

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 25

ttgaggtgct cggctcgctt gttg 24

<210> 26

<211> 37

<212> DNA

<213> 人工序列

<220>

<223> 合成DNA

<400> 26

cgagccgagc acctcaaagc agagcgtagc aaatttg 37

<210> 27

<211> 2006

<212> PRT

<213> 深海发光杆菌（Photobacterium profundum）

<400> 27

Met His Cys Pro Val Asn Tyr Ala Pro Asn Thr Ala Val Thr Phe Ser

1 5 10 15

Pro Ala Ser Arg Val Arg Arg Thr Ser Glu Cys Thr Glu Ile Thr Gln

20 25 30

Cys Ile Val Ser Glu Tyr His Thr Glu His Tyr Thr Arg Arg Ala Ser

35 40 45

Val Ser Ser Gln His Ser Thr Ala Ser Gln Ser Asn Lys Ile Ala Ile

50 55 60

Val Gly Leu Ala Asn Gln Tyr Pro Asp Ala Asp Thr Pro Lys Asp Phe

65 70 75 80

Trp Gln Asn Leu Leu Ala Lys Lys Asp Ser Arg Thr Thr Leu Ser Pro

85 90 95

Asp Lys Leu Gly Ala Asn Pro Asp Ala Tyr Gln Gly Ile Gln Gly Glu

100 105 110

Ser Asp Arg Phe Tyr Cys Asp Lys Gly Gly Tyr Ile Gln Asn Phe Ser

115 120 125

Phe Asp Ser Asn Gly Tyr Arg Leu Pro Ala Glu Thr Phe Glu Gly Leu

130 135 140

Asp Glu Ser Phe Leu Trp Ala Leu Asp Thr Ser Arg Lys Ala Leu Ala

145 150 155 160

Asp Ala Gly Ile Pro Leu Asp Asp Ala Val Leu Glu Arg Thr Gly Val

165 170 175

Ile Met Gly Ala Leu Ser Phe Pro Thr Lys Arg Ser Asn Asp Leu Phe

180 185 190

Leu Pro Ile Tyr His Ser Ala Val Glu Lys Ala Leu Gln Thr Lys Leu

195 200 205

Gly Asn Glu His Phe Thr Leu Thr Pro Ser Asn Ala Asn Ile Thr Ser

210 215 220

Leu Asn Pro Ala Asn Gly Ser Ala Ala His Asn Ala Ser Arg Leu Val

225 230 235 240

Ala Asp Ala Leu Gly Leu Gly Ser Val Gln Leu Ser Leu Asp Ala Ala

245 250 255

Cys Ala Ser Ser Val Tyr Ser Leu Lys Leu Ala Cys Asp Tyr Leu Asn

260 265 270

Thr Gly Lys Ala Asp Met Met Leu Ala Gly Ala Val Ser Gly Ala Asp

275 280 285

Pro Phe Phe Ile Asn Met Gly Phe Ser Ile Phe His Ala Tyr Pro Asp

290 295 300

His Gly Val Ser Val Pro Phe Asp Thr Asn Ser Lys Gly Leu Phe Ala

305 310 315 320

Gly Glu Gly Ala Gly Val Leu Ile Leu Lys Arg Leu Glu Asp Ala Glu

325 330 335

Arg Asp Gly Asp Asn Ile Tyr Ala Val Val Ser Gly Ile Gly Leu Ser

340 345 350

Asn Asp Gly Arg Gly Gln Phe Val Leu Ser Pro Asn Ser Lys Gly Gln

355 360 365

Val Gln Ala Phe Glu Arg Ala Tyr Glu Ala Thr Asp Leu Ser Pro Glu

370 375 380

Ser Ile Glu Val Ile Glu Cys His Ala Thr Gly Thr Pro Leu Gly Asp

385 390 395 400

Lys Val Glu Met Thr Ser Met Glu Arg Phe Phe Ala Asp Lys Leu Asn

405 410 415

Gly Ser Gln Ala Pro Leu Ile Gly Ser Ala Lys Ser Asn Leu Gly His

420 425 430

Leu Leu Thr Ala Ala Gly Met Pro Gly Ile Met Lys Met Ile Phe Ala

435 440 445

Met Lys Glu Gly Val Leu Pro Pro Ser Ile Asn Leu Ser Thr Pro Leu

450 455 460

Ser Ser Pro Glu Gly Leu Phe Gly Ser His Thr Leu Pro Thr Gln Val

465 470 475 480

Gln Ala Trp Pro Asp Lys Ala Gly Asn Thr Glu Arg Cys Ala Gly Val

485 490 495

Ser Val Phe Gly Phe Gly Gly Cys Asn Ala His Leu Leu Leu Glu Ala

500 505 510

His Ser Ala Asn Ser Ala Arg Asn Ser Pro Ala Ala Asn Ser Pro Ala

515 520 525

Lys Pro Ala Val Ser Ala Pro Leu Lys Val Thr Gly Leu Ala Ser His

530 535 540

Phe Gly Ser Leu Lys Thr Ile Asn Ala Leu His Asn Ala Ile Thr Thr

545 550 555 560

Gly Ala Asp Ala Phe Val Ala Leu Pro Lys Lys Arg Trp Lys Gly Leu

565 570 575

Asp Gln His Pro Glu Leu Leu Ser Gln Phe Gly Leu Asp Ala Ile Pro

580 585 590

His Gly Ala Tyr Ile Asp Gln Phe Glu Leu Asp Phe Leu Arg Phe Lys

595 600 605

Val Pro Pro Asn Glu Asp Asp Arg Leu Ile Ser Gln Gln Leu Leu Leu

610 615 620

Met Lys Val Ala Asp Glu Ala Ile Arg Asp Ala Lys Leu Glu Val Gly

625 630 635 640

Gln Lys Val Ala Val Leu Val Ala Met Glu Thr Glu Leu Glu Met His

645 650 655

Gln Phe Arg Gly Arg Val Asn Leu His Thr Gln Leu Ala Asp Ser Leu

660 665 670

Glu Asn Met Gly Val Gln Leu Thr Asp Ser Glu Tyr Gln Ala Leu Glu

675 680 685

Ala Ile Ala Met Asp Ser Val Leu Asp Ala Ala Lys Leu Asn Gln Tyr

690 695 700

Thr Ser Phe Ile Gly Asn Ile Met Ala Ser Arg Ile Ala Ser Leu Trp

705 710 715 720

Asp Phe Asn Gly Pro Ala Phe Thr Ile Ser Ala Ala Glu Gln Ser Val

725 730 735

Ala Arg Cys Ile Asp Val Ala Gln Asn Leu Met Ser Gln Glu Ser Leu

740 745 750

Asp Ala Val Val Ile Ala Ala Val Asp Leu Ser Gly Ser Val Glu Gln

755 760 765

Ile Ile Leu Lys Asn Ser Val Thr Pro Val Ala Leu His Pro Gln Asp

770 775 780

Ser Gly Trp Asn Val Gly Glu Gly Ala Gly Ala Ile Val Leu Val Asp

785 790 795 800

Thr Asp Asn Ala Ser Thr Lys Asn Ser Tyr Gly Glu Ile Thr Ala Leu

805 810 815

Asp Phe Gly Ser Val Ala Gln Ser Asn Ile Thr Ser Asp Arg Leu Leu

820 825 830

Thr Thr Ala Gly Ile Thr Ala Asn Asn Val Ser Leu Leu Glu Leu Asn

835 840 845

Gln Ala Pro Glu Ser Val Glu Thr Val Gln Phe Pro Leu Pro Ser Ala

850 855 860

Thr His Ile Gln Ala Asn Gln Arg Leu Gly His Cys Tyr Ala Ala Ser

865 870 875 880

Gly Met Ala Ser Ile Leu His Gly Leu Leu Ser Leu Asn Ala Ile Pro

885 890 895

Lys Gln Thr Thr Ile Pro Ser Leu Asn Thr Ser Val Thr Ala Ala Val

900 905 910

Thr Lys Ala Ala Ile Val Ala Asn Val Ser Glu Asn Gln Cys Ser Gln

915 920 925

Leu Leu Leu Thr Gln Thr Ser Thr Glu Thr Gln Ser Leu Thr Ala Arg

930 935 940

Leu Asn Ser Glu Leu Ala Asn Asp Ser Lys Arg Gln Leu Ile Lys Gln

945 950 955 960

Val Thr Leu Gly Gly Arg Asp Ile Tyr Gln His Ile Val Thr Ala Glu

965 970 975

Leu Ser Asp Ile Gln His Ile Gln Gln Lys Val Ala Asn Thr Lys Pro

980 985 990

Leu Val Gln Lys Gln Asn Ile Lys Gln Pro Arg Ile Gln Ala Ile Ala

995 1000 1005

Lys Pro Val Ala Gln Pro Gln Ser Ile Ala Gln Pro Thr Ala Ile

1010 1015 1020

Pro Ser Val Ser Pro Ile Leu Thr Thr Pro Arg Gln Thr Pro Ile

1025 1030 1035

Thr Gly Ile Gln Ser Asn Met Thr Asn Val Leu Ser Ala Lys Asn

1040 1045 1050

Lys His Asp Leu Thr Ala Phe Gln Ser Ser Ala Phe Val Glu Asn

1055 1060 1065

Gln Gln Leu Ala Gln Gln Val His Gln Ala Phe Leu Gln Asn Arg

1070 1075 1080

Glu Gln Gly Leu Lys Met Ala Asp Ala Leu Leu Lys Ala Gln Leu

1085 1090 1095

Asn Glu Val Thr Ala Gln Met Asn Ala Ala Thr Gly Gln Val Phe

1100 1105 1110

Asp His Gln Leu Ala Pro Ser Pro Ala Leu Thr Glu Ala Ser Thr

1115 1120 1125

Ser Val Pro Val Asn Val Ala Ala Thr Pro Ala Ile Val Asn Pro

1130 1135 1140

Ile Arg Lys Pro Cys Ile Trp Asp Tyr Glu Asp Leu Val Glu Tyr

1145 1150 1155

Ala Glu Gly Asp Ile Ala Asn Val Phe Gly Pro Asp Tyr Ala Val

1160 1165 1170

Ile Asp Asn Tyr Ser Arg Arg Val Arg Leu Pro Thr Thr Asp Tyr

1175 1180 1185

Leu Leu Val Ser Arg Val Thr Lys Leu Asp Ala Thr Met Leu Glu

1190 1195 1200

Tyr Lys Pro Ser Thr Met Thr Thr Glu Tyr Asp Ile Pro Val Asp

1205 1210 1215

Ala Pro Tyr Leu Val Asp Gly Gln Ile Pro Trp Ala Val Ala Val

1220 1225 1230

Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr Leu Gly Ile

1235 1240 1245

Asp Phe Glu Asn Lys Gly Glu Arg Val Tyr Arg Leu Leu Asp Cys

