CN117441008A - 重组微生物和c6化合物的制造方法 - Google Patents
重组微生物和c6化合物的制造方法 Download PDFInfo
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Abstract
本发明提供具有2,3‑脱氢己二酰‑CoA还原酶活性的重组微生物、以及C6化合物的制造方法,并且提供能够生产己二酸或己二酸衍生物的重组微生物、以及己二酸或己二酸衍生物的制造方法。一种重组微生物,其包含编码具有将2,3‑脱氢己二酰‑CoA还原而转换成己二酰CoA的酶活性的蛋白质的外源基因、以及编码3‑羟基己二酰CoA脱水酶的外源基因中的至少一者。
Description
技术领域
本发明涉及重组微生物以及C6化合物的制造方法。本发明还涉及重组微生物以及己二酸或其衍生物的制造方法。
背景技术
己二酸、六亚甲基二胺、1,6-己二醇、6-氨基己酸、6-氨基-1-己醇和6-羟基己酸之类的C6化合物被用于聚酰胺、聚氨酯原料等,在化学产业中被定位为重要的化合物。例如,己二酸(CAS No.124-04-9)是作为以尼龙-66等为代表的聚酰胺原料、氨基甲酸酯、以及增塑剂原料使用的单体化合物。己二酸目前的常见制造方法为化学合成法,例如有将单独的环己醇、或者环己醇与环己酮的混合物(K/A油)利用硝酸氧化的方法。
近年来,化石燃料面临着枯竭的危险,并且被认为是地球温室化的原因之一,因此在化学品制造工艺中,希望由化石燃料来源的原料过渡到可再生的原料、例如过渡到生物物质来源的原料,提出了基于通过基因重组修饰了代谢的微生物的发酵生产的制造方法。
例如已报告了用于己二酸、6-氨基己酸或己内酰胺生物合成的微生物(专利文献1)。作为一例,公开了对包含下述酶的微生物进行培养的方法:将琥珀酰-CoA和乙酰-CoA转换成3-氧代己二酰-CoA的“琥珀酰-CoA:乙酰-CoA酰基转移酶”;将3-氧代己二酰-CoA转换成3-羟基己二酰-CoA的“3-羟基酰基-CoA脱氢酶”;将3-羟基己二酰-CoA转换成5-羧基-2-戊烯酰基-CoA的“3-羟基己二酰-CoA脱水酶”;将5-羧基-2-戊烯酰基-CoA转换成己二酰-CoA的“5-羧基-2-戊烯酰基-CoA还原酶”;以及将己二酰-CoA转换成己二酸酯的“磷酸己二酰转移酶”和“己二酸激酶”,作为各酶的例示,仅止步于给出有可能促进反应的基因,而针对这些酶的基因,对于其能够由生物物质来源的碳源有效地生产C6化合物的情况并未得到证实。
另外还提出了包括以酶方式将2,3-脱氢己二酸硫酯转换成己二酸硫酯的工序的己二酸硫酯的制备方法(专利文献2),但限于热带念珠菌(Candida tropicalis)、细小裸藻(Euglena gracilis)、拜氏梭菌(Clostridium beijerinckii)和解脂亚罗酵母(Yarrowialipolytica)之类的生物种来源的烯酰CoA还原酶等一部分显示出酶活性,并未示出证实了C6化合物的发酵生产的示例。
此外还报告了,使用使大肠杆菌来源的酶PaaJ、PaaH和PaaF、以及贝氏不动杆菌(Acinetobacter baylyi)来源的酶DcaA和TesB共表达的基因重组大肠杆菌的培养,能够以葡萄糖作为碳源生产36mg/L的己二酸(非专利文献1)。
已证实了来源于多种生物种的反式烯酰CoA还原酶的酶活性,进而还提出了通过对表达出来源于热带念珠菌(Candida tropicalis)或Drosophila melanogaster的反式烯酰CoA还原酶或者这些酶的突变体的大肠杆菌进行培养而生产出己二酸和6-氨基己酸的示例(专利文献3)。
但是,基于上述现有技术的发酵生产的示例未必满足充分的生产率来贡献于工业利用,要求改善微生物的生产能力。
另外,关于己二酸,作为使用生物物质来源的原料的制造方法,还提出了使用基因重组微生物将生物物质来源的碳源转换成顺,顺-粘康酸,之后将所得到的顺,顺-粘康酸氢化来生产己二酸的方法(专利文献4)。另外还提出了使用基因重组体由α-酮戊二酸生产己二酸的方法(专利文献5)。
此外还提出了由生物物质来源的原料直接进行的己二酸的发酵生产方法(专利文献6)。该方法中,作为能够生产己二酸的非天然存在的微生物,举出了包含下述酶的非天然存在的微生物:将琥珀酰-CoA和乙酰-CoA转换成3-氧代己二酰-CoA的“琥珀酰-CoA:乙酰-CoA酰基转移酶”;将3-氧代己二酰-CoA转换成3-羟基己二酰-CoA的“3-羟基酰基-CoA脱氢酶”;将3-羟基己二酰-CoA转换成5-羧基-2-戊烯酰基-CoA的“3-羟基己二酰-CoA脱水酶”;将5-羧基-2-戊烯酰基-CoA转换成己二酰-CoA的“5-羧基-2-戊烯酰基-CoA还原酶”;以及将己二酰-CoA转换成己二酸酯的“磷酸己二酰转移酶”/“己二酸激酶”,但是关于各酶,仅止步于给出编码有可能促进反应的酶的基因,关于通过使用这些基因能够由生物物质来源的碳原料收率良好地生产己二酸的情况并未得到证实。
现有技术文献
专利文献
专利文献1:日本专利第5951990号说明书
专利文献2:日本特表2011-512868号公报
专利文献3:国际公开第2020/219859号
专利文献4:国际公开第1995/007996号
专利文献5:国际公开第2010/104391号
专利文献6:日本特开2011-515111号公报
非专利文献
非专利文献1:Kallscheuer,Nicolai,et al.“Improved production of adipatewith Escherichia coli by reversal ofβ-oxidation.”Applied microbiology andbiotechnology101.6(2017):2371-2382.
发明内容
发明所要解决的课题
本发明的目的在于提供具有2,3-脱氢己二酰-CoA还原酶活性的重组微生物、以及C6化合物的有效的制造方法。
本发明进一步的目的在于提供能够生产己二酸或己二酸衍生物的重组微生物、以及己二酸或己二酸衍生物的制造方法。
用于解决课题的手段
本发明人为了解决上述现有技术的课题进行了深入研究,结果发现,通过在宿主微生物中表达外源性的特定的酶,能够取得2,3-脱氢己二酰-CoA还原酶活性良好的重组微生物,进而通过对该重组微生物进行培养,能够有效地生产C6化合物,从而实现了本发明。
即,本发明提供下述方案。
[1]一种重组微生物,其包含外源基因,该外源基因编码具有将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性的蛋白质,其中,
上述外源基因为:
(i)编码由与序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列具有80%以上的序列一致性的氨基酸序列构成的蛋白质的DNA;
(ii)编码由在序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的蛋白质的DNA;
(iii)由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列具有80%以上的序列一致性的多核苷酸序列构成的DNA;
(iv)编码由在利用序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列编码的蛋白质的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的蛋白质的DNA;
(v)与由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列互补的多核苷酸序列构成的DNA在严谨条件下杂交的DNA;或者
(vi)由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列的简并异构体构成的DNA;
[2]如[1]中所述的重组微生物,其中,上述外源基因为:
(xi)编码由序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列构成的蛋白质的DNA;或者
(xiii)由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列构成的DNA;
[3]如[1]或[2]中所述的重组微生物,其中,上述外源基因来源于选自耳念珠菌(Candida auris)、马克斯克鲁维酵母(Kluyveromyces marxianus)、库德里阿兹威毕赤酵母(Pichia kudriavzevii)、喜热梭孢壳(Thermothelomyces thermophilus)、太瑞斯梭孢壳霉(Thermothielavioides terrestris)、嗜热毛壳菌(Chaetomium thermophilum)、丝状真菌柄孢霉(Podospora anserina)、淡紫拟青霉菌(Purpureocillium lilacinum)和圆核腔菌(Pyrenophora teres)中的至少一者;
[4]如[1]~[3]中任一项所述的重组微生物,其中,
上述重组微生物具有C6化合物的生产路径,
上述C6化合物为选自由己二酸、六亚甲基二胺、1,6-己二醇、6-氨基己酸、6-氨基-1-己醇和6-羟基己酸组成的组中的至少一者;
[5]如[1]~[4]中任一项所述的重组微生物,其中,上述重组微生物属于埃希氏菌(Escherichia)属、芽孢杆菌(Bacillus)属、棒状杆菌(Corynebacterium)属、节杆菌(Arthrobacter)属、短杆菌(Brevibacterium)属、梭菌(Clostridium)属、发酵单胞菌(Zymomonas)属、假单胞菌(Pseudomonas)属、伯克霍尔德菌(Burkholderia)属、链霉菌(Streptomyces)属、红球菌(Rhodococcus)属、集胞藻(Synechocystis)属、嗜碱盐芽孢杆菌(Alkalihalobacillus)属、酵母菌(Saccharomyces)属、裂殖酵母(Schizosaccharomyces)属、亚罗酵母(Yarrowia)属、念珠菌(Candida)属、毕赤酵母(Pichia)属、或曲霉(Aspergillus)属;
[6]如[1]~[5]中任一项所述的重组微生物,其中,上述重组微生物为大肠杆菌(Escherichia coli);
[7]如[1]~[6]中任一项所述的重组微生物,其进一步包含选自由
·编码3-氧代己二酰CoA硫解酶的基因、
·编码3-羟基己二酰CoA脱氢酶的基因、
·编码3-羟基己二酰CoA脱水酶的基因、
·编码羧酸还原酶的基因、
·编码醇脱氢酶的基因、以及
·编码转氨酶(氨基转移酶)的基因
组成的组中的至少一者;
[8]一种C6化合物的制造方法,其包括对[1]~[7]中任一项所述的重组微生物进行培养的培养工序,其中,上述C6化合物为选自由己二酸、六亚甲基二胺、1,6-己二醇、6-氨基己酸、6-氨基-1-己醇和6-羟基己酸组成的组中的至少一者;
[9]一种重组蛋白,其中,
(a)具有将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性;
(b)为下述任一者:
(i)由与序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列具有80%以上的序列一致性的氨基酸序列构成;
(ii)由在序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成;
(iii)利用由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列具有80%以上的序列一致性的多核苷酸序列构成的DNA进行编码;
(iv)由在利用序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列编码的蛋白质的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成;
(v)利用与由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列互补的多核苷酸序列构成的DNA在严谨条件下杂交的DNA进行编码;以及
(vi)利用由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列的简并异构体构成的DNA进行编码;
[10]一种重组蛋白,其中,
(xi)由序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列构成;或者
(xiii)利用由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列构成的DNA进行编码;
[11]一种C6化合物的制造方法,其包括使用[9]或[10]中所述的重组蛋白的步骤。
此外,本发明人为了解决上述课题反复进行了深入研究,结果发现,通过对于表达新近由自然界分离出的微生物伯克霍尔德菌属(Burkholderia sp.)LEBP-3株(保藏编号:NITE BP-03334)所具有的规定的酶的基因重组微生物进行培养,能够有效地生产己二酸类,从而完成了本发明。
即,本发明进一步提供下述方案:
[12]一种重组微生物,其包含编码3-羟基己二酰CoA脱水酶的外源基因和编码2,3-脱氢己二酰CoA还原酶的外源基因中的至少一者;
[13]如[12]中所述的重组微生物,其中,上述3-羟基己二酰CoA脱水酶和2,3-脱氢己二酰CoA还原酶中的至少一者来源于伯克霍尔德菌属(Burkholderiasp.)LEBP-3株(保藏编号:NITE BP-03334);
[14]如[12]或[13]中所述的重组微生物,其中,
上述3-羟基己二酰CoA脱水酶被与下述PCR扩增物的序列一致性为80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码,
该DNA具有626~940bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(1a)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(1b)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的;
