CN112601808A - 生产二十碳五烯酸的微生物和二十碳五烯酸的制造方法 - Google Patents

生产二十碳五烯酸的微生物和二十碳五烯酸的制造方法 Download PDF

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CN112601808A
CN112601808A CN201980053538.XA CN201980053538A CN112601808A CN 112601808 A CN112601808 A CN 112601808A CN 201980053538 A CN201980053538 A CN 201980053538A CN 112601808 A CN112601808 A CN 112601808A
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大利徹
佐藤康治
林祥平
中真以
氏原哲朗
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Abstract

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。

Description

生产二十碳五烯酸的微生物和二十碳五烯酸的制造方法
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]
Figure BDA0002942082320000261
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]
Figure BDA0002942082320000281
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]
Figure BDA0002942082320000311
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]
Figure BDA0002942082320000321
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
atg gcc tct cgc aag aat gtg agc gct gct cac gaa atg cac gac gag 48
Met Ala Ser Arg Lys Asn Val Ser Ala Ala His Glu Met His Asp Glu
1 5 10 15
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Lys Arg Ile Ala Val Val Gly Met Ala Val Gln Tyr Ala Gly Cys Lys
20 25 30
gac aag gaa gag ttc tgg aaa gta gtc atg ggc ggt gag gct gca tgg 144
Asp Lys Glu Glu Phe Trp Lys Val Val Met Gly Gly Glu Ala Ala Trp
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act aag att agc gat aaa cgc ctc gga tcc aac aag cga gcc gag cac 192
Thr Lys Ile Ser Asp Lys Arg Leu Gly Ser Asn Lys Arg Ala Glu His
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ttc aaa gca gag cgt agc aaa ttt gca gat acc ttt tgc aac gag aac 240
Phe Lys Ala Glu Arg Ser Lys Phe Ala Asp Thr Phe Cys Asn Glu Asn
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tac ggc tgc gtc gat gac tcc gtc gat aac gaa cac gag ctt ctc ctc 288
Tyr Gly Cys Val Asp Asp Ser Val Asp Asn Glu His Glu Leu Leu Leu
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aag ctc tcc aag aag gct ctc tcc gag aca tcg gtc tcc gac tct aca 336
Lys Leu Ser Lys Lys Ala Leu Ser Glu Thr Ser Val Ser Asp Ser Thr
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agg tgc ggt att gtg agc gga tgc ctg tcc ttt ccc atg gac aac ctc 384
Arg Cys Gly Ile Val Ser Gly Cys Leu Ser Phe Pro Met Asp Asn Leu
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cag ggc gaa ctc ctc aat gtg tac caa aac cac gtc gaa aag aaa ctc 432
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
245 250 255
cag ggt ctc acc cct ggt gaa ggt ggt gcc att atg gtt ctc aag cgc 816
Gln Gly Leu Thr Pro Gly Glu Gly Gly Ala Ile Met Val Leu Lys Arg
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ctt gac gac gct att cgc gat gga gac cac att tac ggt act ctg ctc 864
Leu Asp Asp Ala Ile Arg Asp Gly Asp His Ile Tyr Gly Thr Leu Leu
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ggt gct acc atc agc aat gct ggc tgt ggt ctt ccc ctc aag cca cac 912
Gly Ala Thr Ile Ser Asn Ala Gly Cys Gly Leu Pro Leu Lys Pro His
290 295 300
ttg ccc agc gag aag tcc tgc ctc att gat acc tac aag cgc gtc aac 960
Leu Pro Ser Glu Lys Ser Cys Leu Ile Asp Thr Tyr Lys Arg Val Asn
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gtg cac ccg cac aag atc cag tac gtc gag tgc cac gca acg ggt act 1008
Val His Pro His Lys Ile Gln Tyr Val Glu Cys His Ala Thr Gly Thr
325 330 335
ccc cag gga gac cgc gtt gag att gat gcc gtc aag gct tgc ttc gag 1056
Pro Gln Gly Asp Arg Val Glu Ile Asp Ala Val Lys Ala Cys Phe Glu
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ggc aag gtg cct cgc ttt gga agc tcc aag ggt aac ttt ggc cac aca 1104
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
385 390 395 400