1250 1255 1260

Thr Leu Thr Phe Leu Gly Asp Leu Pro Arg Gly Gly Asp Thr Leu

1265 1270 1275

Arg Tyr Asp Ile Ser Ile Asn Asn Phe Ala Arg Asn Gly Asp Thr

1280 1285 1290

Leu Leu Phe Phe Phe Ser Tyr Glu Cys Phe Val Gly Asp Lys Met

1295 1300 1305

Val Leu Lys Met Asp Asn Gly Cys Ala Gly Phe Phe Thr Asp Glu

1310 1315 1320

Glu Leu Ser Asp Gly Lys Gly Val Ile Arg Thr Glu Asp Glu Ile

1325 1330 1335

Lys Ser Arg Asn Leu Ala Val Lys Gln Arg Phe Asn Pro Leu Leu

1340 1345 1350

His Cys Gln Lys Thr Gln Phe Asp Tyr Gln Thr Leu His Asn Leu

1355 1360 1365

Leu Asp Ala Asn Ile Ala Gly Cys Phe Gly Glu Ser His Ile Ser

1370 1375 1380

Asp Arg His Gln Pro Ser Leu Cys Phe Ser Ser Asp Lys Phe Met

1385 1390 1395

Met Ile Glu Gln Ile Ser His Val Asp Pro Gln Gly Gly Thr Trp

1400 1405 1410

Gly Leu Gly Leu Ile Glu Gly His Lys Gln Leu Glu Ala Asp His

1415 1420 1425

Trp Tyr Phe Pro Cys His Phe Lys Asp Asp Ser Val Met Ala Gly

1430 1435 1440

Ser Leu Met Ala Glu Gly Cys Gly Gln Leu Leu Gln Phe Phe Met

1445 1450 1455

Met Tyr Leu Gly Met His Thr Gln Val Glu Asn Gly Arg Phe Gln

1460 1465 1470

Pro Leu Glu Asn Ala Pro Gln Gln Val Arg Cys Arg Gly Gln Val

1475 1480 1485

Leu Pro Gln Ser Ala Val Leu Thr Tyr Arg Met Glu Val Thr Glu

1490 1495 1500

Ile Gly Leu Ser Pro Arg Pro Tyr Ala Lys Ala Asn Ile Asp Ile

1505 1510 1515

Leu Leu Asp Gly Lys Val Val Val Asp Phe Gln Asn Leu Gly Val

1520 1525 1530

Met Ile Lys Glu Glu Ser Glu Cys Thr Arg Tyr Leu Gly Ser Ser

1535 1540 1545

Asp Phe Asp Ser Ala Ser Leu Ala Ser Ser Ser Tyr Val Ala Glu

1550 1555 1560

Ser Ala Pro Ala Gln Ala Asp Val Ile Thr Pro Val Glu Ala Pro

1565 1570 1575

Ile Ser Gln Gln Ala Ser Ala Asn Ala Pro Leu Met Ala Gln Ile

1580 1585 1590

Pro Asp Leu Asn Thr Ala Pro Asn Lys Gly Val Ile Pro Leu Gln

1595 1600 1605

His Ile Glu Ala Pro Ile Val Pro Asp Tyr Gln Asn Arg Thr Pro

1610 1615 1620

Asp Thr Val Pro Phe Thr Pro Tyr His Met Phe Glu Phe Ala Thr

1625 1630 1635

Gly Asp Ile Glu Lys Cys Phe Gly Pro Asp Phe Ser Ile Tyr Arg

1640 1645 1650

Gly Met Ile Pro Pro Arg Thr Pro Cys Gly Asp Leu Gln Leu Thr

1655 1660 1665

Thr Arg Val Ile Glu Val Asn Gly Thr Arg Gly Asp Phe Lys Thr

1670 1675 1680

Pro Ser Ser Cys Ile Ala Glu Tyr Glu Val Pro Glu Asn Ala Trp

1685 1690 1695

Tyr Phe Asp Glu Asn Ser His Ser Ser Leu Met Pro Tyr Ser Val

1700 1705 1710

Leu Met Glu Ile Ser Leu Gln Pro Asn Gly Phe Ile Ser Gly Tyr

1715 1720 1725

Met Gly Thr Thr Leu Gly Phe Pro Gly Leu Glu Leu Phe Phe Arg

1730 1735 1740

Asn Leu Asp Gly Ser Gly Lys Met Leu Arg Asn Val Asp Leu Arg

1745 1750 1755

Gly Lys Thr Ile Val Asn Asp Ser Arg Leu Leu Ser Thr Val Met

1760 1765 1770

Met Gly Thr Asn Ile Val Gln Ser Phe Ser Phe Glu Leu Ser Thr

1775 1780 1785

Asp Gly Val Pro Phe Tyr Glu Gly Thr Ala Val Phe Gly Tyr Phe

1790 1795 1800

Lys Gly Ala Ala Leu Lys Asp Gln Leu Gly Leu Asp Asn Gly Gln

1805 1810 1815

Val Thr Tyr Pro Trp His Val Asn Asn Asn Arg Thr Pro Asp Val

1820 1825 1830

Ser Ile Asn Leu Leu Asp Lys Glu Ser Arg Tyr Phe Asn Ala Pro

1835 1840 1845

Leu Ser Ala Thr Gly Glu Ala Gln Pro His Tyr Gln Leu Ala Gly

1850 1855 1860

Gly Arg Leu Asn Phe Ile Asp Lys Val Asp Ile Thr Ser Asp Gly

1865 1870 1875

Gly Lys Ala Gly Leu Gly Tyr Leu Tyr Ala Glu Arg Thr Ile Asp

1880 1885 1890

Pro Ser Asp Trp Phe Phe Gln Phe His Phe His Gln Asp Pro Val

1895 1900 1905

Met Pro Gly Ser Leu Gly Val Glu Ala Ile Ile Glu Leu Met Gln

1910 1915 1920

Thr Tyr Ala Leu Asn Lys Asp Leu Gly Ala Gly Phe Arg Ser Pro

1925 1930 1935

Lys Phe Gly Gln Ile Gln Ser Glu Val Lys Trp Lys Tyr Arg Gly

1940 1945 1950

Gln Ile Asn Pro Leu Asn Lys Gln Met Ser Leu Asp Val His Ile

1955 1960 1965

Thr Ala Ile Lys Asp Glu Asp Gly Lys Arg Ile Ile Val Gly Asp

1970 1975 1980

Ala Asn Leu Ser Lys Asp Gly Leu Arg Ile Tyr Glu Val Lys Asp

1985 1990 1995

Ile Ala Ile Cys Ile Glu Glu Ala

2000 2005

<210> 28

<211> 1963

<212> PRT

<213> 奥奈达希瓦氏菌（Shewanella oneidensis）

<400> 28

Met Ser Ser Gln Met His Thr His Pro Thr Leu Gln Asp Ser Ala Ala

1 5 10 15

Val Pro Asn Asp Gln Arg Gln Thr Leu Lys Ala Met Pro Lys Ile Ala

20 25 30

Ile Val Gly Leu Ala Val Gln Tyr Pro Asp Ala Asp Thr Pro Glu Gln

35 40 45

Phe Trp Gln Asn Leu Leu Asp Lys Lys Asp Ser Arg Ser Gln Ile Asp

50 55 60

Ala Ala Lys Leu Asn Ala Asn Pro Ala Asp Tyr Gln Gly Ile Gln Gly

65 70 75 80

Gln Ala Asp Arg Phe Tyr Cys Asp Lys Gly Gly Tyr Ile Arg Asn Phe

85 90 95

Arg Phe Asp Pro Gln Gly Tyr Gln Leu Leu Pro Ala Thr Phe Ala Gly

100 105 110

Leu Asp Glu Ser Phe Leu Trp Ala Leu Asp Cys Ser Lys Lys Ala Leu

115 120 125

Leu Asn Ala Gly Val Asp Leu Thr Ala Pro Leu Leu Glu Arg Thr Gly

130 135 140

Ile Val Met Gly Thr Leu Ser Phe Pro Thr Ala Arg Ser Asn Glu Leu

145 150 155 160

Phe Leu Pro Ile Tyr His Gln Ala Val Glu Lys Ala Leu Lys Thr Lys

165 170 175

Leu Asn Gln Pro Gln Phe Ala Leu Ala Pro Phe Ala Asn Ala Ser Ile

180 185 190

Ala Gly Ser Gln Leu Ala Ala Asn Gly Val Ile Ala His Thr Ala Ser

195 200 205

Lys Leu Leu Ser Asp Ala Leu Gly Leu Gly Gly Ala Gln Leu Ser Leu

210 215 220

Asp Ala Ala Cys Ala Ser Ser Val Tyr Ala Leu Lys Leu Ala Cys Asp

225 230 235 240

Tyr Leu Thr Thr Gly Lys Ala Asp Met Met Leu Ala Gly Ala Val Ser

245 250 255

Gly Ala Asp Pro Phe Phe Ile Asn Met Gly Phe Ser Ile Phe His Ala

260 265 270

Tyr Pro Asp His Gly Ile Ser Ala Pro Phe Asp Ser Asn Ser Lys Gly

275 280 285

Leu Phe Ala Gly Glu Gly Ala Gly Val Leu Val Leu Lys Arg Leu Glu

290 295 300

Asp Ala Glu Arg Asp Gly Asp Asn Ile Tyr Ala Val Val Ser Gly Ile

305 310 315 320

Gly Leu Ser Asn Asp Gly Lys Gly Gln Phe Val Leu Ser Pro Asn Ser

325 330 335

Lys Gly Gln Val Gln Ala Phe Glu Arg Ala Tyr Ala Ala Ala Asn Thr

340 345 350

His Pro Ser Asn Ile Glu Val Ile Glu Cys His Ala Thr Gly Thr Pro

355 360 365

Leu Gly Asp Lys Val Glu Leu Thr Ser Met Glu Arg Phe Phe Glu Asp

370 375 380

Lys Leu Asp Gly Thr Lys Ala Pro Leu Ile Gly Ser Ala Lys Ser Asn

385 390 395 400

Leu Gly His Leu Leu Thr Ala Ala Gly Met Pro Gly Ile Met Lys Met

405 410 415

Ile Phe Ala Met Arg Ser Gly His Leu Pro Pro Ser Ile Asn Leu Thr

420 425 430

Ala Pro Ile Ser Ser Pro Lys Gly Leu Phe Ser Val Asn Asn Leu Pro

435 440 445

Thr Gln Arg Gln Ala Trp Pro Asp Lys Ala Gly Asn Asp Arg Arg His

450 455 460

Ala Gly Val Ser Val Phe Gly Phe Gly Gly Cys Asn Ala His Leu Leu

465 470 475 480

Leu Glu Ser Tyr Gln Pro Thr Ala His Ser Ala Glu Lys Gln Ala Asn

485 490 495

Lys Pro Val Tyr Gln Gln Gln Ala Leu Thr Val Ile Gly Met Ala Ser

500 505 510

His Phe Gly Pro Leu Ala Ser Ile Asn Ala Leu Asp Lys Ala Leu Ile

515 520 525