[15]如[12]~[14]中任一项所述的重组微生物,其中,
上述2,3-脱氢己二酰CoA还原酶被与下述PCR扩增物的序列一致性为80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码,
该DNA具有924~1386bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(2a)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(2b)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的;
[16]如[12]或[13]中所述的重组微生物,其中,
上述3-羟基己二酰CoA脱水酶被作为下述PCR扩增物的DNA所编码,
该DNA具有783或784bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(1a)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(1b)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的;
[17]如[12]~[14]中任一项所述的重组微生物,其中,
上述2,3-脱氢己二酰CoA还原酶被作为下述PCR扩增物的DNA所编码,
该DNA具有1155或1156bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(2a)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(2b)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的;
[18]如[12]~[17]中任一项所述的重组微生物,其中,上述重组微生物属于选自由埃希氏菌属、棒状杆菌属、芽孢杆菌属、不动杆菌属、伯克霍尔德菌属、假单胞菌属、梭菌属、酵母菌属、裂殖酵母属、亚罗酵母属、念珠菌属、毕赤酵母属和曲霉属组成的组中的属;
[19]如[12]~[18]中任一项所述的重组微生物,其具有己二酸生产路径;
[20]如[12]~[19]中任一项所述的重组微生物,其具有六亚甲基二胺生产路径;
[21]如[12]~[19]中任一项所述的重组微生物,其具有1,6-己二醇生产路径;
[22]如[12]~[19]中任一项所述的重组微生物,其具有6-氨基-1-己醇生产路径;
[23]一种目标化合物的制造方法,其包括对[12]~[18]中任一项所述的重组微生物进行培养的培养工序,其中,
上述目标化合物选自由己二酸、己二酸衍生物、六亚甲基二胺、1,6-己二醇和6-氨基-1-己醇组成的组,
上述己二酸衍生物选自由氧代己二酸、乙酰丙酸、3-羟基己二酸和2,3-脱氢己二酸组成的组;
[24]一种己二酸的制造方法,其包括对[19]中所述的重组微生物进行培养的培养工序;
[25]一种六亚甲基二胺的制造方法,其包括对[20]中所述的重组微生物进行培养的培养工序;
[26]一种1,6-己二醇的制造方法,其包括对[21]中所述的重组微生物进行培养的培养工序;
[27]一种6-氨基-1-己醇的制造方法,其包括对[22]中所述的重组微生物进行培养的培养工序;
[28]一种重组多肽,其是下述(A-1)、(A-2)或(A-3)的重组多肽:
(A-1)被与下述PCR扩增物的序列一致性为80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码的重组多肽,
具有3-羟基己二酰CoA脱水酶活性,
上述DNA具有626~940bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(a1)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(a2)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的;
(A-2)由在利用作为下述PCR扩增物的DNA所编码的多肽的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的重组多肽,
具有3-羟基己二酰CoA脱水酶活性,
上述DNA具有626~940bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(a1)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(a2)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的;
(A-3)被由下述PCR扩增物的简并异构体构成的DNA所编码的重组多肽,
具有3-羟基己二酰CoA脱水酶活性,
上述DNA具有626~940bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(a1)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(a2)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的;
[29]一种重组多肽,其是下述(B-1)、(B-2)或(B-3)的重组多肽:
(B-1)被与下述PCR扩增物的序列一致性为80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码的重组多肽,
具有2,3-脱氢己二酰CoA还原酶活性,
上述DNA具有924~1386bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(b1)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(b2)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的;
(B-2)由在利用作为下述PCR扩增物的DNA所编码的多肽的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的重组多肽,
具有2,3-脱氢己二酰CoA还原酶活性,
上述DNA具有924~1386bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(b1)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(b2)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的;
(B-3)被由下述PCR扩增物的简并异构体构成的DNA所编码的重组多肽,
具有2,3-脱氢己二酰CoA还原酶活性,
上述DNA具有924~1386bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(b1)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(b2)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的。
发明的效果
根据本发明,可提供具有2,3-脱氢己二酰-CoA还原酶活性的重组微生物、以及C6化合物的制造方法。
另外,根据本发明,可提供能够生产己二酸或己二酸衍生物的重组微生物、以及己二酸或己二酸衍生物的制造方法。
附图说明
图1是示出由乙酰-CoA和琥珀酰-CoA合成C6化合物的生物合成路径的实例的图。
图2是示出各酶的氨基酸序列以及引物的碱基序列的图。
图3是示出各酶的氨基酸序列的图。
图4是示出各酶的氨基酸序列的图。
图5是示出各酶的氨基酸序列的图。
图6是示出编码各酶的碱基序列、以及引物的碱基序列的图。
图7是示出编码各酶的碱基序列、以及引物的碱基序列的图。
图8是示出编码各酶的碱基序列、以及引物的碱基序列的图。
图9是示出编码各酶的碱基序列的图。
图10是示出编码各酶的碱基序列的图。
图11是示出各引物的碱基序列的图。
图12是示出编码各酶的碱基序列、以及各引物的碱基序列的图。
图13是示出各引物的碱基序列的图。
图14是示出编码各酶的碱基序列、以及各引物的碱基序列的图。
图15是示出各酶的氨基酸序列、编码各酶的碱基序列、以及各引物的碱基序列的图。
图16示出本发明的重组微生物所具有的己二酸、己二酸衍生物、六亚甲基二胺、1,6-己二醇和6-氨基-1-己醇生产路径的实例。
图17示出PCR中的扩增对象的基因和所使用的引物。
图18示出各酶的氨基酸序列。
图19A示出编码各酶的碱基序列。
图19B示出编码各酶的碱基序列。
图20A示出PCR中的扩增对象和所使用的引物。
图20B示出PCR中的扩增对象和所使用的引物。
图21示出酶的氨基酸序列。
图22A示出酶的氨基酸序列。
图22B示出酶的氨基酸序列。
图22C示出酶的氨基酸序列。
图22D示出酶的氨基酸序列。
图22E示出酶的氨基酸序列。
图23A示出酶的氨基酸序列。
图23B示出酶的氨基酸序列。
图23C示出酶的氨基酸序列。
图23D示出酶的氨基酸序列。
图24A示出酶的氨基酸序列。
图24B示出酶的氨基酸序列。
图24C示出酶的氨基酸序列。
图24D示出酶的氨基酸序列。
图24E示出酶的氨基酸序列。
图25示出酶的氨基酸序列。
具体实施方式
以下对本发明的实施方式进行详细说明。需要说明的是,本发明并不限定于以下的实施方式,可以在其要点的范围内进行各种变形来实施。另外,只要没有特别载明,本说明书中所述的DNA的取得、载体的制备和转化等基因操作可通过Molecular Cloning 4thEdition(Cold Spring Harbor Laboratory Press,2012)、Current Protocols inMolecular Biology(Greene Publishing Associates and Wiley-Interscience)以及基因工程实验笔记(遺伝子工学実験ノート)(羊土社田村隆明)等公知的文献中记载的方法来进行。本说明书中,只要不特别声明,核苷酸序列以从5’方向朝向3’方向来记载。本说明书中,“多肽”和“蛋白质”的术语能够互换使用。
本说明书中,使用“~”表示的数值范围表示包含“~”的前后记载的数值分别作为最小值和最大值的范围。本说明书中阶段性地记载的数值范围中,某一阶段的数值范围的上限值或下限值可以与其他阶段的数值范围的上限值或下限值任意地组合。
本说明书中,“内源”或“内源性”这一术语用于表示下述含义:未进行所提及的基于基因重组的修饰的宿主微生物无论是否将所提及的基因或由其编码的蛋白质(代表性地为酶)以可在该宿主细胞内进行显性的生物化学反应的程度进行了功能性表达,均包含在宿主微生物中。
本说明书中,“外源”或“外源性”这一术语用于表示下述含义:在基因重组前的宿主微生物不具有要由本发明导入的基因的情况下;在实质上不表达基于该基因的酶的情况下;以及在该酶的氨基酸序列由该基因或者不同的基因编码、但在基因重组后不表达相匹配的内源性酶活性的情况下,向宿主中导入基于本发明的基因或核酸序列。
<1>发明A:重组微生物和C6化合物的制造方法
本发明中的重组微生物包含编码具有将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性的蛋白质的外源基因。将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的反应如图1的步骤D所示。“具有将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性的蛋白质”也被称为“2,3-脱氢己二酰CoA还原酶”。另外,将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性也被称为“2,3-脱氢己二酰CoA还原酶活性”或“2,3-脱氢己二酰CoA还原酶酶活性”,这些用语可互换使用。
本发明中的重组微生物中所包含的外源基因选自下述基因:
(i)编码由与序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列具有80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的序列一致性的氨基酸序列构成的蛋白质的DNA;
(ii)编码由在序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列中缺失、置换、插入和/或添加1~10个、1~7个、1~5个或1~3个氨基酸而成的氨基酸序列构成的蛋白质的DNA;
(iii)由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列具有80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的序列一致性的多核苷酸序列构成的DNA;
(iv)编码由在利用序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列编码的蛋白质的氨基酸序列中缺失、置换、插入和/或添加1~10个、1~7个、1~5个或1~3个氨基酸而成的氨基酸序列构成的蛋白质的DNA;
(v)与由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列互补的多核苷酸序列构成的DNA在严谨条件下杂交的DNA;
(vi)由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列的简并异构体构成的DNA。
本发明中的重组微生物中所包含的外源基因优选选自下述基因:
(i)编码由与序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列具有80%以上的序列一致性的氨基酸序列构成的蛋白质的DNA;
(ii)编码由在序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的蛋白质的DNA;
(iii)由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列具有80%以上的序列一致性的多核苷酸序列构成的DNA;