atg gac ccg ctt gtg gtc tct gag ccc atc cca tgg ccc gac act gag 1248
Met Asp Pro Leu Val Val Ser Glu Pro Ile Pro Trp Pro Asp Thr Glu
405 410 415
ggc gag ccc aag cgc gct ggt ctc tcc gct ttc ggc ttt ggt ggc acc 1296
Gly Glu Pro Lys Arg Ala Gly Leu Ser Ala Phe Gly Phe Gly Gly Thr
420 425 430
aac gcc cac gca gtc ttt gag gag ttt gac cgc tcc aag gct gcc tgt 1344
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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ggc aac atg cgc att gct att acc ggt atg gat gcc acc ttc ggc tcc 1440
Gly Asn Met Arg Ile Ala Ile Thr Gly Met Asp Ala Thr Phe Gly Ser
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ctc aag ggc ctg gac gcc ttt gag cgt gcc atc tac aat ggc caa cat 1488
Leu Lys Gly Leu Asp Ala Phe Glu Arg Ala Ile Tyr Asn Gly Gln His
485 490 495
ggt gct gtg cca ttg cct gag aag cgc tgg cgt ttc ctt ggt aaa gac 1536
Gly Ala Val Pro Leu Pro Glu Lys Arg Trp Arg Phe Leu Gly Lys Asp
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aag gac ttt ttg gac ctg tgc ggc gtc aag gag gtg ccc cac gga tgc 1584
Lys Asp Phe Leu Asp Leu Cys Gly Val Lys Glu Val Pro His Gly Cys
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Tyr Ile Glu Asp Val Glu Val Asp Phe Ser Arg Leu Arg Thr Pro Met
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Thr Pro Asp Asp Met Leu Arg Pro Met Gln Leu Leu Ala Val Thr Thr
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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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tac aca tcc tac atc ggc aac ctc gtg gcc acc cgc gtg tct tca caa 1920
Tyr Thr Ser Tyr Ile Gly Asn Leu Val Ala Thr Arg Val Ser Ser Gln
625 630 635 640
tgg ggt ttc gag ggt cct tct ttc acc atc aca gag ggc aac aac tcc 1968
Trp Gly Phe Glu Gly Pro Ser Phe Thr Ile Thr Glu Gly Asn Asn Ser
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gtc tac cgt tgc gca gag ttg ggc aag tac ttg ctc gag act ggc gag 2016
Val Tyr Arg Cys Ala Glu Leu Gly Lys Tyr Leu Leu Glu Thr Gly Glu
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gtc gag gcc gta gtg atc gcc ggt gtg gat ctt tgc gcc agc gct gag 2064
Val Glu Ala Val Val Ile Ala Gly Val Asp Leu Cys Ala Ser Ala Glu
675 680 685
aat ctc tac gtg aag tcg cgt cgt ttc aag gtc tcg gag cag gag agc 2112
Asn Leu Tyr Val Lys Ser Arg Arg Phe Lys Val Ser Glu Gln Glu Ser
690 695 700
ccg cgg gcc agc ttc gac tcc ggc gct gac ggc tac ttt gtt ggt gag 2160
Pro Arg Ala Ser Phe Asp Ser Gly Ala Asp Gly Tyr Phe Val Gly Glu
705 710 715 720
gga tgt ggt gcc ctc gtc ctc aag cgc gag agc gac tgc acc aag gac 2208
Gly Cys Gly Ala Leu Val Leu Lys Arg Glu Ser Asp Cys Thr Lys Asp
725 730 735
gaa cgc att tac gcc tgc atg gac gct atc gtg ccc ggc aac atg ccg 2256
Glu Arg Ile Tyr Ala Cys Met Asp Ala Ile Val Pro Gly Asn Met Pro
740 745 750
gca gcc tgc atg gag gag gct ctc gcc cag gct cgc gtc aac ccc aag 2304
Ala Ala Cys Met Glu Glu Ala Leu Ala Gln Ala Arg Val Asn Pro Lys
755 760 765
gac gtt gag atg ctc gag ctc tcc gct gac tct gcc cgc cac ctc aag 2352
Asp Val Glu Met Leu Glu Leu Ser Ala Asp Ser Ala Arg His Leu Lys
770 775 780
aac ccc tcc gtt ctg cct aag gaa ctc act gct gag gag gaa atc cgc 2400
Asn Pro Ser Val Leu Pro Lys Glu Leu Thr Ala Glu Glu Glu Ile Arg
785 790 795 800
ggc att gag gcc att ctc agc cag cgc tct agc aac gaa gct gtg gag 2448
Gly Ile Glu Ala Ile Leu Ser Gln Arg Ser Ser Asn Glu Ala Val Glu
805 810 815
ccc cac aac gtc gct gtc agc agc gtc aag tcc act gtc ggt gac acc 2496
Pro His Asn Val Ala Val Ser Ser Val Lys Ser Thr Val Gly Asp Thr
820 825 830
ggc tac gcc tca gga gct gcc agt ctc atc aag acg gct ctc tgt ctg 2544
Gly Tyr Ala Ser Gly Ala Ala Ser Leu Ile Lys Thr Ala Leu Cys Leu
835 840 845
tac aac cgc tac ttg ccc tca aac ggc gcc tcc tgg gag gag cct gca 2592