Ala Gln Thr Asp Ala Phe Ile Pro Leu Pro Pro Lys Arg Trp Lys Gly

530 535 540

Leu Asp Lys His Pro Asp Ile Leu Gln Gln Phe Gly Leu Asn Arg Ala

545 550 555 560

Pro Lys Gly Ala Tyr Ile Glu Gln Phe Asp Phe Asp Phe Leu Arg Phe

565 570 575

Lys Val Pro Pro Asn Glu Asp Asp Arg Leu Ile Ser Gln Gln Leu Leu

580 585 590

Leu Ile Lys Val Ala Asp Glu Ala Ile Arg Asp Ala Lys Leu Thr Ala

595 600 605

Gly Ser Lys Val Ala Val Leu Val Ala Met Glu Thr Glu Leu Glu Leu

610 615 620

His Gln Phe Arg Gly Arg Val Asn Leu His Thr Gln Leu Ala Asp Ser

625 630 635 640

Leu Lys Lys Gln Gly Val His Leu Ser Asn Asp Glu Tyr Leu Ala Leu

645 650 655

Glu Ala Ile Ala Met Asp Ser Val Leu Asp Ala Ala Lys Leu Asn Gln

660 665 670

Tyr Thr Ser Phe Ile Gly Asn Ile Met Ala Ser Arg Ile Ala Ser Leu

675 680 685

Trp Asp Phe Asn Gly Pro Ala Phe Thr Ile Ser Ala Ala Glu Gln Ser

690 695 700

Val Ala Arg Cys Ile Asp Val Ala Gln Asn Leu Leu Ser Lys Glu Ala

705 710 715 720

Leu Asp Gly Val Val Ile Ala Ala Val Asp Leu Ser Gly Ser Val Glu

725 730 735

Gln Val Ile Leu Lys Asn Ala Gln Val Ala Val Asp Leu Asp Ala Asn

740 745 750

Ser Ala Asn Pro Gln Trp Lys Val Gly Glu Gly Ala Gly Ala Ile Val

755 760 765

Leu Thr Asn Gln Gln Ala Ser Asn Ser Gln Gln Ala Gly Tyr Gly Gln

770 775 780

Ile Arg Gly Gln Ala Phe Gly Thr Asn His Gln Leu Pro Lys Leu Leu

785 790 795 800

Asp Ser Leu Ile Thr Glu Thr Ala Ile Ala Asn Pro Ser Met Pro Thr

805 810 815

Ala Ile His Met Ile Glu Gln Cys Ile Ala Pro Glu Glu Gln Leu Pro

820 825 830

Ala Glu His Leu Leu Ala Gln Leu Asn Leu Leu Gly Thr Ser Cys Asn

835 840 845

Arg Val Ala Asn Thr Leu Gly His Asn Phe Ala Ala Ala Gly Met Ala

850 855 860

Ser Leu Leu Ser Ala Leu Leu Ser Leu Lys Asn Arg Ser Ala Asn Ser

865 870 875 880

Asp Lys Asn Ala Glu Lys Gln Ala Leu Val Ser Thr Gln Ser Gln Gly

885 890 895

Val Ser Ser Leu Leu Leu Leu Ser Gln Thr Ala Thr Gln Ala Ala Gln

900 905 910

Leu Glu Leu Arg Leu Ala Gln Asp Leu Thr Leu Ser Glu Gln Lys His

915 920 925

Leu Ile Lys Pro Val Thr Leu Gly Gly Arg Asp Ile Tyr Gln His Ile

930 935 940

Val Asp Thr Pro Leu Pro Ala Leu Ala Ala Ile Gln Gly Lys Met Arg

945 950 955 960

Gln Leu Gln Pro Leu Ala Ser Gln Ala Thr Gln Thr Lys Pro Ala Val

965 970 975

Gly Ala Ala Leu Asp Ile Thr Ala Glu Asn Ala Thr Pro Leu Ala Ala

980 985 990

Glu Ser Gly Met Ser Ser Asn Ala Pro Leu Gln Phe Glu Thr Thr Ala

995 1000 1005

Ser Ala Gln Asp Ser Ala Ala Leu Leu Gln Asn Gln Gln Leu Ala

1010 1015 1020

Arg Glu Ala His Leu Ala Phe Leu Gln Ser Arg Glu Gln Gly Leu

1025 1030 1035

Lys Leu Ala Asp Ala Leu Leu Lys Ala Gln Leu Ser Gln Thr Thr

1040 1045 1050

Gln Met Gly Ala Val Ala Ala His Val Ala Thr Ser Ala Asn Val

1055 1060 1065

Ala Glu Thr Lys Ala Gln Gln Ala Val Ser Ile Pro Glu Leu Met

1070 1075 1080

Pro Asn His Ala Pro Asn His Ala Arg Val Pro Pro Tyr Thr Pro

1085 1090 1095

Pro Ile Pro Ala Ala Lys Pro Cys Ile Trp Asn Tyr Gln Asp Leu

1100 1105 1110

Val Glu Tyr Ala Glu Gly Asp Ile Ala Lys Val Phe Gly Ala Asp

1115 1120 1125

Tyr Ala Ile Ile Asp Ser Tyr Ala Arg Arg Val Arg Leu Pro Thr

1130 1135 1140

Ser Asp Tyr Leu Leu Val Ser Arg Val Thr Lys Leu Asn Ala Gln

1145 1150 1155

Met Asn Arg Tyr Gln Pro Ser Ser Met Thr Thr Glu Tyr Asp Ile

1160 1165 1170

Pro Val Asp Ala Pro Phe Leu Val Asp Gly Gln Ile Pro Trp Ala

1175 1180 1185

Val Ala Val Glu Ser Gly Gln Cys Asp Leu Met Leu Ile Ser Tyr

1190 1195 1200

Leu Gly Ile Asp Phe Glu Asn Lys Gly Glu Arg Val Tyr Arg Leu

1205 1210 1215

Leu Asp Cys Thr Leu Thr Phe Leu Gly Asp Leu Pro Arg Gly Gly

1220 1225 1230

Asp Thr Leu Arg Tyr Asp Ile Ser Ile Asn His Phe Ala Arg Asn

1235 1240 1245

Gly Asp Thr Leu Leu Phe Phe Phe Ser Tyr Glu Cys Phe Val Gly

1250 1255 1260

Asp Lys Leu Ile Leu Lys Met Asp Gly Gly Cys Ala Gly Phe Phe

1265 1270 1275

Thr Asp Lys Glu Leu Ala Asp Gly Lys Gly Val Ile Arg Thr Glu

1280 1285 1290

Val Glu Ile Lys Val Arg Glu Gln Ala Gln Ile Ala Leu Ala Asn

1295 1300 1305

Glu Tyr Thr Arg Asn Gly Asn Lys Pro Arg Phe Thr Pro Leu Leu

1310 1315 1320

Asn Cys Ala Gln Thr Ala Phe Ser Tyr Gly Gln Ile His Arg Leu

1325 1330 1335

Leu Ser Ala Asp Ile Gly Gly Cys Phe Gly Gly Glu His Ala Ala

1340 1345 1350

His Gln Ala Lys Phe Gly Leu Gln Pro Ser Leu Cys Phe Ala Ser

1355 1360 1365

Glu Lys Phe Leu Met Ile Glu Gln Val Ser Lys Leu Glu Val His

1370 1375 1380

Gly Gly Ala Trp Gly Leu Gly Leu Ile Glu Gly His Lys Gln Leu

1385 1390 1395

Ala Pro Asp His Trp Tyr Phe Pro Cys His Phe Lys Gly Asp Gln

1400 1405 1410

Val Met Ala Gly Ser Leu Met Ala Glu Gly Cys Gly Gln Leu Leu

1415 1420 1425

Gln Phe Phe Met Leu His Ile Gly Met His Ala Asn Thr Gln Ala

1430 1435 1440

Gly Gly Val Thr Asn Gly Arg Phe Gln Pro Leu Glu Asn Ala Ser

1445 1450 1455

Gln Lys Val Arg Cys Arg Gly Gln Val Leu Pro Gln Ser Gly Thr

1460 1465 1470

Leu Thr Tyr Arg Met Glu Val Thr Glu Ile Gly Met Ser Pro Arg

1475 1480 1485

Pro Tyr Ala Lys Ala Asn Ile Asp Ile Leu Leu Asn Gly Lys Val

1490 1495 1500

Val Val Asp Phe Gln Asn Leu Gly Val Met Ile Lys Glu Glu Ala

1505 1510 1515

Asp Cys Thr Arg Tyr Ser Gln Ser His Ser Ser Gln Gly Asn His

1520 1525 1530

Thr Gln Ala Ala Asn Ile Glu Ser Leu Ala Glu Gln Ala Pro Leu

1535 1540 1545

Met Ala Gln Ile Pro Asp Val Ala Ala Pro Val Asn Lys Gly Val

1550 1555 1560

Val Pro Leu Lys His Val Ser Ala Pro Ile Ala Pro Ala Gly Ser

1565 1570 1575

Lys Tyr Ala Asn Arg Val Pro Asp Thr Leu Pro Phe Thr Pro Tyr

1580 1585 1590

His Leu Phe Glu Phe Ala Thr Gly Asp Ile Glu Asn Cys Phe Gly

1595 1600 1605

Pro Asp Phe Ser Ile Tyr Arg Gly Leu Ile Pro Pro Arg Thr Pro

1610 1615 1620

Cys Gly Asp Leu Gln Leu Thr Thr Arg Val Val Ala Ile Glu Gly

1625 1630 1635

Lys Arg Gly Glu Leu Lys Lys Pro Ser Thr Cys Ile Ala Glu Tyr

1640 1645 1650

Glu Val Pro Ser Asn Ala Trp Tyr Tyr Arg Lys Thr Ser His Pro

1655 1660 1665

Ser Val Met Pro Tyr Ser Val Leu Met Glu Ile Ser Leu Gln Pro

1670 1675 1680

Asn Gly Phe Ile Ser Gly Tyr Met Gly Thr Thr Leu Gly Phe Pro

1685 1690 1695

Gly Gln Glu Leu Phe Phe Arg Asn Leu Asp Gly Ser Gly Lys Leu

1700 1705 1710

Leu Arg Glu Val Asp Leu Arg Gly Lys Thr Ile Val Asn Asp Ser

1715 1720 1725

Arg Leu Leu Ser Thr Val Ile Ala Gly Ser Asn Ile Ile Gln Asn

1730 1735 1740

Phe Ser Phe Glu Leu Ser Cys Asp Gly Glu Pro Phe Tyr Arg Gly

1745 1750 1755

Asn Ala Val Phe Gly Tyr Phe Lys Ala Asp Ala Leu Lys Asn Gln

1760 1765 1770

Leu Gly Ile Asp Asn Gly Lys Ile Thr Gln Ala Trp His Leu Glu

1775 1780 1785

Arg Gly Ile Lys Ala Asp Cys Gln Ile Asn Leu Leu Asp Lys Asn

1790 1795 1800

Gly Arg Ser Phe Val Ala Pro Leu Gly Lys Pro His Tyr Arg Leu

1805 1810 1815