(iv)编码由在利用序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列编码的蛋白质的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的蛋白质的DNA;
(v)与由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列互补的多核苷酸序列构成的DNA在严谨条件下杂交的DNA;
(vi)由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列的简并异构体构成的DNA。
本发明中重组微生物中所包含的外源基因更优选为下述基因:
(xi)编码由序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列构成的蛋白质的DNA;或者
(xiii)由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列构成的DNA。
上述外源基因例如来源于选自耳念珠菌(Candida auris)、马克斯克鲁维酵母(Kluyveromyces marxianus)、库德里阿兹威毕赤酵母(Pichia kudriavzevii)、喜热梭孢壳(Thermothelomyces thermophilus)、太瑞斯梭孢壳霉(Thermothielavioidesterrestris)、嗜热毛壳菌(Chaetomium thermophilum)、丝状真菌柄孢霉(Podosporaanserina)、淡紫拟青霉菌(Purpureocillium lilacinum)和圆核腔菌(Pyrenophorateres)中的至少一者。
通过包含上述外源基因,重组微生物具有良好的2,3-脱氢己二酰-CoA还原酶活性。通过使用具有该酶活性的重组微生物,能够制造出目标化合物。作为目标化合物,例如可以举出C6化合物。
本说明书中,“C6化合物”中包括:
·己二酸(CAS No.124-04-9)、
·六亚甲基二胺(CAS No.124-09-4)、
·1,6-己二醇(CAS No.629-11-8)、
·6-氨基己酸(CAS No.60-32-2)、
·6-氨基-1-己醇(CAS No.4048-33-3)、以及
·6-羟基己酸(CAS No.1191-25-9),
“C6化合物”是指选自由这些化合物组成的组中的1种或2种以上的化合物。
如本领域技术人员所理解,本说明书中的“C6化合物”可采取包括任意的盐形态的中性或电离形态,并且该形态取决于pH。
如上所述,本发明的微生物是导入有外源性酶基因的基因重组微生物。“基因重组微生物”也被简称为“重组微生物”。
就本发明而言,导入目标外源基因的宿主微生物没有特别限定,可以为原核生物和真核生物中的任一者。可以从已经分离保存的微生物、新近从自然界分离出的微生物、以及进行了基因修饰的微生物中任意地选择。宿主微生物例如属于埃希氏菌(Escherichia)属、芽孢杆菌(Bacillus)属、棒状杆菌(Corynebacterium)属、节杆菌(Arthrobacter)属、短杆菌(Brevibacterium)属、梭菌(Clostridium)属、发酵单胞菌(Zymomonas)属、假单胞菌(Pseudomonas)属、伯克霍尔德菌(Burkholderia)属、链霉菌(Streptomyces)属、红球菌(Rhodococcus)属、集胞藻(Synechocystis)属、嗜碱盐芽孢杆菌(Alkalihalobacillus)属、酵母菌(Saccharomyces)属、裂殖酵母(Schizosaccharomyces)属、亚罗酵母(Yarrowia)属、念珠菌(Candida)属、毕赤酵母(Pichia)属、或曲霉(Aspergillus)属。宿主微生物优选属于埃希氏菌(Escherichia)属,更优选为大肠杆菌(Escherichia coli)。
因此,在上述宿主微生物在导入了外源基因的本发明中的重组微生物例如属于埃希氏菌(Escherichia)属、芽孢杆菌(Bacillus)属、棒状杆菌(Corynebacterium)属、节杆菌(Arthrobacter)属、短杆菌(Brevibacterium)属、梭菌(Clostridium)属、发酵单胞菌(Zymomonas)属、假单胞菌(Pseudomonas)属、伯克霍尔德菌(Burkholderia)属、链霉菌(Streptomyces)属、红球菌(Rhodococcus)属、集胞藻(Synechocystis)属、嗜碱盐芽孢杆菌(Alkalihalobacillus)属、酵母菌(Saccharomyces)属、裂殖酵母(Schizosaccharomyces)属、亚罗酵母(Yarrowia)属、念珠菌(Candida)属、毕赤酵母(Pichia)属、或曲霉(Aspergillus)属。本发明中的重组微生物优选属于埃希氏菌(Escherichia)属,更优选为大肠杆菌(Escherichia coli)。
本说明书中,关于某一化合物“具有生产路径”,是指本发明中的基因重组微生物可表达出足以进行该化合物的生产路径的各反应阶段的量的酶,能够生物合成该化合物。本发明的重组微生物可以使用本来具有该化合物的生产能力的宿主微生物,也可以对本来不具有该化合物的生产能力的宿主微生物进行修饰以使其具有该化合物的生产能力。
本说明书中,“来源于”是指所提及的特定生物种内源性地具有基因或由其编码的蛋白质(代表性地为酶)。
本发明的基因重组微生物包含编码具有将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性(即2,3-脱氢己二酰CoA还原酶活性)的蛋白质的外源基因,由此表达出足以进行反应的量的酶(即2,3-脱氢己二酰CoA还原酶)。该酶参与图1的步骤D的反应。
另外,通过进一步表达催化C6化合物生产路径的反应阶段的酶,可构成具有C6化合物生产路径的基因重组微生物。
在一个方式中,本发明中的重组微生物进一步包含选自由下述基因组成的组中的至少一者:
·编码3-氧代己二酰CoA硫解酶基因、
·编码3-羟基己二酰CoA脱氢酶的基因、
·编码3-羟基己二酰CoA脱水酶的基因、
·编码磷酸己二酰转移酶的基因与编码己二酸激酶的基因的组合、
·编码己二酰-CoA水解酶的基因、
·编码己二酸CoA转移酶的基因、
·编码脱氢酶的基因、
·编码羧酸还原酶的基因、
·编码转氨酶(氨基转移酶)的基因、
·编码醇脱氢酶的基因、以及
·编码CoA转移酶的基因、
·编码酸硫醇连接酶的基因。
·3-氧代己二酰CoA硫解酶催化图1的步骤A的反应、
·3-羟基己二酰CoA脱氢酶催化图1的步骤B的反应、
·3-羟基己二酰CoA脱水酶催化图1的步骤C的反应、
·磷酸己二酰转移酶己二酸激酶的组合、己二酰-CoA水解酶、己二酸CoA转移酶催化图1的步骤E的反应、
·脱氢酶催化图1的步骤F、U和V中的至少一者的反应、
·羧酸还原酶催化图1的步骤G、I、K和N中的至少一者的反应、
·转氨酶(氨基转移酶)催化图1的步骤J、M、P和R中的至少一者的反应、
·醇脱氢酶催化图1的步骤H、L、O和Q中的至少一者的反应、
·CoA转移酶或酸硫醇连接酶催化步骤S和T的至少一者的反应。
在优选的一个方式中,本发明中的重组微生物进一步包含选自由下述基因组成的组中的至少一者:
·编码3-氧代己二酰CoA硫解酶的基因、
·编码3-羟基己二酰CoA脱氢酶的基因、
·编码3-羟基己二酰CoA脱水酶的基因、
·编码羧酸还原酶的基因、
·编码醇脱氢酶的基因、以及
·编码转氨酶(氨基转移酶)的基因。
需要说明的是,本发明的基因重组微生物即使不通过外源基因的导入也可表达出足以催化C6化合物生产路径的反应阶段的量的酶的情况下,可通过由内源基因编码的酶进行反应。
将本发明中的基因重组微生物可以具有的C6化合物生产路径的一例示于图1。以下参照图1对催化各反应阶段的酶进行说明。
图1的步骤A中,琥珀酰-CoA与乙酰-CoA缩合,转换成3-氧代己二酰-CoA。作为可催化该转换的酶的示例,可以举出β-酮硫解酶。进而,还可例示出例如被分类在EC 2.3.1.9(乙酰基乙酰CoA硫解酶)、EC 2.3.1.16(3-酮酰-CoA硫解酶)和EC 2.3.1.174(3-氧代己二酰-CoA硫解酶)等组中的酶作为可对该转换具有活性的酶。作为可在该转换中使用的酶,只要对该转换具有活性就没有限定,例如可以举出琥珀酰CoA:乙酰CoA酰基转移酶和3-氧代己二酰CoA硫解酶。在一个方式中,使用由序列编号1中记载的氨基酸序列构成的大肠杆菌来源的PaaJ。
在图1的步骤B中,3-氧代己二酰-CoA转换成3-羟基己二酰-CoA。作为可催化该转换的酶的示例,可以举出被分类在EC 1.1.1组中的氧化还原酶。例如可例示出被分类在EC1.1.1.35(3-羟基酰基-CoA脱氢酶)、EC 1.1.1.36(乙酰基乙酰-CoA脱氢酶)、EC 1.1.1.157(3-羟基丁酰基-CoA脱氢酶)、EC 1.1.1.211(长链3-羟基酰基-CoA脱氢酶)、EC 1.1.1.259(3-羟基庚二酰-CoA脱氢酶)之类的组中的酶作为可对该转换具有活性的酶。可在本发明中使用的酶只要具有对该转换的活性就没有限定,例如为3-羟基己二酰CoA脱氢酶。在一个方式中,使用由序列编号2中记载的氨基酸序列构成的大肠杆菌来源的PaaH。
在图1的步骤C中,3-羟基己二酰-CoA被转换成2,3-脱氢己二酰-CoA。作为可催化该转换的酶的示例,可以举出被分类在EC 4.2.1组中的氢化裂解酶。例如可例示出被分类在EC 4.2.1.17(烯酰基-CoA水合酶)、EC 4.2.1.55(3-羟基丁酰-CoA脱水酶)、EC 4.2.1.74(长链烯酰基-CoA水合酶)之类的组中的酶作为可对该转换具有活性的酶。可在本发明中使用的酶只要对该转换具有活性就没有限定,例如为3-羟基己二酰CoA脱水酶。在一个方式中,使用由序列编号3中记载的氨基酸序列构成的大肠杆菌来源的PaaF。另外,作为其他方式,作为该酶,使用被下述PCR扩增物的碱基序列(783bp)所编码的3-羟基己二酰CoA脱水酶PaaF(L3),该PCR扩增物是使用序列编号4所示的核苷酸和序列编号46所示的核苷酸作为引物、以伯克霍尔德菌属(Burkholderiasp.)LEBP-3株的染色体DNA作为模板得到的。伯克霍尔德菌属(Burkholderiasp.)LEBP-3株(下文中也简称为“L3株”)于2020年12月4日向独立行政法人制品评价技术基础机构专利微生物保藏中心(NPMD)(地址:千叶县木更津市かずさ镰足2-5-8 122号室)提交了保藏申请(受理号:NITE ABP-03334),以“保藏号:NITE BP-03334”进行了国际保藏。
此处,作为PCR扩增物的制备中使用的DNA聚合酶,使用该领域中已知的酶,例如可以举出Taq DNA聚合酶、进行了热启动调整的DNA聚合酶、与聚合酶活性分开地具有3’-5’核酸外切酶活性的校对DNA聚合酶等,但并不限定于这些。在PCR扩增物的制备条件的一个优选方式中,使用各1μM的特定的核苷酸作为正向引物和反向引物,并且作为酶使用PrimeSTAR Max DNA Polymerase(产品名,Takara Bio制),以25μL的液量在热处理98℃10秒、退火55℃15秒、延伸72℃5秒/kb的条件下实施30次循环的处理,由此制备该PCR扩增物。
在图1的步骤D中,2,3-脱氢己二酰-CoA被转换成己二酰-CoA。作为可催化该转换的酶的示例,可以举出被分类在EC 1.3.1组中的氧化还原酶。例如可例示出被分类在EC1.3.1.8(酰基-CoA脱氢酶(NADP+))、EC 1.3.1.9(烯酰基-ACP还原酶(NADH))、EC 1.3.1.38(反式-2-烯酰基-CoA还原酶(NADP+))、EC 1.3.1.44(反式-2-烯酰基-CoA还原酶(NAD+))、EC1.3.1.86(巴豆基-CoA还原酶)、EC 1.3.1.93(长链酰基-CoA还原酶)、EC 1.3.1.104(烯酰基-ACP还原酶(NADPH))之类的组中的酶作为可对该转换具有活性的酶。本发明中,步骤D的转换中使用上述2,3-脱氢己二酰CoA还原酶。
本发明中所使用的2,3-脱氢己二酰CoA还原酶例如为来源于下述生物种的酶:耳念珠菌(Candida auris)(序列编号8)、马克斯克鲁维酵母(Kluyveromyces marxianus)(序列编号9)、库德里阿兹威毕赤酵母(Pichia kudriavzevii)(序列编号10)、喜热梭孢壳(Thermothelomyces thermophilus)(序列编号11)、太瑞斯梭孢壳霉(Thermothielavioides terrestris)(序列编号12)、嗜热毛壳菌(Chaetomiumthermophilum)(序列编号13)、丝状真菌柄孢霉(Podospora anserina)(序列编号14)、淡紫拟青霉菌(Purpureocillium lilacinum)(序列编号15)、圆核腔菌(Pyrenophora teres)(序列编号16)。优选使用由序列编号8~16中记载的氨基酸序列构成的酶中的至少一者,更优选使用由序列编号11、12、13、14和16中记载的氨基酸序列构成的酶中的至少一者(图3)。
图1的步骤E中,己二酰-CoA被转换成己二酸。作为可催化该转换的酶的示例,可以举出被分类在EC 3.1.2组中的硫酯水解酶。例如可例示出被分类在EC 3.1.2.1(乙酰-CoA水解酶)、EC 3.1.2.20(酰基-CoA水解酶)之类的组中的酶作为可对该转换具有活性的酶。可在本发明中使用的酶只要对该转换具有活性就没有限定,例如为己二酰-CoA水解酶。
另外,作为可催化图1的步骤E的反应的其他酶的示例,还可以举出被分类在EC2.8.3组中的CoA-转移酶。例如可例示出被分类在EC 2.8.3.5(3-含氧酸CoA-转移酶)、EC2.8.3.6(3-氧代己二酸CoA-转移酶)、EC 2.8.3.18(琥珀酰-CoA:乙酰-CoA-转移酶)之类的组中的酶作为可对该转换具有活性的酶。可在本发明中使用的酶只要对该转换具有活性就没有限定,例如为己二酸CoA转移酶。