Tyr Asn Arg Tyr Leu Pro Ser Asn Gly Ala Ser Trp Glu Glu Pro Ala
850 855 860
cct gag aca cag tgg ggc aag tct ctg tac gcg tgc cag tcc tcg cgg 2640
Pro Glu Thr Gln Trp Gly Lys Ser Leu Tyr Ala Cys Gln Ser Ser Arg
865 870 875 880
gcc tgg ttg aag aac cct gga gct cgc cgc cac gca gct gtc tca ggt 2688
Ala Trp Leu Lys Asn Pro Gly Ala Arg Arg His Ala Ala Val Ser Gly
885 890 895
gtt tcc gag acc cgt tca tgc tac acg gtg ctg ctc tct gat gtg gag 2736
Val Ser Glu Thr Arg Ser Cys Tyr Thr Val Leu Leu Ser Asp Val Glu
900 905 910
ggc cac cac gag acc aag agc cgc att tcg ctc gat gac gat gcc gtc 2784
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
930 935 940
gtt gac aag ctc cgc gag cgc ctc gcc cag cct agc gct aat gta cgt 2880
Val Asp Lys Leu Arg Glu Arg Leu Ala Gln Pro Ser Ala Asn Val Arg
945 950 955 960
ctt gct ttt atg gag ttg ctc ggc gag agc att gcc cag gag acc aag 2928
Leu Ala Phe Met Glu Leu Leu Gly Glu Ser Ile Ala Gln Glu Thr Lys
965 970 975
acc ccg ttg ccg gcc ttc gct ctg tgc ctg gtg acc tct cct agt aag 2976
Thr Pro Leu Pro Ala Phe Ala Leu Cys Leu Val Thr Ser Pro Ser Lys
980 985 990
ctc cag aag gag ctt gaa ctc gcc tcc aag ggc atc ccg cgg agt ctt 3024
Leu Gln Lys Glu Leu Glu Leu Ala Ser Lys Gly Ile Pro Arg Ser Leu
995 1000 1005
aag atg ggc cgc gac tgg aca tca ccc tcg ggc agc cac ttt gca 3069
Lys Met Gly Arg Asp Trp Thr Ser Pro Ser Gly Ser His Phe Ala
1010 1015 1020
ccc aag cca ctg tca agc gat cgc gtt gcg ttt atg tac ggc gaa 3114
Pro Lys Pro Leu Ser Ser Asp Arg Val Ala Phe Met Tyr Gly Glu
1025 1030 1035
ggc cga agc cct tac tat ggt atc ggc ctt gac att cac cgc atc 3159
Gly Arg Ser Pro Tyr Tyr Gly Ile Gly Leu Asp Ile His Arg Ile
1040 1045 1050
tgg ccc gaa ctt cac gag ttt gta aac gcc aag acc aac aag ctt 3204
Trp Pro Glu Leu His Glu Phe Val Asn Ala Lys Thr Asn Lys Leu
1055 1060 1065
tgg gat caa ggc gac aga tgg ttg atc ccg cgc gcc tcg acg aag 3249
Trp Asp Gln Gly Asp Arg Trp Leu Ile Pro Arg Ala Ser Thr Lys
1070 1075 1080
gag gag ctt aag gcg cag gaa gat gag ttc agc cgc aac cag gtg 3294
Glu Glu Leu Lys Ala Gln Glu Asp Glu Phe Ser Arg Asn Gln Val
1085 1090 1095
gag atg ttc cga ctc ggt att ctc atg tcc atg tgc ttc acc cac 3339
Glu Met Phe Arg Leu Gly Ile Leu Met Ser Met Cys Phe Thr His
1100 1105 1110
atc gct cgt gac gtg ctt ggc atc cag ccc aag gct gct ttc gga 3384
Ile Ala Arg Asp Val Leu Gly Ile Gln Pro Lys Ala Ala Phe Gly
1115 1120 1125
ctg agc ctt gga gag att tcc atg gtt ttt gcc ttt tct gag aag 3429
Leu Ser Leu Gly Glu Ile Ser Met Val Phe Ala Phe Ser Glu Lys
1130 1135 1140
aac ggc ctt gtc tct gag gag ctg aca act aaa ctc cgc aac tcg 3474
Asn Gly Leu Val Ser Glu Glu Leu Thr Thr Lys Leu Arg Asn Ser
1145 1150 1155
gag gtc tgg cgt aag gcc ctc gct gtt gag ttt gac gcc ctc cgc 3519
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
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Ala Leu Val Ala Ser Ile Asp Asp Thr Tyr Val Pro Ala Glu Lys
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Leu

Claims (8)

1.一种微生物，其是具有生产二十二碳六烯酸(以下称为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.如权利要求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.如权利要求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.如权利要求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)β-酮脂酰-ACP合酶(以下称为KS)结构域；
(b)丙二酰辅酶A：ACP酰基转移酶(以下称为MAT)结构域；
(c)ACP结构域；
(d)酮还原酶(以下称为KR)结构域；
(e)聚酮合酶脱水酶(以下称为PS-DH)结构域；
(f)链延伸因子(以下称为CLF)结构域；
(g)酰基转移酶(以下称为AT)结构域；
(h)FabA样β-羟酰-ACP脱水酶(以下称为FabA-DH)结构域；
(i)烯酰ACP还原酶(以下称为ER)结构域；
(j)磷酸泛酰巯基乙胺基转移酶(以下称为PPT)结构域。
6.如权利要求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，将权利要求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。
CN201980053538.XA 2018-08-10 2019-08-09 生产二十碳五烯酸的微生物和二十碳五烯酸的制造方法 Pending CN112601808A (zh)

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