Ala Gly Gly Gln Leu Asn Phe Ile Asp Lys Ala Glu Ile Val Lys

1820 1825 1830

Thr Gly Gly Lys Lys Gly Leu Gly Tyr Leu Tyr Ala Glu Arg Thr

1835 1840 1845

Ile Asp Pro Ser Asp Trp Phe Phe Gln Phe His Phe His Gln Asp

1850 1855 1860

Pro Val Met Pro Gly Ser Leu Gly Val Glu Ala Ile Ile Glu Leu

1865 1870 1875

Leu Gln Thr Tyr Ala Ile Asp Gln Asp Leu Gly Ala Gly Phe Asn

1880 1885 1890

Asn Pro Lys Phe Gly Gln Ile Leu Ser Glu Ile Lys Trp Lys Tyr

1895 1900 1905

Arg Gly Gln Ile Asn Pro Leu Asn Lys Gln Met Ser Leu Asp Val

1910 1915 1920

His Ile Thr Ser Ile Glu Asp Lys Asp Gly Lys Arg Ile Ile Lys

1925 1930 1935

Gly Asp Ala Asn Leu Ser Lys Asp Gly Leu Arg Ile Tyr Glu Val

1940 1945 1950

Thr Asp Ile Ala Ile Cys Ile Glu Glu Ala

1955 1960

<210> 29

<211> 2011

<212> PRT

<213> 海摩替亚氏菌（Moritella marina）

<400> 29

Met Glu Asn Ile Ala Val Val Gly Ile Ala Asn Leu Phe Pro Gly Ser

1 5 10 15

Gln Ala Pro Asp Gln Phe Trp Gln Gln Leu Leu Glu Gln Gln Asp Cys

20 25 30

Arg Ser Lys Ala Thr Ala Val Gln Met Gly Val Asp Pro Ala Lys Tyr

35 40 45

Thr Ala Asn Lys Gly Asp Thr Asp Lys Phe Tyr Cys Val His Gly Gly

50 55 60

Tyr Ile Ser Asp Phe Asn Phe Asp Ala Ser Gly Tyr Gln Leu Asp Asn

65 70 75 80

Asp Tyr Leu Ala Gly Leu Asp Asp Leu Asn Gln Trp Gly Leu Tyr Val

85 90 95

Thr Lys Gln Ala Leu Thr Asp Ala Gly Tyr Trp Gly Ser Thr Ala Leu

100 105 110

Glu Asn Cys Gly Val Ile Leu Gly Asn Leu Ser Phe Pro Thr Lys Ser

115 120 125

Ser Asn Gln Leu Phe Met Pro Leu Tyr His Gln Val Val Asp Asn Ala

130 135 140

Leu Lys Ala Val Leu His Pro Asp Phe Gln Leu Thr His Tyr Thr Ala

145 150 155 160

Pro Lys Lys Thr His Ala Asp Asn Ala Leu Val Ala Gly Tyr Pro Ala

165 170 175

Ala Leu Ile Ala Gln Ala Ala Gly Leu Gly Gly Ser His Phe Ala Leu

180 185 190

Asp Ala Ala Cys Ala Ser Ser Cys Tyr Ser Val Lys Leu Ala Cys Asp

195 200 205

Tyr Leu His Thr Gly Lys Ala Asn Met Met Leu Ala Gly Ala Val Ser

210 215 220

Ala Ala Asp Pro Met Phe Val Asn Met Gly Phe Ser Ile Phe Gln Ala

225 230 235 240

Tyr Pro Ala Asn Asn Val His Ala Pro Phe Asp Gln Asn Ser Gln Gly

245 250 255

Leu Phe Ala Gly Glu Gly Ala Gly Met Met Val Leu Lys Arg Gln Ser

260 265 270

Asp Ala Val Arg Asp Gly Asp His Ile Tyr Ala Ile Ile Lys Gly Gly

275 280 285

Ala Leu Ser Asn Asp Gly Lys Gly Glu Phe Val Leu Ser Pro Asn Thr

290 295 300

Lys Gly Gln Val Leu Val Tyr Glu Arg Ala Tyr Ala Asp Ala Asp Val

305 310 315 320

Asp Pro Ser Thr Val Asp Tyr Ile Glu Cys His Ala Thr Gly Thr Pro

325 330 335

Lys Gly Asp Asn Val Glu Leu Arg Ser Met Glu Thr Phe Phe Ser Arg

340 345 350

Val Asn Asn Lys Pro Leu Leu Gly Ser Val Lys Ser Asn Leu Gly His

355 360 365

Leu Leu Thr Ala Ala Gly Met Pro Gly Met Thr Lys Ala Met Leu Ala

370 375 380

Leu Gly Lys Gly Leu Ile Pro Ala Thr Ile Asn Leu Lys Gln Pro Leu

385 390 395 400

Gln Ser Lys Asn Gly Tyr Phe Thr Gly Glu Gln Met Pro Thr Thr Thr

405 410 415

Val Ser Trp Pro Thr Thr Pro Gly Ala Lys Ala Asp Lys Pro Arg Thr

420 425 430

Ala Gly Val Ser Val Phe Gly Phe Gly Gly Ser Asn Ala His Leu Val

435 440 445

Leu Gln Gln Pro Thr Gln Thr Leu Glu Thr Asn Phe Ser Val Ala Lys

450 455 460

Pro Arg Glu Pro Leu Ala Ile Ile Gly Met Asp Ser His Phe Gly Ser

465 470 475 480

Ala Ser Asn Leu Ala Gln Phe Lys Thr Leu Leu Asn Asn Asn Gln Asn

485 490 495

Thr Phe Arg Glu Leu Pro Glu Gln Arg Trp Lys Gly Met Glu Ser Asn

500 505 510

Ala Asn Val Met Gln Ser Leu Gln Leu Arg Lys Ala Pro Lys Gly Ser

515 520 525

Tyr Val Glu Gln Leu Asp Ile Asp Phe Leu Arg Phe Lys Val Pro Pro

530 535 540

Asn Glu Lys Asp Cys Leu Ile Pro Gln Gln Leu Met Met Met Gln Val

545 550 555 560

Ala Asp Asn Ala Ala Lys Asp Gly Gly Leu Val Glu Gly Arg Asn Val

565 570 575

Ala Val Leu Val Ala Met Gly Met Glu Leu Glu Leu His Gln Tyr Arg

580 585 590

Gly Arg Val Asn Leu Thr Thr Gln Ile Glu Asp Ser Leu Leu Gln Gln

595 600 605

Gly Ile Asn Leu Thr Val Glu Gln Arg Glu Glu Leu Thr Asn Ile Ala

610 615 620

Lys Asp Gly Val Ala Ser Ala Ala Gln Leu Asn Gln Tyr Thr Ser Phe

625 630 635 640

Ile Gly Asn Ile Met Ala Ser Arg Ile Ser Ala Leu Trp Asp Phe Ser

645 650 655

Gly Pro Ala Ile Thr Val Ser Ala Glu Glu Asn Ser Val Tyr Arg Cys

660 665 670

Val Glu Leu Ala Glu Asn Leu Phe Gln Thr Ser Asp Val Glu Ala Val

675 680 685

Ile Ile Ala Ala Val Asp Leu Ser Gly Ser Ile Glu Asn Ile Thr Leu

690 695 700

Arg Gln His Tyr Gly Pro Val Asn Glu Lys Gly Ser Val Ser Glu Cys

705 710 715 720

Gly Pro Val Asn Glu Ser Ser Ser Val Thr Asn Asn Ile Leu Asp Gln

725 730 735

Gln Gln Trp Leu Val Gly Glu Gly Ala Ala Ala Ile Val Val Lys Pro

740 745 750

Ser Ser Gln Val Thr Ala Glu Gln Val Tyr Ala Arg Ile Asp Ala Val

755 760 765

Ser Phe Ala Pro Gly Ser Asn Ala Lys Ala Ile Thr Ile Ala Ala Asp

770 775 780

Lys Ala Leu Thr Leu Ala Gly Ile Ser Ala Ala Asp Val Ala Ser Val

785 790 795 800

Glu Ala His Ala Ser Gly Phe Ser Ala Glu Asn Asn Ala Glu Lys Thr

805 810 815

Ala Leu Pro Thr Leu Tyr Pro Ser Ala Ser Ile Ser Ser Val Lys Ala

820 825 830

Asn Ile Gly His Thr Phe Asn Ala Ser Gly Met Ala Ser Ile Ile Lys

835 840 845

Thr Ala Leu Leu Leu Asp Gln Asn Thr Ser Gln Asp Gln Lys Ser Lys

850 855 860

His Ile Ala Ile Asn Gly Leu Gly Arg Asp Asn Ser Cys Ala His Leu

865 870 875 880

Ile Leu Ser Ser Ser Ala Gln Ala His Gln Val Ala Pro Ala Pro Val

885 890 895

Ser Gly Met Ala Lys Gln Arg Pro Gln Leu Val Lys Thr Ile Lys Leu

900 905 910

Gly Gly Gln Leu Ile Ser Asn Ala Ile Val Asn Ser Ala Ser Ser Ser

915 920 925

Leu His Ala Ile Lys Ala Gln Phe Ala Gly Lys His Leu Asn Lys Val

930 935 940

Asn Gln Pro Val Met Met Asp Asn Leu Lys Pro Gln Gly Ile Ser Ala

945 950 955 960

His Ala Thr Asn Glu Tyr Val Val Thr Gly Ala Ala Asn Thr Gln Ala

965 970 975

Ser Asn Ile Gln Ala Ser His Val Gln Ala Ser Ser His Ala Gln Glu

980 985 990

Ile Ala Pro Asn Gln Val Gln Asn Met Gln Ala Thr Ala Ala Ala Val

995 1000 1005

Ser Ser Pro Leu Ser Gln His Gln His Thr Ala Gln Pro Val Ala

1010 1015 1020

Ala Pro Ser Val Val Gly Val Thr Val Lys His Lys Ala Ser Asn

1025 1030 1035

Gln Ile His Gln Gln Ala Ser Thr His Lys Ala Phe Leu Glu Ser

1040 1045 1050

Arg Leu Ala Ala Gln Lys Asn Leu Ser Gln Leu Val Glu Leu Gln

1055 1060 1065

Thr Lys Leu Ser Ile Gln Thr Gly Ser Asp Asn Thr Ser Asn Asn

1070 1075 1080

Thr Ala Ser Thr Ser Asn Thr Val Leu Thr Asn Pro Val Ser Ala

1085 1090 1095

Thr Pro Leu Thr Leu Val Ser Asn Ala Pro Val Val Ala Thr Asn

1100 1105 1110

Leu Thr Ser Thr Glu Ala Lys Ala Gln Ala Ala Ala Thr Gln Ala

1115 1120 1125

Gly Phe Gln Ile Lys Gly Pro Val Gly Tyr Asn Tyr Pro Pro Leu

1130 1135 1140

Gln Leu Ile Glu Arg Tyr Asn Lys Pro Glu Asn Val Ile Tyr Asp

1145 1150 1155