此外,作为可催化图1的步骤E的反应的其他酶转换的示例,还可例示出下述路径:通过被分类在EC 2.3.1组中的酰基转移酶将己二酰-CoA的己二酰基转移至磷酸而生成己二酰磷酸后,利用被分类在EC 2.7.2组中的磷酸转移酶进行脱磷酸化。例如,作为酰基转移酶,可例示出被分类在EC 2.3.1.8(磷酸乙酰基转移酶)、EC 2.3.1.19(磷酸丁酰基转移酶)之类的组中的酶作为可对该转换具有活性的酶。作为磷酸转移酶,可例示出被分类在EC2.7.2.1(乙酸激酶)、EC 2.7.2.7(丁酸激酶)之类的组中的酶作为可对该转换具有活性的酶。可在本发明中使用的酶只要对该转换具有活性就没有限定,例如为磷酸己二酰转移酶和己二酸激酶。
图1的步骤F、U和V中,酰基-CoA被转换成醛。作为可催化该转换的酶的示例,可以举出被分类在EC 1.2.1组中的酶。出于与该转换同样地催化CoA脱离而生成醛的转换反应的原因,例如可例示出被分类在EC 1.2.1.10(乙醛脱氢酶(乙酰化))、EC 1.2.1.17(乙醛酸脱氢酶(酰化))、EC 1.2.1.42(十六醛脱氢酶(酰化))、EC 1.2.1.44(肉桂酰-CoA还原酶(酰化))、EC 1.2.1.75(丙二酰-CoA还原酶(形成丙二酸半醛))、EC1.2.1.76(琥珀酸半醛脱氢酶(酰化))之类的组中的酶作为对该转换也可具有活性的酶。可在本发明中使用的酶只要对该转换具有活性就没有限定,例如可使用由序列编号17中记载的氨基酸序列构成的克氏梭菌(Clostridium kluyveri)来源的sucD(图4)。
在图1的步骤G、I、K和N中,将羧基转换成醛。作为可催化该转换的酶,例如可以举出羧酸还原酶(Carboxylic Acid Reductase;CAR)。出于与该转换同样地可催化由羧酸生成醛的转换反应的原因,可例示出例如被分类在EC 1.2.1.30(羧酸还原酶(NADP+))、EC1.2.1.31(L-氨基己二酸半醛脱氢酶)、EC 1.2.1.95(L-2-氨基己二酸还原酶)、EC1.2.99.6(羧酸还原酶)之类的组中的酶作为对该转换也可具有活性的酶。关于作为酶的来源的生物种的代表示例,可以举出艾阿华诺卡氏菌(Nocardia iowensis)、星形诺卡氏菌(Nocardia asteroides)、巴西诺卡氏菌(Nocardia brasiliensis)、皮疽诺卡氏菌(Nocardia farcinica)、褶皱慢反应脂肪酸菌(Segniliparus rugosus)、光滑脂肪酸慢出菌(Segniliparus rotundus)、酪酸冢村菌(Tsukamurella paurometabola)、海洋分枝杆菌(Mycobacterium marinum)、新金分枝杆菌(Mycobacterium neoaurum)、脓肿分枝杆菌(Mycobacterium abscessus)、鸟分枝杆菌(Mycobacterium avium)、龟分枝杆菌(Mycobacterium chelonae)、免疫原分枝杆菌(Mycobacterium immunogenum)、耻垢分枝杆菌(Mycobacterium smegmatis)、干朽菌(Serpula lacrymans)、多年异担子菌(Heterobasidion annosum)、灰拟鬼伞(Coprinopsis cinerea)、黄曲霉菌(Aspergillusflavus)、土曲霉(Aspergillus terreus)、粗糙脉孢菌(Neurospora crassa)、酿酒酵母(Saccharomyces cerevisiae),但并不限定于这些。可在本发明中使用的酶只要对该转换具有活性就没有限定,例如可以使用由序列编号18~22的任一者中记载的氨基酸序列构成的酶的至少一种,优选使用由序列编号20中记载的氨基酸序列构成的脓肿分枝杆菌(Mycobacterium abscessus)来源的酶MaCar、由序列编号22中记载的氨基酸序列构成的作为MaCar突变体的MaCar(m)中的至少一者,更优选使用由序列编号22中记载的氨基酸序列构成的MaCar(m)(图4)。
另外,羧酸还原酶可通过磷酸泛酰巯基乙胺基化而转换成活性型全酶(Venkitasubramanian et al.,Journal of Biological Chemistry,Vol.282,No.1,478-485(2007))。磷酸泛酰巯基乙胺基化通过磷酸泛酰巯基乙胺基转移酶(Phosphopantetheinyl Transferase;PT)进行催化。作为可催化该反应的酶,例如可以举出被分类在EC 2.7.8.7中的酶。因此,本发明的微生物可以按照进一步增大磷酸泛酰巯基乙胺基转移酶的活性的方式进行修饰。作为增大磷酸泛酰巯基乙胺基转移酶的活性的方法,可以举出导入外源性磷酸泛酰巯基乙胺基转移酶基因的方法、增强内源性磷酸泛酰巯基乙胺基转移酶基因的表达的方法,但并不限定于这些。可在本发明中使用的酶只要具有磷酸泛酰巯基乙胺基转移活性,并不限定于这些,作为代表性的酶,例如可以举出大肠杆菌的EntD、枯草杆菌(Bacillus subtilis)的Sfp、艾阿华诺卡氏菌(Nocardia iowensis)的Npt(Venkitasubramanian et al.,Journal of Biological Chemistry,Vol.282,No.1,478-485(2007))、酿酒酵母(Saccharomyces cerevisiae)的Lys5(Ehmann et al.,Biochemistry 38.19(1999):6171-6177.)。可在本发明中使用的酶只要对该转换具有活性就没有限定,例如可以使用由序列编号23~26中记载的氨基酸序列构成的酶中的至少任一种,优选使用由序列编号24中记载的氨基酸序列构成的艾阿华诺卡氏菌(Nocardiaiowensis)来源的艾阿华诺卡氏菌(Nocardia iowensis)的Npt(图5)。
图1的步骤J、M、P和R的转换为氨基转移反应。作为可催化该转换的酶的示例,可以举出被分类在EC 2.6.1组中的转氨酶(氨基转移酶)。例如可例示出被分类在EC 2.6.1.19(4-氨基丁酸-2-氧代戊二酸转氨酶)、EC 2.6.1.29(二胺转氨酶)、EC 2.6.1.48(5-氨基戊酸转氨酶)之类的组中的酶作为对该转换也可具有活性的酶。可在本发明中使用的酶只要具有各步骤的转换活性就没有特别限定,例如可以使用已报告了对尸胺、亚精胺进行氨基转移的作为大肠杆菌的腐胺氨基转移酶的YgjG(Samsonova.,et al.,BMC microbiology3.1(2003):2.)、作为假单胞菌属的腐胺氨基转移酶的SpuC(Lu et al.,Journal ofbacteriology 184.14(2002):3765-3773.、Galman et al.,Green Chemistry 19.2(2017):361-366.)、大肠杆菌的GABA氨基转移酶GabT、PuuE。此外,帕氏鲁杰氏菌(Ruegeriapomeroyi)、紫色色杆菌(Chromobacterium violaceum)、柠檬节杆菌(Arthrobactercitreus)、嗜热球杆菌(Sphaerobacter thermophilus)、费希尔曲霉(Aspergillusfischeri)、河流弧菌(Vibrio fluvialis)、根癌农杆菌(Agrobacterium tumefaciens)、百脉根中生根瘤菌(Mesorhizobium loti)等生物种来源的ω-转氨酶也被报告具有对1,8-二氨基辛烷、1,10-二氨基癸烷等二胺化合物的氨基转移活性,可在本发明中使用(Sung etal.,Green Chemistry 20.20(2018):4591-4595.、Sattler et al.,Angewandte Chemie124.36(2012):9290-9293.)。可在本发明中使用的酶只要对该转换具有活性就没有限定,例如可以使用由序列编号27~31和158中记载的氨基酸序列构成的酶中的至少任一种(图5和图15)。作为代表性的氨基供体,可以举出L-谷氨酸、L-丙氨酸和甘氨酸,但并不限定于这些。
在图1的步骤H、L、O和Q中,醛转换成醇。作为可催化该转换的酶的示例,可以举出被分类在EC 1.1.1组中的氧化还原酶。出于与该转换同样地催化由醛向醇的转换反应的原因,例如可例示出被分类在EC 1.1.1.1(醇脱氢酶)、EC 1.1.1.2(醇脱氢酶(NADP+))、EC1.1.1.71(醇脱氢酶[NAD(P)+])之类的组中的酶作为对该转换也可具有活性的酶。可在本发明中使用的酶只要对该转换具有活性就没有限定,例如为序列编号32中记载的大肠杆菌来源的Ahr(图5)。
在图1的步骤S和T的反应中,在羧基上加成CoA。作为可催化该转换的酶的示例,可以举出被分类在EC 2.8.3组中的CoA转移酶、被分类在EC 6.2.1组中的酸硫醇连接酶。本发明中所使用的酶只要具有对该转换的活性就没有限定。
编码可用于本发明的上述酶的基因可以来源于所例示的生物以外,并且也可以为人工合成的基因,只要在宿主微生物细胞内实质上可表达出酶活性即可。
本发明中的重组微生物中,宿主微生物中的任意基因可被适宜地破坏。靶基因的破坏可通过该领域中公知的方法来进行。
另外,在可用于本发明的上述酶的氨基酸序列、或者编码酶的基因的碱基序列中,只要可在上述宿主微生物细胞中实质上表达出酶活性,可具有在自然界中可能发生的全部的突变、人工导入的突变以及修饰,例如可以含有缺失、置换、插入、添加之类的突变。例如可以包含在上述酶的氨基酸序列中缺失、置换、插入和/或添加1或2个以上、优选1~20个、更优选1~10个、进一步优选1~7个、进一步更优选1~5个、特别优选1~3个氨基酸而成的氨基酸序列。
此外,在编码可用于本发明的上述酶的基因的碱基序列中,只要可在上述宿主微生物细胞中实质上表达出酶活性,也可利用与具有互补的碱基序列的DNA在严谨条件下杂交的DNA。“严谨条件”例如为“1×SSC、0.1%SDS、60℃”的程度的条件,作为更严谨的条件,为“0.1×SSC、0.1%SDS、60℃”的程度的条件,作为进一步严谨的条件,为“0.1×SSC、0.1%SDS、68℃”的程度的条件。
此外已知在编码特定氨基酸的各种密码子中存在冗余密码子,因此在本发明中,也可利用最终被翻译成同一氨基酸的替代密码子。即,由于基因编码被简并,因此为了编码某一特定的氨基酸,可以使用多种密码子,因此氨基酸序列可利用任意的1组类似的DNA寡核苷酸进行编码。需要说明的是,已知大部分生物优先使用特定密码子(最佳密码子)的子集(Gene,Vol.105,pp.61-72,1991等),因而根据宿主微生物进行“密码子最佳化”在本发明中也可以是有用的。
因此,对于本发明中的基因重组微生物,可以以能够表达出酶活性作为条件,包含与上述酶基因的碱基序列具有例如80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的序列一致性的碱基序列。或者可以包含编码包含与上述酶的氨基酸序列具有例如80%以上、85%以上、88%以上、90%以上、95%以上、97%以上、98%以上或99%以上的序列一致性的氨基酸序列的蛋白质的基因。
需要说明的是,本说明书中,比较氨基酸序列相对于参照氨基酸序列的“序列一致性”的比例(%)如下定义:按照2个序列间的一致性最大的方式将序列排列整齐,必要时在2个序列中的一方或两方中插入间隙,将比较序列中的氨基酸残基与参照序列中的氨基酸残基相同的百分数定义为该比例。此时,保守置换不被视为序列一致性的一部分。序列一致性可以通过使用能够公知获得的计算机软件来确定,例如可以使用BLAST(注册商标。以下省略)(Basic Local Alignment Search Tool)等比对搜索工具来确定。本领域技术人员在比对中可以确定适当的参数以得到比较序列最大的比对。关于核苷酸序列的“序列一致性”,也可利用相同的方法来确定。
本发明中,通过将上述C6化合物生物合成酶基因作为“表达盒”导入至宿主微生物细胞内,能够更稳定地得到高水平的酶活性。本说明书中,“表达盒”是指包含与表达对象的核酸或表达对象的基因功能性结合的、对于转录和翻译进行调节的核酸序列的核苷酸。代表性地,本发明的表达盒以功能性结合的状态在编码序列的5’上游含有启动子序列、在3’下游含有终止子序列、根据情况进一步含有通常的调节元件,这种情况下,表达对象的核酸或表达对象的基因被导入至宿主微生物中。
关于启动子,不论是结构表达型启动子还是诱导表达型启动子,均被定义为使RNA聚合酶与DNA结合、引发RNA合成的DNA序列。强启动子是指以高频率引发mRNA合成的启动子,也适用于本发明中。在大肠杆菌中,能够利用lac系、trp系、tac或trc系、λ噬菌体的主要操纵子和启动子区域、fd外壳蛋白的调节区域、针对糖酵解系酶(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、T7噬菌体来源的RNA聚合酶的启动子区域等。在谷氨酸棒杆菌(Corynebacterium glutamicum)中,可以利用HCE(高水平组成型表达)启动子、cspB启动子、sodA启动子、延伸因子(EF-Tu)启动子等。作为终止子,可以利用T7终止子、rrnBT1T2终止子、lac终止子等。除了启动子和终止子序列以外,可以作为其他调节元件的示例举出的序列为选择标记、扩增信号、复制起点等。适宜的调节序列例如记载于“Gene Expression Technology:Methods in Enzymology 185”,Academic Press(1990)。
上述说明的表达盒嵌入到例如由质粒、噬菌体、转座子、IS元件、噬粒、粘粒、或者线状或环状DNA等构成的载体中而被插入到宿主微生物中。优选质粒和噬菌体。这些载体在宿主微生物中可自体复制,也可通过染色体复制。适宜的质粒例如为:大肠杆菌的pLG338、pACYC184、pBR322、pUC18、pUC19、pKC30、pRep4、pHS1、pKK223-3、pDHE19.2、pHS2、pPLc236、pMBL24、pLG200、pUR290、pIN-III113-B1、λgt11或pBdCI;杆菌的pUB110、pC194或pBD214;棒状杆菌属的pSA77或pAJ667等。在芽孢杆菌属等的杆菌中可以举出pUB110、pC194或pBD214等。除了这些以外还能够使用的质粒等记载于“Gene Cloning and DNA analysis 7thedition”,Wiley-Blackwell(2016)中。表达盒向载体中的导入可通过包括利用适当的限制酶切割、克隆化以及连接的惯用方法进行。各个表达盒可以配置在1个载体上,也可以配置在2个或其以上的载体上。