Gln Ala Asp Leu Val Glu Phe Ala Glu Gly Asp Ile Gly Lys Val

1160 1165 1170

Phe Gly Ala Glu Tyr Asn Ile Ile Asp Gly Tyr Ser Arg Arg Val

1175 1180 1185

Arg Leu Pro Thr Ser Asp Tyr Leu Leu Val Thr Arg Val Thr Glu

1190 1195 1200

Leu Asp Ala Lys Val His Glu Tyr Lys Lys Ser Tyr Met Cys Thr

1205 1210 1215

Glu Tyr Asp Val Pro Val Asp Ala Pro Phe Leu Ile Asp Gly Gln

1220 1225 1230

Ile Pro Trp Ser Val Ala Val Glu Ser Gly Gln Cys Asp Leu Met

1235 1240 1245

Leu Ile Ser Tyr Ile Gly Ile Asp Phe Gln Ala Lys Gly Glu Arg

1250 1255 1260

Val Tyr Arg Leu Leu Asp Cys Glu Leu Thr Phe Leu Glu Glu Met

1265 1270 1275

Ala Phe Gly Gly Asp Thr Leu Arg Tyr Glu Ile His Ile Asp Ser

1280 1285 1290

Tyr Ala Arg Asn Gly Glu Gln Leu Leu Phe Phe Phe His Tyr Asp

1295 1300 1305

Cys Tyr Val Gly Asp Lys Lys Val Leu Ile Met Arg Asn Gly Cys

1310 1315 1320

Ala Gly Phe Phe Thr Asp Glu Glu Leu Ser Asp Gly Lys Gly Val

1325 1330 1335

Ile His Asn Asp Lys Asp Lys Ala Glu Phe Ser Asn Ala Val Lys

1340 1345 1350

Ser Ser Phe Thr Pro Leu Leu Gln His Asn Arg Gly Gln Tyr Asp

1355 1360 1365

Tyr Asn Asp Met Met Lys Leu Val Asn Gly Asp Val Ala Ser Cys

1370 1375 1380

Phe Gly Pro Gln Tyr Asp Gln Gly Gly Arg Asn Pro Ser Leu Lys

1385 1390 1395

Phe Ser Ser Glu Lys Phe Leu Met Ile Glu Arg Ile Thr Lys Ile

1400 1405 1410

Asp Pro Thr Gly Gly His Trp Gly Leu Gly Leu Leu Glu Gly Gln

1415 1420 1425

Lys Asp Leu Asp Pro Glu His Trp Tyr Phe Pro Cys His Phe Lys

1430 1435 1440

Gly Asp Gln Val Met Ala Gly Ser Leu Met Ser Glu Gly Cys Gly

1445 1450 1455

Gln Met Ala Met Phe Phe Met Leu Ser Leu Gly Met His Thr Asn

1460 1465 1470

Val Asn Asn Ala Arg Phe Gln Pro Leu Pro Gly Glu Ser Gln Thr

1475 1480 1485

Val Arg Cys Arg Gly Gln Val Leu Pro Gln Arg Asn Thr Leu Thr

1490 1495 1500

Tyr Arg Met Glu Val Thr Ala Met Gly Met His Pro Gln Pro Phe

1505 1510 1515

Met Lys Ala Asn Ile Asp Ile Leu Leu Asp Gly Lys Val Val Val

1520 1525 1530

Asp Phe Lys Asn Leu Ser Val Met Ile Ser Glu Gln Asp Glu His

1535 1540 1545

Ser Asp Tyr Pro Val Thr Leu Pro Ser Asn Val Ala Leu Lys Ala

1550 1555 1560

Ile Thr Ala Pro Val Ala Ser Val Ala Pro Ala Ser Ser Pro Ala

1565 1570 1575

Asn Ser Ala Asp Leu Asp Glu Arg Gly Val Glu Pro Phe Lys Phe

1580 1585 1590

Pro Glu Arg Pro Leu Met Arg Val Glu Ser Asp Leu Ser Ala Pro

1595 1600 1605

Lys Ser Lys Gly Val Thr Pro Ile Lys His Phe Glu Ala Pro Ala

1610 1615 1620

Val Ala Gly His His Arg Val Pro Asn Gln Ala Pro Phe Thr Pro

1625 1630 1635

Trp His Met Phe Glu Phe Ala Thr Gly Asn Ile Ser Asn Cys Phe

1640 1645 1650

Gly Pro Asp Phe Asp Val Tyr Glu Gly Arg Ile Pro Pro Arg Thr

1655 1660 1665

Pro Cys Gly Asp Leu Gln Val Val Thr Gln Val Val Glu Val Gln

1670 1675 1680

Gly Glu Arg Leu Asp Leu Lys Asn Pro Ser Ser Cys Val Ala Glu

1685 1690 1695

Tyr Tyr Val Pro Glu Asp Ala Trp Tyr Phe Thr Lys Asn Ser His

1700 1705 1710

Glu Asn Trp Met Pro Tyr Ser Leu Ile Met Glu Ile Ala Leu Gln

1715 1720 1725

Pro Asn Gly Phe Ile Ser Gly Tyr Met Gly Thr Thr Leu Lys Tyr

1730 1735 1740

Pro Glu Lys Asp Leu Phe Phe Arg Asn Leu Asp Gly Ser Gly Thr

1745 1750 1755

Leu Leu Lys Gln Ile Asp Leu Arg Gly Lys Thr Ile Val Asn Lys

1760 1765 1770

Ser Val Leu Val Ser Thr Ala Ile Ala Gly Gly Ala Ile Ile Gln

1775 1780 1785

Ser Phe Thr Phe Asp Met Ser Val Asp Gly Glu Leu Phe Tyr Thr

1790 1795 1800

Gly Lys Ala Val Phe Gly Tyr Phe Ser Gly Glu Ser Leu Thr Asn

1805 1810 1815

Gln Leu Gly Ile Asp Asn Gly Lys Thr Thr Asn Ala Trp Phe Val

1820 1825 1830

Asp Asn Asn Thr Pro Ala Ala Asn Ile Asp Val Phe Asp Leu Thr

1835 1840 1845

Asn Gln Ser Leu Ala Leu Tyr Lys Ala Pro Val Asp Lys Pro His

1850 1855 1860

Tyr Lys Leu Ala Gly Gly Gln Met Asn Phe Ile Asp Thr Val Ser

1865 1870 1875

Val Val Glu Gly Gly Gly Lys Ala Gly Val Ala Tyr Val Tyr Gly

1880 1885 1890

Glu Arg Thr Ile Asp Ala Asp Asp Trp Phe Phe Arg Tyr His Phe

1895 1900 1905

His Gln Asp Pro Val Met Pro Gly Ser Leu Gly Val Glu Ala Ile

1910 1915 1920

Ile Glu Leu Met Gln Thr Tyr Ala Leu Lys Asn Asp Leu Gly Gly

1925 1930 1935

Lys Phe Ala Asn Pro Arg Phe Ile Ala Pro Met Thr Gln Val Asp

1940 1945 1950

Trp Lys Tyr Arg Gly Gln Ile Thr Pro Leu Asn Lys Gln Met Ser

1955 1960 1965

Leu Asp Val His Ile Thr Glu Ile Val Asn Asp Ala Gly Glu Val

1970 1975 1980

Arg Ile Val Gly Asp Ala Asn Leu Ser Lys Asp Gly Leu Arg Ile

1985 1990 1995

Tyr Glu Val Lys Asn Ile Val Leu Ser Ile Val Glu Ala

2000 2005 2010

<210> 30

<211> 2241

<212> PRT

<213> 海洋金色螺旋菌（Aureispira marina）

<400> 30

Met Lys Ile Ala Ile Ile Gly Leu Ser Gly Leu Phe Pro Gly Ser Ser

1 5 10 15

Thr Asn Glu Glu Phe Trp Gln Asn Leu Leu Asp Glu Lys Asp Leu Thr

20 25 30

Asn Leu Ala Asn Leu Glu Asp Phe Gly Ala Asp Pro Ala Leu Phe Tyr

35 40 45

Glu Asp Lys Lys Gly Ala Val Asp Arg Cys Tyr Ser Leu Arg Gly Gly

50 55 60

Tyr Ile Arg Asp Phe Asp Phe Asp Pro Thr Gly Tyr Gln Leu Ser Ala

65 70 75 80

Asp Phe Leu Ala Gln Gln Asp Lys Leu Tyr Gln Trp Ser Leu Tyr Val

85 90 95

Ala Lys Thr Ala Leu Glu Glu Ser Gly Tyr Ala His Asn Lys Glu Val

100 105 110

Leu Ala Lys Cys Gly Leu Ile Leu Gly Asn Leu Ser Phe Pro Thr Gly

115 120 125

Ser Ser His Lys Leu Leu Ala Asp Leu Tyr Thr Lys Thr Thr Glu Lys

130 135 140

Ala Leu Gln Glu Leu Leu Glu Asp Lys Asn Phe Lys Ile Pro Ala Ser

145 150 155 160

Gln Leu Pro Ile Pro Asn Asn Glu Val Leu Ala Asp Thr Pro Ser Gln

165 170 175

Met Val Ala Lys Gly Leu Gly Leu Gly Gly Gly His Tyr Ala Leu Asp

180 185 190

Ala Ala Cys Ala Thr Ser Leu Tyr Ala Ile Lys Leu Ala Cys Asp Glu

195 200 205

Leu Ile Thr Gly Lys Ala Asp Leu Met Leu Ala Gly Ala Val Cys Gly

210 215 220

Ser Asp Gln Leu Phe Ile His Met Gly Phe Ser Ile Phe His Ala Tyr

225 230 235 240

Ala Pro His Gly Glu Lys Phe Ala Pro Leu Asp Lys Ala Ser Gly Gly

245 250 255

Leu Val Ser Ala Glu Gly Ala Gly Met Val Val Leu Lys Arg Leu Glu

260 265 270

Asp Ala Glu Arg Asp Gly Asp Asn Ile Leu Gly Leu Ile Gly Gly Ile

275 280 285

Gly Leu Ser Asn Asp Gly Ser Gly Lys Phe Leu Leu Ser Pro Asn Pro

290 295 300

Lys Gly Gln Arg Leu Ala Phe Glu Arg Ala Tyr Asp Leu Glu Glu Val

305 310 315 320

Leu Pro Gln Asn Thr Ser Tyr Leu Glu Cys His Ala Thr Gly Thr Pro

325 330 335

Leu Gly Asp Val Thr Glu Met Asn Ser Ile Ser Asp Phe Phe Ala Gln

340 345 350

His Gln Thr Lys Pro Leu Leu Gly Ser Val Lys Ser Asn Met Gly His

355 360 365

Leu Leu Thr Ala Ala Gly Met Ser Gly Leu Phe Lys Val Leu Leu Ser

370 375 380

Met Gln Lys Gly Ile Ile Pro Pro Asn Ile Asn Leu Glu Ser Ala Val

385 390 395 400

Gln Ala Asn Asn Gln Trp Ile Gln Asp Glu Gln Ile Ile Lys Lys Thr

405 410 415

Thr Pro Trp Lys Gly Asp Gln Ala Gly Ile Asn Ser Phe Gly Phe Gly

420 425 430

Gly Thr Asn Ala His Met Val Val Gln Lys Pro Thr Ser Ser Thr Leu

435 440 445

Lys Glu Lys Lys Ala Tyr Gln Ala Gln Glu Leu Leu Pro Leu Ala Ile

450 455 460

Val Gly Met Asp Ala His Phe Gly Ser Cys Glu Asn Leu Glu Asp Phe

465 470 475 480

Tyr Ala Ala Ile Tyr Asn Gly Asn Gln Asp Phe Lys Pro Leu Pro Pro

485 490 495

Lys Arg Trp Lys Gly Phe Asp Ala Asp Gln Asp Leu Leu Lys Arg Tyr

500 505 510

Gly Phe Lys Asp Gly Leu Ala Pro Lys Gly Ala Tyr Ile Asp Gln Phe

515 520 525

Asp Ile Asp Leu Leu Arg Tyr Lys Ile Gln Pro Lys Glu Ala Glu Thr

530 535 540

Leu Glu Pro Gln Gln Ala Leu Ile Leu Lys Val Ala Asp Lys Ala Leu

545 550 555 560

Gln Asp Ala Gln Ile Ser Pro Ser Gln Asn Ile Ala Val Leu Ile Ala

565 570 575

Met Glu Ser Glu Leu Ala Ile His His Tyr Leu Ala Arg Trp Asp Ser

580 585 590

Val Trp Gln Leu Asp Lys Ala Leu Glu Gln Ser Gly Leu Ser Leu Ser

595 600 605

Glu Glu Lys Lys Thr Ala Leu Lys Glu Tyr Ser Lys Asn Ala Leu Tyr

610 615 620

Phe Arg Glu Gly Ser Gln Thr Pro Ser Gln His Thr Ser Phe Val Gly

625 630 635 640

Asn Ile Met Ala Ser Arg Ile Ala Ala Leu Trp Asp Phe Ser Gly Pro

645 650 655

Ala Phe Thr Val Ser Cys Gly Asp Asn Ala Val Phe Lys Ala Leu Glu

660 665 670

Val Ala Gln Asn Ile Leu Ser Leu Gly Glu Val Asp Ala Val Val Val

675 680 685

Gly Gly Val Asp Phe Cys Gly Gly Leu Glu Asn Val Leu Leu Arg Gln

690 695 700

Glu Lys Glu Ala Ser Ser Gln Asn Ile Ala Pro Ser Leu Ser Leu Asn

705 710 715 720

Gln Gly Gln Lys Gly Trp Leu Val Gly Glu Gly Ala Gly Ala Val Val

725 730 735

Leu Lys Arg Gln Ile Asp Leu Gln Lys Gln Asp Asn Val Tyr Ala Val

740 745 750

Leu Glu His Ile Gly Gln Ala Ser Glu Gln Leu Asn Val Gly Tyr Gln

755 760 765

Glu Leu Val Ser Ser Gly Tyr Ala Ala Gln Asp His Gln Glu Leu Lys

770 775 780

Gln Leu Leu Ala Thr Gln Leu Glu Gln Lys Thr Ala Leu Gly Ser Val

785 790 795 800

Lys Thr Ser Phe Gly His Thr Gly Ala Ala Ser Gly Ile Ala Ala Leu

805 810 815

Ile Lys Thr Ala Leu Cys Leu His His Lys Phe Ile Pro Gly Ile Pro

820 825 830

Asn Trp Glu Ala Pro Gln Glu Ala Thr Ala Phe Ala Lys Thr Lys Tyr

835 840 845

Tyr Phe Pro Met Ala Ser Arg Pro Trp Leu Leu Asn Ala Gly Glu Lys

850 855 860

Arg Lys Ala Ala Ile Asn Gly Leu Glu Gly Leu Gln Ile His Leu Ser

865 870 875 880

Glu Gly Val Arg Ser Ser Pro Ala Pro Ser Pro Leu Leu Gln Gly Arg

885 890 895

Val Gly Ser Leu Phe Val Leu Lys Gly Asn Thr Glu Thr Ala Leu Arg

900 905 910

Glu Ala Leu Ala Leu Leu Leu Glu Asp Leu Ala Gly Lys Ser Ser Leu

915 920 925

Pro Glu Leu Ala Ala Arg Leu Tyr Tyr Asn His Gln Ala Lys Pro Ser

930 935 940

Ser Tyr Thr Ile Val Leu Leu Ala Asn Ser Lys Lys Asn Leu Gln Gln

945 950 955 960

Glu Ile Arg Phe Met Gln Val Gly Leu Glu Ala Ala Leu Thr Glu Asn

965 970 975

Lys Val Leu Lys Thr Pro Arg Gly Ser Tyr Phe Thr Ala Lys Pro Leu

980 985 990

Gly Lys Thr Gly Lys Ile Ala Phe Ser Tyr Pro Gly Ser Ala Thr Ala

995 1000 1005

Tyr Arg Gly Leu Gly Gln Asp Ile Phe Gln Leu Phe Pro Ser Leu

1010 1015 1020

His Glu His Phe Gly Gln Lys Leu Glu Asp Ile Ala Asp Phe Val

1025 1030 1035

Gly Ser Ser Tyr Leu His Pro Lys Leu Gln Ser Arg Gln Glu Glu

1040 1045 1050

Ala Pro Ser Ile Gln Thr Asp Ala Val Ser Met Met Cys Ala Gly

1055 1060 1065

Val Phe Ser Ser Ala Ile Tyr Thr His Leu Leu Lys Asp Lys Phe

1070 1075 1080

Gly Leu Lys Pro Asp Leu Ala Phe Gly Tyr Ser Met Gly Glu Ser

1085 1090 1095

Ala Gly Met Trp Tyr Ser Phe Asp Val Trp Asn Pro Asp Asn Thr

1100 1105 1110

Ala Val Phe Arg Asn Ser Asp Leu Phe Ala Asn Gln Leu Ser Gly

1115 1120 1125

Asp Leu Arg Leu Leu Ala Glu Thr Trp Gly Ile Ser Ser Glu Glu

1130 1135 1140

Ala Lys Ala Arg Trp Ile Ser Leu Ile Leu Leu Ala Asp Lys Glu

1145 1150 1155

Ala Val Gln Asn Leu Val Ala Gln Glu Asp Arg Cys Tyr Leu Ser

1160 1165 1170

Phe Ile Asn Thr Pro Gln Glu Val Ile Ile Ser Gly Asp Lys Glu

1175 1180 1185

Ala Cys Asn Arg Val Val Gln Gln Leu Gly Cys Pro Ala Val Glu

1190 1195 1200

Val Pro Phe Gln Asn Val Ile His His Asp Phe Cys Lys Lys Val

1205 1210 1215

Gln Glu Glu Leu Tyr Asp Met His His Phe Pro Leu Glu Thr Gln

1220 1225 1230

Pro Asn Ile Asp Phe Tyr Ser Ser Leu Ser Leu Ala Pro Leu Pro

1235 1240 1245

Met Asp Ser Gly Val Ile Ala Gln Asn Ser Thr Gln Val Cys Phe

1250 1255 1260

Gln Pro Val Asp Tyr Pro Thr Thr Ile Gln Gln Leu Tyr Asn Asp

1265 1270 1275

Gly Ala Arg Ile Phe Ile Glu Leu Gly Ala Gly Asn Thr Cys Thr

1280 1285 1290

Gln Trp Thr Ser Ser Ile Leu Gly Gln Gln Ala His Leu Ala Val

1295 1300 1305

Ser Cys Thr Gln Lys Gly Lys Pro Glu Gly Thr Ala Leu Leu Gln

1310 1315 1320

Ala Leu Ala Gln Leu Leu Ser His Gly Val Ala Leu Asp Leu Gln

1325 1330 1335

Pro Leu Phe Ala Ala Asp Leu Leu Ala Pro Ser Pro Arg Ala Phe

1340 1345 1350

Tyr Lys Ala Ile Val Ser Gly Gly Ala Arg Ile Phe Asp Tyr Leu

1355 1360 1365

Leu Gln Pro Gln Thr Lys Lys Gln Phe Ala Gly Val Thr Lys Thr

1370 1375 1380

Ala Leu Val Gln Gln Leu Glu Pro Ala Leu Ala Ser Asn Ser Arg

1385 1390 1395

Glu Tyr Ser Phe Thr Ser Thr Lys Thr Thr Thr Val Asp Thr Thr

1400 1405 1410

Gln Ser Ile Pro Ser Pro Ser Gln Lys Val Leu Leu Gly Glu Asn

1415 1420 1425

Gly Leu Lys Leu Gln Asp Phe Asn Asp Pro Asn His Leu Gln Gly

1430 1435 1440

Lys Thr Ile Ile Phe Ser Gln Glu Asp Leu Glu Glu Phe Ala Thr

1445 1450 1455

Gly Lys Ile Ala Lys Val Phe Gly Glu Glu Tyr Ser Ile Ile Asp

1460 1465 1470

Thr Tyr Lys Arg Arg Val Met Leu Pro Met Ala Pro Tyr Leu Leu

1475 1480 1485

Val Ser Arg Val Thr Gly Leu Asp Ala Lys Arg Gly Glu Phe Lys

1490 1495 1500

Pro Ser Thr Met Gln Thr Glu Tyr Asp Ile Pro Tyr Asn Ala Trp

1505 1510 1515

Phe Thr Thr Asp Gly Gln Ile Pro Trp Ala Val Ser Val Glu Ser

1520 1525 1530

Gly Gln Cys Asp Leu Leu Leu Ile Ser Tyr Leu Gly Ile Asp Phe

1535 1540 1545

Glu Asn Lys Gly Asp Leu Val Tyr Arg Leu Leu Asp Cys Thr Leu

1550 1555 1560

Thr Phe Val Asp Asp Leu Pro Phe Glu Gly Gln Thr Leu Arg Tyr

1565 1570 1575

Asp Ile Ser Ile Asn Ser Phe Val Arg Asn Gly Asp Asn Leu Leu

1580 1585 1590

Phe Phe Phe Ser Tyr Asn Cys Tyr Val Glu Asp Arg Leu Val Leu

1595 1600 1605

Lys Met Arg Asn Gly Cys Ala Gly Phe Phe Thr Asp Glu Gln Leu

1610 1615 1620

Glu Glu Gly Leu Gly Val Val Tyr Ser Lys Glu Glu Leu Glu Ala

1625 1630 1635

Lys Thr Asn Ala Lys Lys Pro Ala Phe Thr Pro Leu Leu Asn Thr

1640 1645 1650

Lys Lys Thr Ser Phe Ser Lys Glu Asp Leu His His Leu Ile Glu

1655 1660 1665

Gly Asn Met Glu Leu Cys Phe Asp Ser Pro Ala Tyr Phe Ala Asn

1670 1675 1680

Gly Arg Asn Pro Ser Leu Arg Leu Pro Pro Glu Gln Ile Leu Met

1685 1690 1695

Ile Asp Arg Ile Val Ser Val Asp Leu Lys Gly Gly Ala Tyr Gly

1700 1705 1710

Leu Gly Tyr Val Ile Ala Glu Lys Asp Leu Ala Pro Glu Asp Trp