在如上所述构建出本发明的具有表达盒的载体后,作为将该载体导入至宿主微生物中时能够适用的方法,可以使用惯用的方法。例如可以举出氯化钙法、电穿孔法、接合传递法、原生质体融合法等,但并不限定于这些,可以选择适合于宿主微生物的方法。
本发明的第二方面涉及目标化合物的制造方法,其包括对上述重组微生物进行培养的培养工序。
此处,该化合物优选为C6化合物。C6化合物为选自由己二酸、六亚甲基二胺、1,6-己二醇、6-氨基己酸、6-氨基-1-己醇和6-羟基己酸组成的组中的至少一者。
C6化合物的制造方法包括对本发明中的基因重组微生物进行培养的培养工序。在培养工序中,通过利用含有碳源和氮源的培养基对重组微生物进行培养,可得到包含菌体的培养物。本发明的基因重组微生物在适于C6化合物生产、以及微生物的生长-维持的条件下进行培养,适宜的培养基组成、培养时间、培养条件可由本领域技术人员容易地设定。
作为碳源,可以举出D-葡萄糖、蔗糖、乳糖、果糖、麦芽糖、低聚糖、多糖、淀粉、纤维素、米糠、废糖蜜、油脂(例如大豆油、葵花籽油、花生油、椰子油等)、脂肪酸(例如棕榈酸、亚油酸、油酸、亚麻酸等)、醇(例如甘油、乙醇等)、有机酸(例如乙酸、乳酸、琥珀酸等)、玉米分解液、纤维素分解液。优选为D-葡萄糖、蔗糖或甘油。这些碳源可以单独使用或者以混合物的形式使用。
使用生物物质来源的原料制造的C6化合物可通过基于ISO16620-2或ASTM D6866中规定的碳-14(放射性碳)分析的生物基碳含量的测定,与例如来自石油、天然气、煤炭等的合成原料明确区分。
作为氮源,可以举出含氮有机化合物(例如蛋白胨、酪蛋白氨基酸、胰蛋白胨、酵母提取物、肉提取物、麦芽提取物、玉米浆、大豆粉、氨基酸和脲等)、或无机化合物(例如氨水溶液、硫酸铵、氯化铵、磷酸铵、碳酸铵、硝酸钠、硝酸铵等)。这些氮源可以单独使用或者以混合物的形式使用。
另外,在重组微生物表达有用的附加特性的情况下、例如具有针对抗生素的抗性标记的情况下,培养基可以包含相对应的抗生素。由此,培养时的杂菌所致的污染风险降低。作为抗生素,可以举出氨苄青霉素等β-内酰胺系抗生素、卡那霉素等氨基糖苷系抗生素、红霉素等大环内酯系抗生素、四环素系抗生素、氯霉素等,但并不限定于这些。
培养可以为分批式、也可以为连续式。另外,在任一情况下,均可以为在培养的适当时刻追加补给上述碳源等的形式。进而,关于培养,可以一边控制合适的温度、氧浓度、pH等条件一边进行培养。来源于一般的微生物宿主细胞的转化体的合适的培养温度通常为15℃~55℃、优选为25℃~40℃的范围。宿主微生物为好氧性的情况下,为了确保发酵中的适当的氧浓度,可以进行振荡(烧瓶培养等)、搅拌/通气(发酵罐培养等)。这些培养条件可由本领域技术人员容易地设定。
培养工序中可以包括得到重组微生物的培养物和/或培养物的提取物的步骤。
本发明中的化合物的制造方法可以进一步包括将上述培养物和/或上述培养物的提取物与底物化合物混合而得到混合液的混合工序。底物化合物可以根据酶和目标化合物适宜地选择。
本发明中的化合物的制造方法可以包括对作为目标化合物的C6化合物进行分离和/或精制的工序。该分离和/或精制的工序中可以举出任意方法,例如离心分离、过滤、膜分离、析晶、提取、蒸馏、吸附、相分离、离子交换和各种色谱法等,但并不限定于这些。在分离和/或精制工序中,可以选择一种方法,并且也可以将多种方法组合。
本发明的又一方面涉及蛋白质。具体地说,该蛋白质为下述重组蛋白:
(a)具有将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性;
(b)为下述任一者:
(i)由与序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列具有80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的序列一致性的氨基酸序列构成;
(ii)由在序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列中缺失、置换、插入和/或添加1~10个、1~7个、1~5个或1~3个氨基酸而成的氨基酸序列构成;
(iii)利用由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列具有80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的序列一致性的多核苷酸序列构成的DNA进行编码;
(iv)由在利用序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列编码的蛋白质的氨基酸序列中缺失、置换、插入和/或添加1~10个、1~7个、1~5个或1~3个氨基酸而成的氨基酸序列构成;
(v)利用与由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列互补的多核苷酸序列构成的DNA在严谨条件下杂交的DNA进行编码;以及
(vi)利用由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列的简并异构体构成的DNA进行编码。
该蛋白质优选为下述重组蛋白:
(a)具有将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性,
(b)为下述任一者:
(i)由与序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列具有80%以上的序列一致性的氨基酸序列构成;
(ii)由在序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成;
(iii)利用由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列具有80%以上的序列一致性的多核苷酸序列构成的DNA进行编码;
(iv)由在利用序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列编码的蛋白质的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成;
(v)利用与由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列互补的多核苷酸序列构成的DNA在严谨条件下杂交的DNA进行编码;以及
(vi)利用由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列的简并异构体构成的DNA进行编码。
在优选的一个方式中,重组蛋白:
(xi)由序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列构成;或者
(xiii)利用由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列构成的DNA进行编码。
本发明的又一方面涉及C6化合物的制造方法,其包括使用上述蛋白质的步骤。
如以上所说明,根据本发明,通过在宿主微生物中导入编码上述具有2,3-脱氢己二酰CoA还原酶活性的蛋白质的基因,能够得到2,3-脱氢己二酰-CoA还原酶活性良好的重组微生物。另外还提供具有良好的2,3-脱氢己二酰CoA还原酶活性的蛋白质。
通过使用上述重组微生物或上述蛋白质,能够有效地制造出目标C6化合物。另外,通过根据所期望的C6化合物使用追加的酶进行转换,能够得到各种C6化合物。此外,本发明中的重组微生物由于具有良好的C6化合物生产能力,因此期待能够用于工业规模的目标化合物的生产。
<2>发明B:重组微生物以及己二酸或其衍生物的制造方法
本发明中的重组微生物是对宿主微生物进行了基因修饰以能够有效地生产己二酸(Adipic acid;ADA)(CAS No.124-04-9)或己二酸衍生物的微生物。
本说明书中,“己二酸衍生物”是具有碳原子数3~5的脂肪族烃基、以及1个或2个以上的羧基的脂肪族羧酸。己二酸衍生物所具有的脂肪族烃基为饱和或不饱和的。脂肪族烃基中,碳例如可以带有羟基、羧基、酮基、氨基等取代基的取代。“己二酸衍生物”所具有的脂肪族烃基的碳原子数优选为3或4。“己二酸衍生物”所具有的羧基优选为1或2。作为“己二酸衍生物”的具体例,可以举出具有碳原子数4的脂肪族烃基和2个羧酸的3-氧代己二酸(3-Oxoadipic acid;3-OA)、具有碳原子数3的脂肪族烃基和1个羧酸的乙酰丙酸(Levulinicacid;LEV)、具有碳原子数4的脂肪族烃基和2个羧酸的3-羟基己二酸(3-Hydroxyadipicacid;3HA)、以及具有碳原子数4的脂肪族烃基和2个羧酸的2,3-脱氢己二酸(2,3-Dehydroadipic acid;23DA)(参照图16)。
如本领域技术人员所理解,本说明书中的己二酸和己二酸衍生物可采取包括任意的盐形态的中性或电离形态,该形态取决于pH。因此,“己二酸”和“己二酸衍生物”中除了包含游离酸的形态以外,还包含任意的盐形态和电离形态。本说明书中,有时将己二酸和己二酸衍生物合起来总称为“己二酸类”或“己二酸类似物”。
也可以将由本发明中的重组微生物生产的己二酸作为原料来合成6-氨基己酸、六亚甲基二胺、1,6-己二醇和6-氨基-1-己醇。合成方法可以是化学合成法、利用酶或内含有酶的微生物的生物学合成法等。另外,只要为包含用于将己二酸转换成上述化合物的代谢路径的微生物,也可以在细胞内由使用己二酸作为中间体的原料进行代谢转换。
本发明中的基因重组微生物具体地说包含编码3-羟基己二酰CoA脱水酶的外源基因和编码2,3-脱氢己二酰CoA还原酶的外源基因中的至少一者。通过包含这样的外源基因中的至少一者,重组微生物具有己二酸生产路径,能够制造出己二酸或己二酸衍生物。本说明书中,“基因重组微生物”也简称为“重组微生物”。
3-羟基己二酰CoA脱水酶和2,3-脱氢己二酰CoA还原酶中的至少一者来源于伯克霍尔德菌属(Burkholderia sp.)LEBP-3株(保藏号:NITE BP-03334)。优选3-羟基己二酰CoA脱水酶和2,3-脱氢己二酰CoA还原酶这两者来源于伯克霍尔德菌属(Burkholderiasp.)LEBP-3株。
编码3-羟基己二酰CoA脱水酶的外源基因和编码2,3-脱氢己二酰CoA还原酶的外源基因中的至少一者来源于伯克霍尔德菌属(Burkholderia sp.)LEBP-3株(保藏号:NITEBP-03334)。优选编码3-羟基己二酰CoA脱水酶的外源基因和编码2,3-脱氢己二酰CoA还原酶的外源基因这两者来源于伯克霍尔德菌属(Burkholderia sp.)LEBP-3株(保藏号:NITEBP-03334)。
本发明的基因重组微生物被导入编码上述酶的外源基因中的至少一者即可。本发明的基因重组微生物优选被导入编码催化己二酸生产路径的各反应阶段的酶的外源基因中的至少一者,表达外源性酶。另外,本发明的基因重组微生物即使不通过外源基因的导入也可表达出足以催化己二酸生产路径的反应阶段的量的酶的情况下,可通过由内源基因编码的酶进行反应。
本发明中的重组微生物优选表达3-羟基己二酰CoA脱水酶和2,3-脱氢己二酰CoA还原酶中的至少一者。本发明中的重组微生物通过表达这些酶中的至少一者,可进行己二酸生产路径的反应阶段,生产出目标化合物。
更优选本发明中的重组微生物表达3-羟基己二酰CoA脱水酶和2,3-脱氢己二酰CoA还原酶这两者。本发明中的重组微生物通过表达这些酶,能够分别进行己二酸生产路径的反应阶段,生产出目标化合物。
进一步更优选本发明中的重组微生物除了表达3-羟基己二酰CoA脱水酶和2,3-脱氢己二酰CoA还原酶以外还表达氧化还原酶和/或β-酮硫解酶。本发明中的重组微生物通过表达出这些酶,能够分别进行己二酸生产路径的反应阶段,有效地生产出目标化合物(参照图16)。
以下对于本发明中的重组微生物所包含的基因编码的酶分别进行详细说明。
<3-羟基己二酰CoA脱水酶>
3-羟基己二酰CoA脱水酶是催化将3-羟基己二酰-CoA转换成2,3-脱氢己二酰-CoA的反应的酶(图16的步骤C)。
在一个方式中,3-羟基己二酰CoA脱水酶被与下述PCR扩增物的序列一致性为65%以上、70%以上、75%以上、80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码,该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的。此处,该DNA具有例如626~940bp、优选740~862bp、更优选743~823bp、进一步更优选767~799bp、特别更优选775~791bp、最优选778~788bp。关于PCR中使用的引物如下文所述。
在优选的一个方式中,3-羟基己二酰CoA脱水酶被作为PCR扩增物的DNA所编码,该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的。该DNA具有例如626~940bp、优选740~862bp、更优选743~823bp、进一步更优选767~799bp、特别更优选775~791bp、最优选778~788bp。
在另外的方式中,3-羟基己二酰CoA脱水酶选自下述重组多肽:
(A-1)被与下述PCR扩增物的序列一致性为65%以上、70%以上、75%以上、80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码的重组多肽,
具有3-羟基己二酰CoA脱水酶活性,
上述DNA具有上述碱基对数(bp),
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的;
(A-2)由在利用作为下述PCR扩增物的DNA所编码的多肽的氨基酸序列中缺失、置换、插入和/或添加例如1~10个、优选1~7个、更优选1~5个、进一步优选1~3个氨基酸而成的氨基酸序列构成的重组多肽,
具有3-羟基己二酰CoA脱水酶活性,
上述DNA具有上述碱基对数(bp),
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的;
以及
(A-3)被由下述PCR扩增物的简并异构体构成的DNA所编码的重组多肽,
具有3-羟基己二酰CoA脱水酶活性,
上述DNA具有上述碱基对数(bp),
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的。