1715 1720 1725

Tyr Phe Pro Cys His Phe Arg Asp Asp Glu Val Leu Ala Gly Ser

1730 1735 1740

Leu Gln Ala Glu Gly Gly Gly Asn Leu Leu Arg Phe Phe Met Leu

1745 1750 1755

Met Leu Gly Leu Gln Arg Leu Thr Lys Asp Ala Arg Tyr Gln Pro

1760 1765 1770

Ile Phe Asp Leu Pro Gln Lys Val Arg Cys Arg Lys Gln Val Thr

1775 1780 1785

Pro Ser Lys Asp Thr Lys Leu Val Tyr Lys Leu Glu Val Lys Glu

1790 1795 1800

Ile Gly Leu Val Pro Asn Pro Tyr Val Ile Ala Asp Leu Glu Ile

1805 1810 1815

Val Ser Asp Gly Val Ile Thr Val His Phe Glu Asn Leu Gly Leu

1820 1825 1830

Gln Leu Arg Glu Lys Asp Asn Pro Arg Tyr Leu Glu Gln Gln Lys

1835 1840 1845

Gly Val His Ile Ser Pro Arg Ser Lys Asp Ala Leu Leu Thr Glu

1850 1855 1860

Leu Asp Ile Thr Asn Phe Ala Leu Asn Asn Leu Ser Val Ala Phe

1865 1870 1875

Gly Pro Asp Phe Ala Cys Tyr Asp Gly Arg Thr Val Ser Arg Gln

1880 1885 1890

Pro Asn Thr Asp Leu Gln Leu Ile Ser Arg Val Leu Lys Ile Glu

1895 1900 1905

Gly Glu Arg Leu Asn Phe Lys Gln Pro Ser Thr Ile Tyr Ala Glu

1910 1915 1920

Tyr Asp Val Pro Glu Asp Ala Trp Tyr Tyr Gln Gln Asn Ala Ser

1925 1930 1935

Met Thr Met Pro Tyr Ser Val Leu Met Glu Ile Ala Leu Gln Pro

1940 1945 1950

Cys Gly Leu Leu Gly Ala Tyr Leu Gly Ser Thr Leu Pro Phe Ser

1955 1960 1965

Asp Lys Asn Leu Phe Phe Arg Asn Leu Asp Gly Thr Gly Glu Met

1970 1975 1980

Leu Glu Leu Pro Met Gly Thr Asp Trp Arg Gly Lys Thr Ile His

1985 1990 1995

Asn Lys Ala Val Leu Ala Ser Ser Val Ala Leu Gly Gly Thr Val

2000 2005 2010

Leu Gln Asn Tyr Thr Phe Glu Leu Ser Ile Asp Gly Gln Val Phe

2015 2020 2025

Tyr Lys Gly Lys Ser Ser Phe Gly Phe Phe Pro Ala Glu Ala Leu

2030 2035 2040

Ala Gln Gln Val Gly Leu Asp Asn Gly Thr Ala Val Ala Pro Trp

2045 2050 2055

Tyr Gln Gln Gln Asn Leu Ala Gln Lys Asp Tyr Met Ser Ile Lys

2060 2065 2070

Leu Asp Ser Leu Tyr Gly Lys Met Lys Leu Phe Lys Ala Pro Ala

2075 2080 2085

Asn Lys Pro His Tyr His Leu Ser Gly Glu Gln Leu Ser Leu Leu

2090 2095 2100

Asn Asn Leu Lys Ile Val Lys Asp Gly Gly Gln Tyr Gly Lys Gly

2105 2110 2115

Tyr Ile Tyr Gly His Gln Ala Ile Asn Leu Tyr Asp Trp Phe Phe

2120 2125 2130

Thr Cys His Phe Tyr Gln Asp Pro Val Met Pro Gly Ser Leu Gly

2135 2140 2145

Val Glu Ala Ile Leu Gln Ala Met Gln Thr Phe Ala Leu Gln Gln

2150 2155 2160

Asp Leu Gly Lys Asp Phe Lys Ser Pro Arg Phe Val Gln Val Pro

2165 2170 2175

Gln His Thr Thr Val Trp Lys Tyr Arg Gly Gln Ile Leu Gln Gly

2180 2185 2190

Val Glu Asn Met His Cys Glu Val His Phe Lys Ser Ile Glu Lys

2195 2200 2205

Lys Gly Glu Gln Leu Val Ile Val Gly Asp Ala Tyr Leu Trp Asn

2210 2215 2220

Glu Asp Thr Arg Ile Tyr Gln Ile Thr Asp Leu Ala Leu Gly Ile

2225 2230 2235

Glu Glu Ala

2240

<210> 31

<211> 2059

<212> PRT

<213> 裂殖壶菌（Schizochytrium sp）

<400> 31

Met Ala Ala Arg Asn Val Ser Ala Ala His Glu Met His Asp Glu Lys

1 5 10 15

Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys Thr

20 25 30

Lys Asp Glu Phe Trp Glu Val Leu Met Asn Gly Lys Val Glu Ser Lys

35 40 45

Val Ile Ser Asp Lys Arg Leu Gly Ser Asn Tyr Arg Ala Glu His Tyr

50 55 60

Lys Ala Glu Arg Ser Lys Tyr Ala Asp Thr Phe Cys Asn Glu Thr Tyr

65 70 75 80

Gly Thr Leu Asp Glu Asn Glu Ile Asp Asn Glu His Glu Leu Leu Leu

85 90 95

Asn Leu Ala Lys Gln Ala Leu Ala Glu Thr Ser Val Lys Asp Ser Thr

100 105 110

Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu

115 120 125

Gln Gly Glu Leu Leu Asn Val Tyr Gln Asn His Val Glu Lys Lys Leu

130 135 140

Gly Ala Arg Val Phe Lys Asp Ala Ser His Trp Ser Glu Arg Glu Gln

145 150 155 160

Ser Asn Lys Pro Glu Ala Gly Asp Arg Arg Ile Phe Met Asp Pro Ala

165 170 175

Ser Phe Val Ala Glu Glu Leu Asn Leu Gly Ala Leu His Tyr Ser Val

180 185 190

Asp Ala Ala Cys Ala Thr Ala Leu Tyr Val Leu Arg Leu Ala Gln Asp

195 200 205

His Leu Val Ser Gly Ala Ala Asp Val Met Leu Cys Gly Ala Thr Cys

210 215 220

Leu Pro Glu Pro Phe Phe Ile Leu Ser Gly Phe Ser Thr Phe Gln Ala

225 230 235 240

Met Pro Val Gly Thr Gly Gln Asn Val Ser Met Pro Leu His Lys Asp

245 250 255

Ser Gln Gly Leu Thr Pro Gly Glu Gly Gly Ser Ile Met Val Leu Lys

260 265 270

Arg Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu

275 280 285

Leu Gly Ala Asn Val Ser Asn Ser Gly Thr Gly Leu Pro Leu Lys Pro

290 295 300

Leu Leu Pro Ser Glu Lys Lys Cys Leu Met Asp Thr Tyr Thr Arg Ile

305 310 315 320

Asn Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly

325 330 335

Thr Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe

340 345 350

Glu Gly Lys Val Pro Arg Phe Gly Thr Thr Lys Gly Asn Phe Gly His

355 360 365

Thr Leu Val Ala Ala Gly Phe Ala Gly Met Cys Lys Val Leu Leu Ser

370 375 380

Met Lys His Gly Ile Ile Pro Pro Thr Pro Gly Ile Asp Asp Glu Thr

385 390 395 400

Lys Met Asp Pro Leu Val Val Ser Gly Glu Ala Ile Pro Trp Pro Glu

405 410 415

Thr Asn Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly

420 425 430

Gly Thr Asn Ala His Ala Val Phe Glu Glu His Asp Pro Ser Asn Ala

435 440 445

Ala Cys Thr Gly His Asp Ser Ile Ser Ala Leu Ser Ala Arg Cys Gly

450 455 460

Gly Glu Ser Asn Met Arg Ile Ala Ile Thr Gly Met Asp Ala Thr Phe

465 470 475 480

Gly Ala Leu Lys Gly Leu Asp Ala Phe Glu Arg Ala Ile Tyr Thr Gly

485 490 495

Ala His Gly Ala Ile Pro Leu Pro Glu Lys Arg Trp Arg Phe Leu Gly

500 505 510

Lys Asp Lys Asp Phe Leu Asp Leu Cys Gly Val Lys Ala Thr Pro His

515 520 525

Gly Cys Tyr Ile Glu Asp Val Glu Val Asp Phe Gln Arg Leu Arg Thr

530 535 540

Pro Met Thr Pro Glu Asp Met Leu Leu Pro Gln Gln Leu Leu Ala Val

545 550 555 560

Thr Thr Ile Asp Arg Ala Ile Leu Asp Ser Gly Met Lys Lys Gly Gly

565 570 575

Asn Val Ala Val Phe Val Gly Leu Gly Thr Asp Leu Glu Leu Tyr Arg

580 585 590

His Arg Ala Arg Val Ala Leu Lys Glu Arg Val Arg Pro Glu Ala Ser

595 600 605

Lys Lys Leu Asn Asp Met Met Gln Tyr Ile Asn Asp Cys Gly Thr Ser

610 615 620

Thr Ser Tyr Thr Ser Tyr Ile Gly Asn Leu Val Ala Thr Arg Val Ser

625 630 635 640

Ser Gln Trp Gly Phe Thr Gly Pro Ser Phe Thr Ile Thr Glu Gly Asn

645 650 655

Asn Ser Val Tyr Arg Cys Ala Glu Leu Gly Lys Tyr Leu Leu Glu Thr

660 665 670

Gly Glu Val Asp Gly Val Val Val Ala Gly Val Asp Leu Cys Gly Ser

675 680 685

Ala Glu Asn Leu Tyr Val Lys Ser Arg Arg Phe Lys Val Ser Thr Ser

690 695 700

Asp Thr Pro Arg Ala Ser Phe Asp Ala Ala Ala Asp Gly Tyr Phe Val

705 710 715 720

Gly Glu Gly Cys Gly Ala Phe Val Leu Lys Arg Glu Thr Ser Cys Thr

725 730 735

Lys Asp Asp Arg Ile Tyr Ala Cys Met Asp Ala Ile Val Pro Gly Asn

740 745 750

Val Pro Ser Ala Cys Leu Arg Glu Ala Leu Asp Gln Ala Arg Val Lys

755 760 765

Pro Gly Asp Ile Glu Met Leu Glu Leu Ser Ala Asp Ser Ala Arg His