在进一步优选的方式中,3-羟基己二酰CoA脱水酶是被以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的PCR扩增物所编码的重组多肽,该扩增物的碱基对数为783或784bp。
关于3-羟基己二酰CoA脱水酶,作为PCR中使用的引物(正向和反向),可以举出下述引物(参照图17)。
(1a)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;
(1b)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸;
(1c)由将序列编号168所示的碱基序列进行了密码子最佳化的碱基序列构成的核苷酸以及由将序列编号169所示的碱基序列进行了密码子最佳化的碱基序列构成的核苷酸;
(1d)包含将序列编号168所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸以及包含将序列编号169所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸。
上述(1b)的“包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸”具有除了“序列编号168所示的碱基序列”和“序列编号169所示的碱基序列”以外还分别进一步追加了例如1~20bp、优选1~15bp、更优选5~15bp、进一步优选10~15bp之后的核苷酸长度。
上述(1d)的“包含将序列编号168所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸以及包含将序列编号169所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸”具有除了“将序列编号168所示的碱基序列进行了密码子最佳化的碱基序列”和“将序列编号169所示的碱基序列进行了密码子最佳化的碱基序列”以外还分别包含与载体末端相同的序列、例如分别进一步追加了1~20bp、优选1~15bp、更优选5~15bp、进一步优选10~15bp之后的核苷酸长度。作为上述(1d)的“包含将序列编号168所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸、以及包含将序列编号169所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸”的示例,可以举出(1d’)由序列编号198所示的碱基序列构成的核苷酸以及由序列编号199所示的碱基序列构成的核苷酸(图20A)。
关于3-羟基己二酰CoA脱水酶,优选的引物为:
(1a)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;以及
(1b)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸。
作为PCR扩增物(其是使用上述核苷酸作为引物、以伯克霍尔德菌属(Burkholderiasp.)LEBP-3株的染色体DNA作为模板得到的)的DNA优选具有783或784bp。此处,作为PCR中使用的DNA聚合酶,使用该领域中已知的DNA聚合酶,例如可以举出Taq DNA聚合酶、进行了热启动调整的DNA聚合酶、与聚合酶活性分开地具有3’-5’核酸外切酶活性的校对DNA聚合酶等。
<2,3-脱氢己二酰CoA还原酶>
2,3-脱氢己二酰CoA还原酶是催化将2,3-脱氢己二酰-CoA转换成己二酰-CoA的反应的酶(参照图16的步骤D)。
在一个方式中,2,3-脱氢己二酰CoA还原酶被与下述PCR扩增物的序列一致性为65%以上、70%以上、75%以上、80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码,该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的。该DNA例如具有924~1386bp、优选1039~1249bp、更优选1097~1213bp、进一步更优选1131~1179bp、特别更优选1143~1167bp、进一步特别更优选1145~1165bp、最优选1150~1160bp。
在优选的一个方式中,2,3-脱氢己二酰CoA还原酶被作为PCR扩增物的DNA所编码,该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的。该DNA例如具有924~1386bp、优选1039~1249bp、更优选1097~1213bp、进一步更优选1131~1179bp、特别更优选1143~1167bp、进一步特别更优选1145~1165bp、最优选1150~1160bp。
在另外的方式中,2,3-脱氢己二酰CoA还原酶选自下述重组多肽:
(B-1)被与下述PCR扩增物的序列一致性为65%以上、70%以上、75%以上、80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码的重组多肽,
具有2,3-脱氢己二酰CoA还原酶活性,
上述DNA具有上述碱基对数(bp),
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的;
(B-2)由在利用作为下述PCR扩增物的DNA所编码的多肽的氨基酸序列中缺失、置换、插入和/或添加例如1~10个、优选为1~7个、更优选为1~5个、进一步优选为1~3个氨基酸而成的氨基酸序列构成的重组多肽,
具有2,3-脱氢己二酰CoA还原酶活性,
上述DNA具有上述碱基对数(bp),
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的;
以及
(B-3)被由下述PCR扩增物的简并异构体构成的DNA所编码的重组多肽,
具有2,3-脱氢己二酰CoA还原酶活性,
上述DNA具有上述碱基对数(bp),
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的。
在进一步优选的方式中,外源性的2,3-脱氢己二酰CoA还原酶是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板、使用规定的核苷酸作为引物得到的PCR扩增物,该扩增物的碱基对数为1155或1156bp。
关于2,3-脱氢己二酰CoA还原酶,作为PCR中使用的引物(正向和反向),可以举出下述引物(参照图17)。
(2a)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;
(2b)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸;
(2c)由将序列编号170所示的碱基序列进行了密码子最佳化的碱基序列构成的核苷酸以及由将序列编号171所示的碱基序列进行了密码子最佳化的碱基序列构成的核苷酸;
(2d)包含将序列编号170所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸以及包含将序列编号171所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸。
上述(2b)的“包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸”具有除了“序列编号170所示的碱基序列”和“序列编号171所示的碱基序列”以外还分别进一步追加了例如1~20bp、优选1~15bp、更优选5~15bp、进一步优选10~15bp之后的核苷酸长度。
上述(2d)的“包含将序列编号170所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸以及包含将序列编号171所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸”具有除了“将序列编号170所示的碱基序列进行了密码子最佳化的碱基序列”和“将序列编号171所示的碱基序列进行了密码子最佳化的碱基序列”以外还分别包含与载体末端相同的序列、例如分别进一步追加了1~20bp、优选1~15bp、更优选5~15bp、进一步优选10~15bp之后的核苷酸长度。作为上述(2d)的“包含将序列编号170所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸以及包含将序列编号171所示的碱基序列进行了密码子最佳化的碱基序列的核苷酸”的示例,可以举出(2d’)由序列编号210所示的碱基序列构成的核苷酸以及由序列编号211所示的碱基序列构成的核苷酸(图20A)。
关于2,3-脱氢己二酰CoA还原酶,优选的引物为:
(2a)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;以及
(2b)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸。
作为PCR扩增物(其是使用上述核苷酸作为引物、以伯克霍尔德菌属(Burkholderiasp.)LEBP-3株的染色体DNA作为模板得到的)的DNA优选具有1155或1156bp。此处,作为PCR中使用的DNA聚合酶,使用该领域中已知的DNA聚合酶,例如可以举出Taq DNA聚合酶、进行了热启动调整的DNA聚合酶、与聚合酶活性分开地具有3’-5’核酸外切酶活性的校对DNA聚合酶等。
本说明书中,“PCR扩增物”是指以特定的核苷酸作为引物进行PCR从而得到的产物。需要说明的是,只要没有特别示出,本说明书中,在PCR扩增中,使用各1μM的特定的核苷酸作为正向引物和反向引物,并且作为酶使用PrimeSTAR Max DNA Polymerase(产品名、Takara Bio制),以25μL的液量在热处理98℃10秒、退火55℃15秒、延伸72℃5秒/kb的条件下实施30次循环的处理。
就本发明而言,导入目标外源基因的宿主微生物没有特别限定,可以为原核生物和真核生物中的任一者。可以从已经分离保存的微生物、新近从自然界分离出的微生物、以及进行了基因修饰的微生物中任意地选择。宿主微生物例如属于选自由埃希氏菌属、棒状杆菌属、芽孢杆菌属、不动杆菌属、伯克霍尔德菌属、假单胞菌属、梭菌属、酵母菌属、裂殖酵母属、亚罗酵母属、念珠菌属、毕赤酵母属、曲霉属、克雷伯氏菌属、葡糖杆菌属、发酵单胞菌属、乳杆菌属、乳球菌属、链球菌属和链霉菌属组成的组中的属。本发明的宿主微生物优选属于选自由埃希氏菌属、棒状杆菌属、芽孢杆菌属、不动杆菌属、伯克霍尔德菌属、假单胞菌属、梭菌属、酵母菌属、裂殖酵母属、亚罗酵母属、念珠菌属、毕赤酵母属和曲霉属组成的组中的属。宿主微生物更优选为大肠杆菌(Escherichia coli)。
因此,在上述宿主微生物中导入了外源基因的本发明中的重组微生物例如属于选自由埃希氏菌属、棒状杆菌属、芽孢杆菌属、不动杆菌属、伯克霍尔德菌属、假单胞菌属、梭菌属、酵母菌属、裂殖酵母属、亚罗酵母属、念珠菌属、毕赤酵母属、曲霉属、克雷伯氏菌属、葡糖杆菌属、发酵单胞菌属、乳杆菌属、乳球菌属、链球菌属和链霉菌属组成的组中的属,优选属于选自由埃希氏菌属、棒状杆菌属、芽孢杆菌属、不动杆菌属、伯克霍尔德菌属、假单胞菌属、梭菌属、酵母菌属、裂殖酵母属、亚罗酵母属、念珠菌属、毕赤酵母属和曲霉属组成的组中的属。本发明中的重组微生物更优选为大肠杆菌(Escherichia coli)。
以下参照图16对于本发明中的基因重组微生物所具有的己二酸、己二酸衍生物、六亚甲基二胺、1,6-己二醇和6-氨基-1-己醇生产路径以及催化该路径的各反应阶段的酶进行说明。
图16的步骤A的转换中,例如琥珀酰CoA:乙酰CoA酰基转移酶、或者3-氧代己二酰CoA硫解酶参与其中,琥珀酰-CoA与乙酰-CoA发生缩合,转换成3-氧代己二酰-CoA。作为可催化该转换的酶的其他示例,可以举出β-酮硫解酶。进而,例如还可例示出被分类在EC2.3.1.9(乙酰基乙酰CoA硫解酶)、EC 2.3.1.16(3-酮酰-CoA硫解酶)和EC 2.3.1.174(3-氧代己二酰-CoA硫解酶)等组中的酶作为可对该转换具有活性的酶。本发明中所使用的酶只要对该转换具有活性就没有限定,在一个方式中,使用由序列编号172所示的氨基酸序列构成的大肠杆菌来源的PaaJ。在优选的一个方式中,该酶是被作为下述PCR扩增物的DNA序列(1203bp)所编码的琥珀酰CoA:乙酰CoA酰基转移酶,该PCR扩增物是使用序列编号164所示的核苷酸和序列编号165所示的核苷酸作为引物、以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板得到的。
在图16的步骤B的转换中,3-羟基己二酰CoA脱氢酶参与其中,3-氧代己二酰-CoA被转换成3-羟基己二酰-CoA。作为可催化该转换的其他酶的示例,可以举出被分类在EC1.1.1组中的氧化还原酶。具体地说,例如可例示出被分类在EC 1.1.1.35(3-羟基酰基-CoA脱氢酶)、EC 1.1.1.36(乙酰基乙酰-CoA脱氢酶)、EC 1.1.1.157(3-羟基丁酰-CoA脱氢酶)、EC 1.1.1.211(长链3-羟基酰基-CoA脱氢酶)和EC 1.1.1.259(3-羟基庚二酰-CoA脱氢酶)等组中的酶作为可对该转换具有活性的酶。本发明中所使用的酶只要对该转换具有活性就没有限定,例如使用由序列编号173所示的氨基酸序列构成的大肠杆菌来源的PaaH(图18)。在优选的一个方式中,该酶是被PCR扩增物的DNA序列(1521bp)所编码的3-羟基己二酰CoA脱氢酶,该PCR扩增物是使用序列编号166所示的核苷酸和序列编号167所示的核苷酸作为引物、以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板得到的。
在图16的步骤C的转换中,例如3-羟基己二酰CoA脱水酶参与其中,3-羟基己二酰-CoA被转换成2,3-脱氢己二酰-CoA。作为可催化该转换的其他酶的示例,可以举出被分类在EC 4.2.1组中的氢化裂解酶。具体地说,例如可例示出被分类在EC 4.2.1.17(烯酰基-CoA水合酶)、EC 4.2.1.55(3-羟基丁酰-CoA脱水酶)和EC 4.2.1.74(长链烯酰基-CoA水合酶)等组中的酶作为可对该转换具有活性的酶。在优选的一个方式中,该酶为上述3-羟基己二酰CoA脱水酶。