770 775 780

Leu Lys Asp Pro Ser Val Leu Pro Lys Glu Leu Thr Ala Glu Glu Glu

785 790 795 800

Ile Gly Gly Leu Gln Thr Ile Leu Arg Asp Asp Asp Lys Leu Pro Arg

805 810 815

Asn Val Ala Thr Gly Ser Val Lys Ala Thr Val Gly Asp Thr Gly Tyr

820 825 830

Ala Ser Gly Ala Ala Ser Leu Ile Lys Ala Ala Leu Cys Ile Tyr Asn

835 840 845

Arg Tyr Leu Pro Ser Asn Gly Asp Asp Trp Asp Glu Pro Ala Pro Glu

850 855 860

Ala Pro Trp Asp Ser Thr Leu Phe Ala Cys Gln Thr Ser Arg Ala Trp

865 870 875 880

Leu Lys Asn Pro Gly Glu Arg Arg Tyr Ala Ala Val Ser Gly Val Ser

885 890 895

Glu Thr Arg Ser Cys Tyr Ser Val Leu Leu Ser Glu Ala Glu Gly His

900 905 910

Tyr Glu Arg Glu Asn Arg Ile Ser Leu Asp Glu Glu Ala Pro Lys Leu

915 920 925

Ile Val Leu Arg Ala Asp Ser His Glu Glu Ile Leu Gly Arg Leu Asp

930 935 940

Lys Ile Arg Glu Arg Phe Leu Gln Pro Thr Gly Ala Ala Pro Arg Glu

945 950 955 960

Ser Glu Leu Lys Ala Gln Ala Arg Arg Ile Phe Leu Glu Leu Leu Gly

965 970 975

Glu Thr Leu Ala Gln Asp Ala Ala Ser Ser Gly Ser Gln Lys Pro Leu

980 985 990

Ala Leu Ser Leu Val Ser Thr Pro Ser Lys Leu Gln Arg Glu Val Glu

995 1000 1005

Leu Ala Ala Lys Gly Ile Pro Arg Cys Leu Lys Met Arg Arg Asp

1010 1015 1020

Trp Ser Ser Pro Ala Gly Ser Arg Tyr Ala Pro Glu Pro Leu Ala

1025 1030 1035

Ser Asp Arg Val Ala Phe Met Tyr Gly Glu Gly Arg Ser Pro Tyr

1040 1045 1050

Tyr Gly Ile Thr Gln Asp Ile His Arg Ile Trp Pro Glu Leu His

1055 1060 1065

Glu Val Ile Asn Glu Lys Thr Asn Arg Leu Trp Ala Glu Gly Asp

1070 1075 1080

Arg Trp Val Met Pro Arg Ala Ser Phe Lys Ser Glu Leu Glu Ser

1085 1090 1095

Gln Gln Gln Glu Phe Asp Arg Asn Met Ile Glu Met Phe Arg Leu

1100 1105 1110

Gly Ile Leu Thr Ser Ile Ala Phe Thr Asn Leu Ala Arg Asp Val

1115 1120 1125

Leu Asn Ile Thr Pro Lys Ala Ala Phe Gly Leu Ser Leu Gly Glu

1130 1135 1140

Ile Ser Met Ile Phe Ala Phe Ser Lys Lys Asn Gly Leu Ile Ser

1145 1150 1155

Asp Gln Leu Thr Lys Asp Leu Arg Glu Ser Asp Val Trp Asn Lys

1160 1165 1170

Ala Leu Ala Val Glu Phe Asn Ala Leu Arg Glu Ala Trp Gly Ile

1175 1180 1185

Pro Gln Ser Val Pro Lys Asp Glu Phe Trp Gln Gly Tyr Ile Val

1190 1195 1200

Arg Gly Thr Lys Gln Asp Ile Glu Ala Ala Ile Ala Pro Asp Ser

1205 1210 1215

Lys Tyr Val Arg Leu Thr Ile Ile Asn Asp Ala Asn Thr Ala Leu

1220 1225 1230

Ile Ser Gly Lys Pro Asp Ala Cys Lys Ala Ala Ile Ala Arg Leu

1235 1240 1245

Gly Gly Asn Ile Pro Ala Leu Pro Val Thr Gln Gly Met Cys Gly

1250 1255 1260

His Cys Pro Glu Val Gly Pro Tyr Thr Lys Asp Ile Ala Lys Ile

1265 1270 1275

His Ala Asn Leu Glu Phe Pro Val Val Asp Gly Leu Asp Leu Trp

1280 1285 1290

Thr Thr Ile Asn Gln Lys Arg Leu Val Pro Arg Ala Thr Gly Ala

1295 1300 1305

Lys Asp Glu Trp Ala Pro Ser Ser Phe Gly Glu Tyr Ala Gly Gln

1310 1315 1320

Leu Tyr Glu Lys Gln Ala Asn Phe Pro Gln Ile Val Glu Thr Ile

1325 1330 1335

Tyr Lys Gln Asn Tyr Asp Val Phe Val Glu Val Gly Pro Asn Asn

1340 1345 1350

His Arg Ser Thr Ala Val Arg Thr Thr Leu Gly Pro Gln Arg Asn

1355 1360 1365

His Leu Ala Gly Ala Ile Asp Lys Gln Asn Glu Asp Ala Trp Thr

1370 1375 1380

Thr Ile Val Lys Leu Val Ala Ser Leu Lys Ala His Leu Val Pro

1385 1390 1395

Gly Val Thr Ile Ser Pro Leu Tyr His Ser Lys Leu Val Ala Glu

1400 1405 1410

Ala Glu Ala Cys Tyr Ala Ala Leu Cys Lys Gly Glu Lys Pro Lys

1415 1420 1425

Lys Asn Lys Phe Val Arg Lys Ile Gln Leu Asn Gly Arg Phe Asn

1430 1435 1440

Ser Lys Ala Asp Pro Ile Ser Ser Ala Asp Leu Ala Ser Phe Pro

1445 1450 1455

Pro Ala Asp Pro Ala Ile Glu Ala Ala Ile Ser Ser Arg Ile Met

1460 1465 1470

Lys Pro Val Ala Pro Lys Phe Tyr Ala Arg Leu Asn Ile Asp Glu

1475 1480 1485

Gln Asp Glu Thr Arg Asp Pro Ile Leu Asn Lys Asp Asn Ala Pro

1490 1495 1500

Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser

1505 1510 1515

Pro Ser Pro Ala Pro Ser Ala Pro Val Gln Lys Lys Ala Ala Pro

1520 1525 1530

Ala Ala Glu Thr Lys Ala Val Ala Ser Ala Asp Ala Leu Arg Ser

1535 1540 1545

Ala Leu Leu Asp Leu Asp Ser Met Leu Ala Leu Ser Ser Ala Ser

1550 1555 1560

Ala Ser Gly Asn Leu Val Glu Thr Ala Pro Ser Asp Ala Ser Val

1565 1570 1575

Ile Val Pro Pro Cys Asn Ile Ala Asp Leu Gly Ser Arg Ala Phe

1580 1585 1590

Met Lys Thr Tyr Gly Val Ser Ala Pro Leu Tyr Thr Gly Ala Met

1595 1600 1605

Ala Lys Gly Ile Ala Ser Ala Asp Leu Val Ile Ala Ala Gly Arg

1610 1615 1620

Gln Gly Ile Leu Ala Ser Phe Gly Ala Gly Gly Leu Pro Met Gln

1625 1630 1635

Val Val Arg Glu Ser Ile Glu Lys Ile Gln Ala Ala Leu Pro Asn

1640 1645 1650

Gly Pro Tyr Ala Val Asn Leu Ile His Ser Pro Phe Asp Ser Asn

1655 1660 1665

Leu Glu Lys Gly Asn Val Asp Leu Phe Leu Glu Lys Gly Val Thr

1670 1675 1680

Phe Val Glu Ala Ser Ala Phe Met Thr Leu Thr Pro Gln Val Val

1685 1690 1695

Arg Tyr Arg Ala Ala Gly Leu Thr Arg Asn Ala Asp Gly Ser Val

1700 1705 1710

Asn Ile Arg Asn Arg Ile Ile Gly Lys Val Ser Arg Thr Glu Leu

1715 1720 1725

Ala Glu Met Phe Met Arg Pro Ala Pro Glu His Leu Leu Gln Lys

1730 1735 1740

Leu Ile Ala Ser Gly Glu Ile Asn Gln Glu Gln Ala Glu Leu Ala

1745 1750 1755

Arg Arg Val Pro Val Ala Asp Asp Ile Ala Val Glu Ala Asp Ser

1760 1765 1770

Gly Gly His Thr Asp Asn Arg Pro Ile His Val Ile Leu Pro Leu

1775 1780 1785

Ile Ile Asn Leu Arg Asp Arg Leu His Arg Glu Cys Gly Tyr Pro

1790 1795 1800

Ala Asn Leu Arg Val Arg Val Gly Ala Gly Gly Gly Ile Gly Cys

1805 1810 1815

Pro Gln Ala Ala Leu Ala Thr Phe Asn Met Gly Ala Ser Phe Ile

1820 1825 1830

Val Thr Gly Thr Val Asn Gln Val Ala Lys Gln Ser Gly Thr Cys

1835 1840 1845

Asp Asn Val Arg Lys Gln Leu Ala Lys Ala Thr Tyr Ser Asp Val

1850 1855 1860

Cys Met Ala Pro Ala Ala Asp Met Phe Glu Glu Gly Val Lys Leu

1865 1870 1875

Gln Val Leu Lys Lys Gly Thr Met Phe Pro Ser Arg Ala Asn Lys

1880 1885 1890

Leu Tyr Glu Leu Phe Cys Lys Tyr Asp Ser Phe Glu Ser Met Pro

1895 1900 1905

Pro Ala Glu Leu Ala Arg Val Glu Lys Arg Ile Phe Ser Arg Ala

1910 1915 1920

Leu Glu Glu Val Trp Asp Glu Thr Lys Asn Phe Tyr Ile Asn Arg

1925 1930 1935

Leu His Asn Pro Glu Lys Ile Gln Arg Ala Glu Arg Asp Pro Lys

1940 1945 1950

Leu Lys Met Ser Leu Cys Phe Arg Trp Tyr Leu Ser Leu Ala Ser

1955 1960 1965

Arg Trp Ala Asn Thr Gly Ala Ser Asp Arg Val Met Asp Tyr Gln

1970 1975 1980

Val Trp Cys Gly Pro Ala Ile Gly Ser Phe Asn Asp Phe Ile Lys

1985 1990 1995

Gly Thr Tyr Leu Asp Pro Ala Val Ala Asn Glu Tyr Pro Cys Val

2000 2005 2010

Val Gln Ile Asn Lys Gln Ile Leu Arg Gly Ala Cys Phe Leu Arg

2015 2020 2025

Arg Leu Glu Ile Leu Arg Asn Ala Arg Leu Ser Asp Gly Ala Ala

2030 2035 2040

Ala Leu Val Ala Ser Ile Asp Asp Thr Tyr Val Pro Ala Glu Lys

2045 2050 2055

Leu