在图16的步骤D的转换中,例如2,3-脱氢己二酰CoA还原酶参与其中,2,3-脱氢己二酰-CoA被转换成己二酰-CoA。作为可催化该转换的酶的其他示例,可以举出被分类在EC1.3.1组中的氧化还原酶。具体地说,可例示出例如被分类在EC 1.3.1.8(酰基-CoA脱氢酶(NADP+))、EC 1.3.1.9(烯酰基-ACP还原酶(NADH))、EC 1.3.1.38(反式-2-烯酰基-CoA还原酶(NADP+))、EC 1.3.1.44(反式-2-烯酰基-CoA还原酶(NAD+))、EC 1.3.1.86(巴豆基-CoA还原酶)、EC 1.3.1.93(长链酰基-CoA还原酶)和EC 1.3.1.104(烯酰基-ACP还原酶(NADPH))等组中的酶作为可对该转换具有活性的酶。在优选的一个方式中,该酶为上述的2,3-脱氢己二酰CoA还原酶。
在图16的步骤E的转换中,己二酰-CoA被转换成己二酸。作为可催化该转换的酶的示例,可以举出被分类在EC 3.1.2组中的硫酯水合酶。例如可例示出被分类在EC 3.1.2.1(乙酰-CoA水合酶)和EC 3.1.2.20(酰基-CoA水合酶)等组中的酶作为可对该转换具有活性的酶。在优选的一个方式中,该酶只要对该转换具有活性就没有限定,可使用序列编号179所示的不动杆菌ADP1株来源的tesB(Ab)。根据所使用的酶的底物特异性,3-氧代己二酰-CoA、3-羟基己二酰CoA和2,3-脱氢己二酰CoA的CoA脱离,分别生成3-氧代己二酸、3-羟基己二酸、2,3-脱氢己二酸。
另外,作为可催化图16的步骤E的转换的其他酶的示例,还可例示出被分类在EC2.8.3组中的CoA-转移酶。例如可例示出被分类在EC 2.8.3.5(3-含氧酸CoA-转移酶)、EC2.8.3.6(3-氧代己二酸CoA-转移酶)、EC 2.8.3.18(琥珀酰-CoA:乙酸CoA-转移酶)之类的组中的酶作为可对该转换具有活性的酶。
此外,作为可催化图16的步骤E的转换的其他酶转换的示例,还可例示出下述路径:利用被分类在EC 2.3.1组中的酰基转移酶,将己二酰-CoA的己二酰基转移至磷酸而生成己二酰磷酸,之后利用被分类在EC 2.7.2组中的磷酸转移酶进行脱磷酸化。例如,作为酰基转移酶,可例示出被分类在EC 2.3.1.8(磷酸乙酰基转移酶)、EC 2.3.1.19(磷酸丁酰基转移酶)之类的组中的酶作为可对该转换具有活性的酶;对于磷酸转移酶,可例示出被分类在EC 2.7.2.1(乙酸激酶)、EC 2.7.2.7(丁酸激酶)之类的组中的酶作为可对该转换具有活性的酶。
图16的步骤T中,将3-氧代己二酸转换成乙酰丙酸。该转换被酶(3-氧代己二酸脱羧酶)催化、或者自发地产生。
图16的步骤F的转换中,由己二酰-CoA转换成己二酸半醛。作为可催化该转换的酶的示例,可以举出被分类在EC 1.2.1组中的酶。出于与该转换同样地催化CoA脱离而生成醛的转换反应的原因,例如可例示出被分类在EC 1.2.1.10(乙醛脱氢酶(乙酰化))、EC1.2.1.17(乙醛酸脱氢酶(酰化))、EC 1.2.1.42(十六醛脱氢酶(酰化))、EC 1.2.1.44(肉桂酰-CoA还原酶(酰化))、EC 1.2.1.75(丙二酰-CoA还原酶(形成丙二酸半醛))、EC 1.2.1.76(琥珀酸半醛脱氢酶(酰化))之类的组中的酶作为对该转换也可具有活性的酶。本发明所使用的酶只要对该转换具有活性就没有限定,例如使用由序列编号236中记载的氨基酸序列构成的克氏梭菌(Clostridium kluyveri)来源的sucD(图21)。
在图16的步骤G、J、Q的转换中,羧基被转换成醛。作为可催化该转换的酶,例如可以举出羧酸还原酶(Carboxylic Acid Reductase;CAR)。出于与该转换同样地催化由羧酸生成醛的转换反应的原因,例如可例示出被分类在EC 1.2.1.30(羧酸还原酶(NADP+))、EC1.2.1.31(L-氨基己二酸半醛脱氢酶)、EC 1.2.1.95(L-2-氨基己二酸还原酶)、EC1.2.99.6(羧酸还原酶)之类的组中的酶作为对该转换也可具有活性的酶。关于作为酶的来源的生物种的代表示例,可以举出艾阿华诺卡氏菌(Nocardia iowensis)、星形诺卡氏菌(Nocardia asteroides)、巴西诺卡氏菌(Nocardia brasiliensis)、皮疽诺卡氏菌(Nocardia farcinica)、褶皱慢反应脂肪酸菌(Segniliparus rugosus)、光滑脂肪酸慢出菌(Segniliparus rotundus)、酪酸冢村菌(Tsukamurella paurometabola)、海洋分枝杆菌(Mycobacterium marinum)、新金分枝杆菌(Mycobacterium neoaurum)、脓肿分枝杆菌(Mycobacterium abscessus)、鸟分枝杆菌(Mycobacterium avium)、龟分枝杆菌(Mycobacterium chelonae)、免疫原分枝杆菌(Mycobacterium immunogenum)、耻垢分枝杆菌(Mycobacterium smegmatis)、干朽菌(Serpula lacrymans)、多年异担子菌(Heterobasidion annosum)、灰拟鬼伞(Coprinopsis cinerea)、黄曲霉菌(Aspergillusflavus)、土曲霉(Aspergillus terreus)、粗糙脉孢菌(Neurospora crassa)、酿酒酵母(Saccharomyces cerevisiae),但并不限定于这些。本发明所使用的酶只要对该转换具有活性就没有限定,例如可以使用由序列编号237~241的任一者中记载的氨基酸序列构成的酶中的至少一种(图22A~图22E),优选使用由序列编号239中记载的氨基酸序列构成的脓肿分枝杆菌(Mycobacterium abscessus)来源的酶MaCar、由序列编号241中记载的氨基酸序列构成的作为MaCar的突变体的MaCar(m)中的至少一者,更优选使用由序列编号241中记载的氨基酸序列构成的MaCar(m)。
另外,羧酸还原酶可通过磷酸泛酰巯基乙胺基化而转换成活性型的全酶(Venkitasubramanian et al.,Journal of Biological Chemistry,Vol.282,No.1,478-485(2007))。磷酸泛酰巯基乙胺基化通过磷酸泛酰巯基乙胺基转移酶(Phosphopantetheinyl Transferase;PT)进行催化。作为可催化该反应的酶,例如可以举出被分类在EC 2.7.8.7中的酶。因此,本发明的微生物可以按照进一步增大磷酸泛酰巯基乙胺基转移酶的活性的方式进行修饰。作为增大磷酸泛酰巯基乙胺基转移酶的活性的方法,可以举出导入外源性磷酸泛酰巯基乙胺基转移酶基因的方法、增强内源性磷酸泛酰巯基乙胺基转移酶基因的表达的方法,但并不限定于这些。本发明所使用的酶只要具有磷酸泛酰巯基乙胺基转移活性,就不限定于这些,作为代表性的酶,例如可以举出大肠杆菌的EntD、枯草杆菌(Bacillus subtilis)的Sfp、艾阿华诺卡氏菌(Nocardia iowensis)的Npt(Venkitasubramanian et al.,Journal of Biological Chemistry,Vol.282,No.1,478-485(2007))、酿酒酵母(Saccharomyces cerevisiae)的Lys5(Ehmann et al.,Biochemistry 38.19(1999):6171-6177.)。本发明所使用的酶只要对该转换具有活性就没有限定,例如可以使用由序列编号242~245所记载的氨基酸序列构成的酶中的至少任一种(图23A~图23D),优选使用由序列编号243所记载的氨基酸序列构成的艾阿华诺卡氏菌(Nocardia iowensis)来源的艾阿华诺卡氏菌(Nocardia iowensis)的Npt。
图16的步骤M、N、S的转换为氨基转移反应。作为可催化该转换的酶的示例,可以举出被分类在EC 2.6.1组中的转氨酶(氨基转移酶)。例如可例示出被分类在EC 2.6.1.19(4-氨基丁酸-2-氧代戊二酸转氨酶)、EC 2.6.1.29(二胺转氨酶)、EC 2.6.1.48(5-氨基戊酸转氨酶)之类的组中的酶作为对该转换也可具有活性的酶。本发明所使用的酶只要具有各步骤的转换活性就没有特别限定,例如可以使用已报告了对尸胺、亚精胺进行氨基转移的作为大肠杆菌的腐胺氨基转移酶的YgjG(Samsonova.,et al.,BMC microbiology 3.1(2003):2.)、作为假单胞菌属的腐胺氨基转移酶的SpuC(Lu etal.,Journal ofbacteriology 184.14(2002):3765-3773.、Galman et al.,Green Chemistry 19.2(2017):361-366.)、大肠杆菌的GABA氨基转移酶GabT、PuuE。此外,帕氏鲁杰氏菌(Ruegeriapomeroyi)、紫色色杆菌(Chromobacterium violaceum)、柠檬节杆菌(Arthrobactercitreus)、嗜热球杆菌(Sphaerobacter thermophilus)、费希尔曲霉(Aspergillusfischeri)、河流弧菌(Vibrio fluvialis)、根癌农杆菌(Agrobacterium tumefaciens)、百脉根中生根瘤菌(Mesorhizobium loti)等生物种来源的ω-转氨酶也被报告具有对1,8-二氨基辛烷、1,10-二氨基癸烷等二胺化合物的氨基转移活性,可在本发明中使用(Sung etal.,Green Chemistry 20.20(2018):4591-4595.、Sattler et al.,Angewandte Chemie124.36(2012):9290-9293.)。本发明所使用的酶只要对该转换具有活性就没有限定,例如可以使用由序列编号246~250中记载的氨基酸序列构成的酶中的至少任一种(图24A~图24E)。作为代表性的氨基供体,可以举出L-谷氨酸、L-丙氨酸、甘氨酸,但并不限定于这些。
图16的步骤H、L、R的转换中,醛被转换成醇。作为可催化该转换的酶的示例,可以举出被分类在EC 1.1.1组中的氧化还原酶。出于与该转换同样地催化由醛向醇的转换反应的原因,可例示出例如被分类在EC 1.1.1.1(醇脱氢酶)、EC 1.1.1.2(醇脱氢酶(NADP+))、EC 1.1.1.71(醇脱氢酶[NAD(P)+])之类的组中的酶作为对该转换也可具有活性的酶。本发明所使用的酶只要对该转换具有活性就没有限定,例如使用序列编号251所记载的大肠杆菌来源的Ahr(图25)。
图16的步骤I、O的转换中,在羧基上加成CoA。作为可催化该转换的酶的示例,可以举出被分类在EC 2.8.3组中的CoA转移酶、被分类在EC 6.2.1组中的酸硫醇连接酶。本发明所使用的酶只要对该转换具有活性就没有限定。
编码可用于本发明的上述酶的基因可以来源于所例示的生物以外,并且也可以为人工合成的基因,只要在宿主微生物细胞内实质上可表达出酶活性即可。
本发明中的基因重组微生物中,优选除了上述的编码3-羟基己二酰CoA脱水酶和/或2,3-脱氢己二酰CoA还原酶的外源基因以外,还导入有编码选自由琥珀酰CoA:乙酰CoA酰基转移酶和3-羟基己二酰CoA脱氢酶组成的组中的至少一种酶的外源基因。
即,本发明中的重组微生物优选进一步包含编码琥珀酰CoA:乙酰CoA酰基转移酶的外源基因和编码3-羟基己二酰CoA脱氢酶的外源基因中的至少一者。
本发明中的重组微生物中,除了上述外源基因、即3-羟基己二酰CoA脱水酶、2,3-脱氢己二酰CoA还原酶、琥珀酰CoA:乙酰CoA酰基转移酶和3-羟基己二酰CoA脱氢酶以外,还可以导入另外的外源基因。
本发明中的重组微生物中,宿主微生物中的任意基因可被适宜地破坏。靶基因的破坏可依据该领域中公知的方法进行。
另外,可用于本发明的目的的上述酶基因只要在上述宿主微生物细胞内可实质上表达出酶活性,可具有在自然界中可能发生的全部的突变、人工导入的突变以及修饰。例如已知在编码特定氨基酸的各种密码子中存在冗余密码子。因此在本发明中也可利用最终被翻译成同一氨基酸的替代密码子。即,由于基因编码被简并,因此为了编码某一特定的氨基酸,可以使用多个密码子,因此氨基酸序列可利用任意的1组类似的DNA寡核苷酸进行编码。尽管仅该组唯一的成员与天然型酶的基因序列相同,但即使是不匹配的DNA寡核苷酸,在适当的严谨条件下(例如,3×SSC、68℃杂交、2×SSC、0.1%SDS以及68℃清洗)也能够与天然型序列杂交,能够对编码天然型序列的DNA进行鉴定、分离,进一步还能够将这样的基因用于本发明中。特别是已知大部分生物优先使用特定密码子(最佳密码子)的子集(Gene、Vol.105、pp.61-72、1991等),因而根据宿主微生物进行“密码子最佳化”在本发明中也可以是有用的。
因此,对于本发明中的基因重组微生物,可以以实质上能够表达出酶活性作为条件,包含与上述酶基因的碱基序列具有例如65%以上、70%以上、75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上或99%以上的序列一致性的碱基序列。或者,本发明中的基因重组微生物可以包含与编码上述酶的氨基酸序列的碱基序列具有例如65%以上、70%以上、75%以上、80%以上、85%以上、90%以上、95%以上、97%以上、98%以上或99%以上的序列一致性的碱基序列。
本发明中,通过将上述生物合成酶基因作为“表达盒”导入至宿主微生物细胞内,能够更稳定地得到高水平的酶活性。本说明书中,“表达盒”是指包含与表达对象的核酸或表达对象的基因功能性结合的、对于转录和翻译进行调节的核酸序列的核苷酸。代表性地,本发明的表达盒以功能性结合的状态在编码序列的5’上游含有启动子序列、在3’下游含有终止子序列、根据情况进一步含有通常的调节元件,这种情况下,表达对象的核酸或表达对象的基因被导入至宿主微生物中。
本说明书中,关于启动子,不论是结构表达型启动子还是诱导表达型启动子,均被定义为使RNA聚合酶与DNA结合、引发RNA合成的DNA序列。强启动子是指以高频率引发mRNA合成的启动子,也适用于本发明中。在大肠杆菌中,能够利用lac系、trp系、tac或trc系、λ噬菌体的主要操纵子和启动子区域、fd外壳蛋白的控制区域、针对糖酵解系酶(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、T7噬菌体来源的RNA聚合酶的启动子区域等。在谷氨酸棒杆菌(Corynebacterium glutamicum)中,可以利用HCE(高水平组成型表达)启动子、cspB启动子、sodA启动子和延伸因子(EF-Tu)启动子等。
作为终止子,可以利用T7终止子、rrnBT1T2终止子、lac终止子等。
除了启动子和终止子序列以外,作为其他调节元件,可以举出例如选择标记、扩增信号和复制起点等。关于适宜的调节元件,例如记载于”Gene Expression Technology:Methods in Enzymology 185”、Academic Press(1990)。
本发明中使用的宿主微生物可以是编码该微生物本来具有的酶的基因被破坏的微生物。作为编码该酶的基因,例如可以举出编码乳酸脱氢酶的ldh基因、编码乙酰CoA乙酰基转移酶的atoB基因、以及编码琥珀酰CoA合成酶α亚基的sucD基因。通过像这样使用宿主微生物本来具有的酶的基因中的一部分被破坏的宿主微生物,能够避免杂质的混入、抑制目标外反应的进行。具体地说,通过破坏编码乳酸脱氢酶的ldh基因,能够降低作为杂质的乳酸的生成。通过破坏编码乙酰CoA乙酰基转移酶的atoB基因、以及编码琥珀酰CoA合成酶α亚基的sucD基因,能够改善己二酸的收率。
上述说明的表达盒嵌入到例如由质粒、噬菌体、转座子、IS元件、噬粒、粘粒、或者线状或环状DNA等构成的载体中而被插入到宿主微生物中。优选质粒和噬菌体。这些载体在宿主微生物中可自体复制,也可以通过染色体复制。适宜的质粒例如为:大肠杆菌的pLG338、pACYC184、pBR322、pUC18、pUC19、pKC30、pRep4、pHS1、pKK223-3、pDHE19.2、pHS2、pPLc236、pMBL24、pLG200、pUR290、pIN-III113-B1、λgt11或pBdCI;杆菌的pUB110、pC194或pBD214;棒状杆菌属的pSA77或pAJ667等。在芽孢杆菌属等的杆菌中可以举出pUB110、pC194或pBD214等。除了这些以外还能够使用的质粒等记载于“Gene Cloning and DNA nalysis6th edition”、Wiley-Blackwell(2016)中。表达盒向载体中的导入可通过包括利用适当的限制酶切割、克隆化以及连接的惯用方法进行。各个表达盒可以配置在1个载体上,也可以配置在2个或其以上的载体上。
在如上所述构建出本发明的具有表达盒的载体后,作为将该载体导入至宿主微生物中时能够适用的方法,可以使用惯用的方法。例如可以举出氯化钙法、电穿孔法、接合传递法、原生质体融合法等,但并不限定于这些,可以选择适合于宿主微生物的方法。
本发明的另一方面涉及使用了上述重组微生物的目标化合物的制造方法。更具体地说,涉及包括对上述重组微生物进行培养的培养工序的目标化合物的制造方法。
此处,目标化合物选自由己二酸、己二酸衍生物、六亚甲基二胺、1,6-己二醇和6-氨基-1-己醇组成的组。作为己二酸衍生物,如上所述,例如可以举出3-氧代己二酸(3-OA)、乙酰丙酸(LEV)、3-羟基己二酸(3HA)和2,3-脱氢己二酸(23DA)。
在一个方式中,本发明中的重组微生物具有己二酸生产路径。这种情况下,本发明还涉及包括对该重组微生物进行培养的培养工序的己二酸的制造方法。
在一个方式中,本发明中的重组微生物具有六亚甲基二胺生产路径。这种情况下,本发明还涉及包括对该重组微生物进行培养的培养工序的六亚甲基二胺的制造方法。
在一个方式中,本发明中的重组微生物具有1,6-己二醇生产路径。这种情况下,本发明还涉及包括对该重组微生物进行培养的培养工序的1,6-己二醇的制造方法。
在一个方式中,本发明中的重组微生物具有6-氨基-1-己醇生产路径。这种情况下,本发明还涉及包括对该重组微生物进行培养的培养工序的6-氨基-1-己醇的制造方法。
在培养工序中,通过利用含有碳源和氮源的培养基对重组微生物进行培养,可得到包含菌体的培养物。本发明的基因重组微生物在适合于己二酸生产以及微生物的生长和维持的条件下进行培养,适宜的培养基组成、培养时间和培养条件可以由本领域技术人员容易地设定。
碳源可以举出D-葡萄糖、蔗糖、乳糖、果糖、麦芽糖、低聚糖、多糖、淀粉、纤维素、米糠、废糖蜜、油脂(例如大豆油、葵花籽油、花生油、椰子油等)、脂肪酸(例如棕榈酸、亚油酸、油酸、亚麻酸等)、醇(例如甘油、乙醇等)、有机酸(例如乙酸、乳酸、琥珀酸等)、玉米分解液、纤维素分解液、二氧化碳、一氧化碳等。优选为D-葡萄糖、蔗糖或甘油。这些碳源可以单独使用或者以混合物的形式使用。
使用生物物质来源的原料制造的己二酸可通过基于ISO16620-2或ASTM D6866中规定的碳-14(放射性碳)分析的生物基碳含量的测定,与例如来自石油、天然气体、煤炭等的合成原料明确区分。
氮源可以举出含氮有机化合物(例如蛋白胨、酪蛋白氨基酸、胰蛋白胨、酵母提取物、肉提取物、麦芽提取物、玉米浆、大豆粉、氨基酸和脲等)、或无机化合物(例如氨水溶液、硫酸铵、氯化铵、磷酸铵、碳酸铵、硝酸钠、硝酸铵等)。这些氮源可以单独使用或者以混合物的形式使用。
另外,在重组微生物表达有用的附加特性的情况下、例如具有针对抗生素的抗性标记的情况下,培养基可以包含相对应的抗生素。由此,培养时的杂菌所致的污染风险降低。作为抗生素,可以举出氨苄青霉素等β-内酰胺系抗生素、卡那霉素等氨基糖苷系抗生素、红霉素等大环内酯系抗生素、四环素系抗生素、氯霉素等,但并不限定于这些。
培养可以为分批式、也可以为连续式。另外,在任一情况下,均可以为在培养的适当时刻追加补给上述碳源等的形式。进而,关于培养,可以一边控制合适的温度、氧浓度、pH等条件一边进行培养。来源于一般的微生物宿主细胞的转化体的合适的培养温度通常为15℃~45℃、优选为25℃~37℃的范围。宿主微生物为好氧性的情况下,为了确保发酵中的适当的氧浓度,可以进行振荡(烧瓶培养等)、搅拌/通气(发酵罐培养等)。这些培养条件可由本领域技术人员容易地设定。
本发明中的目标化合物的制造方法可以包括由培养物中将生成化合物分离精制出来的分离精制工序。该工序中可以使用任意的方法,例如使用离心分离、膜过滤、膜分离、析晶、提取、蒸馏、吸附、相分离和各种色谱法之类的方法,但并不限定于这些。分离精制工序可由一种工序构成,并且也可以由多个工序的组合构成。
接受了上述说明的本领域技术人员能够充分实施本发明。以下为了进一步进行说明而举出实施例,因此本发明并不限定于该实施例。
实施例
<1>发明A
以下基于实施例对本发明A进行说明,但本发明A并不被这些实施例所限定。
将实施例中使用的试样的氨基酸序列和碱基序列示于图2~15。
<C6化合物生产路径酶基因表达质粒的构建>
在PCR片段的扩增中,使用PrimeSTAR Max DNAPolymerase(产品名、Takara Bio制)或者KOD FX Neo(产品名、东洋纺制),在质粒的制备中使用大肠杆菌JM109株。碱基序列的最佳化使用GeneArt Gene Optimizer(软件名、Thermo Fisher Scientific公司)、或者Eurofins Genomics公司的人工基因合成服务。
由大肠杆菌(Eshcherichia coli)W3110株(NBRC12713)基因组DNA进行克隆,取得编码大肠杆菌(Eshcherichia coli)的PaaJ(Ec)(序列编号1)的多核苷酸。将序列编号34和35的寡核苷酸作为引物进行PCR,得到包含paaJ(Ec)基因的编码区域(序列编号33)的PCR产物。接下来,将pRSFDuet-1(产品名、Merck公司制造)作为模板,以序列编号36和37的寡核苷酸作为引物进行PCR,得到pRSFDuet-1片段。将包含paaJ(Ec)基因编码区域的DNA片段与pRSFDuet-1片段使用In-Fusion(注册商标。以下省略)HD cloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。得到作为PaaJ(Ec)表达质粒的“paaJ(Ec)-pRSFDuet”。
由大肠杆菌(Eshcherichia coli)W3110株(NBRC12713)的基因组DNA进行克隆,取得编码大肠杆菌(Eshcherichia coli)的PaaH(Ec)(序列编号2)的多核苷酸。将序列编号39和40的寡核苷酸作为引物进行PCR,得到包含paaH(Ec)基因的编码区域(序列编号38)的PCR产物。接下来,将“paaJ(Ec)-pRSFDuet”作为模板,以序列编号41和42的寡核苷酸作为引物进行PCR,得到“paaJ(Ec)-pRSFDuet”片段。将包含paaH(Ec)基因编码区域的DNA片段与“paaJ(Ec)-pRSFDuet”片段使用In-Fusion HD cloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。取得作为PaaJ(Ec)、PaaH(Ec)共表达质粒的“paaJ(Ec)-paaH(Ec)-pRSFDuet”。
由大肠杆菌(Eshcherichia coli)W3110株(NBRC12713)基因组DNA进行克隆,取得编码大肠杆菌(Eshcherichia coli)的PaaF(Ec)(序列编号3)的多核苷酸。以序列编号44和45的寡核苷酸作为引物进行PCR,得到包含paaF(Ec)基因的编码区域(序列编号43)的PCR产物。对于编码被使用序列编号4所示的核苷酸和序列编号46所示的核苷酸作为引物、以L3株的染色体DNA作为模板的PCR扩增物的碱基序列所编码的L3株的PaaF(L3)的核苷酸,进行用于大肠杆菌表达的碱基序列的最佳化,利用Eurofins Genomics公司的人工基因合成服务来取得该核苷酸。以序列编号47和48的寡核苷酸作为引物进行PCR,得到包含paaF(L3)基因的编码区域的最佳化序列的PCR产物。以pETDuet-1(产品名、Merck公司制造)作为模板、以序列编号49和50的寡核苷酸作为引物进行PCR,得到pETDuet-1片段。将包含paaF(Ec)、paaF(L3)各自的编码区域的DNA片段和pETDuet-1片段使用In-Fusion HD cloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。作为PaaF(Ec)表达质粒得到“paaF(Ec)-pETDuet”、作为PaaF(L3)表达质粒得到“paaF(L3)-pETDuet”。
对于编码贝氏不动杆菌(Acinetobacter baylyi)的dcaA(Ab)(序列编号5)的多核苷酸,进行用于大肠杆菌表达的碱基序列的最佳化,利用Eurofins Genomics公司的人工基因合成服务来取得该核苷酸。以序列编号52和53的寡核苷酸作为引物进行PCR,得到包含dcaA(Ab)基因的编码区域(序列编号51)的PCR产物。以“paaF(Ec)-pETDuet”作为模板、以序列编号84和85的寡核苷酸作为引物进行PCR,得到“paaF(Ec)-pETDuet”片段。将包含dcaA(Ab)基因的编码区域的DNA片段与“paaF(Ec)-pETDuet”片段使用In-Fusion HD cloningkit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。得到作为PaaF(Ec)、DcaA(Ab)共表达质粒的“paaF(Ec)-dcaA(Ab)-pETDuet”。
对于热带念珠菌(Candida tropicalis)的Ter(Ct)(序列编号6),以将N末端区域除去22个残基的序列(序列编号7)的形式进行克隆。对于所编码的多核苷酸,进行用于大肠杆菌表达的碱基序列的最佳化,利用Eurofins Genomics公司的人工基因合成服务取得该核苷酸。以序列编号55和56的寡核苷酸作为引物进行PCR,得到包含ter(Ct)基因的编码区域(序列编号54)的PCR产物。以“paaF(L3)-pETDuet”作为模板、以序列编号84和85的寡核苷酸作为引物进行PCR,得到“paaF(L3)-pETDuet”片段。将包含ter(Ct)基因的编码区域的DNA片段与“paaF(L3)-pETDuet”片段利用In-FusionHD cloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。得到作为PaaF(L3)、Ter(Ct)共表达质粒的“paaF(L3)-ter(Ct)-pETDuet”。
使用BLAST Search(https://www.genome.jp/tools/blast/)BLASTP,由KEGGGENES数据库中选择推测具有2,3-脱氢己二酰-CoA还原酶的功能的蛋白质。将所选择的候选酶蛋白示于表A-1。
[表A-1]
表A候选2,3-脱氢己二酰CoA还原酶蛋白
对于所选择的编码2,3-脱氢己二酰-CoA还原酶候选蛋白的多核苷酸,进行用于大肠杆菌表达的碱基序列的最佳化,利用Eurofins Genomics公司的人工基因合成服务取得该核苷酸,通过PCR得到包含各酶基因的编码区域的DNA片段。将各酶基因的编码区域的碱基序列与PCR中使用的引物组的碱基序列的序列编号示于表2。以“paaF(L3)-pETDuet”作为模板、以序列编号84和85的寡核苷酸作为引物进行PCR,得到“paaF(L3)-pETDuet”片段。将包含各基因的编码区域的DNA片段与“paaF(L3)-pETDuet”片段使用In-Fusion HD cloningkit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。得到PaaF(L3)与各2,3-脱氢己二酰-CoA还原酶候选蛋白的共表达质粒。
[表A-2]
表A-2:候选2,3-脱氢己二酰
还原酶基因碱基序列和引物组
对于脓肿分枝杆菌(Mycobacterium abscessus)的羧酸还原酶MaCar(序列编号20),以将氨基酸序列的第283位色氨酸残基置换成精氨酸残基、将第303位丙氨酸残基置换成蛋氨酸残基的突变体MaCar(m)的形式使用(序列编号22)。对于编码MaCar(m)的多核苷酸,进行用于大肠杆菌表达的碱基序列的最佳化,利用Eurofins Genomics公司的人工基因合成服务取得该核苷酸。以序列编号87和88的寡核苷酸作为引物进行PCR,得到包含Macar(m)基因的编码区域(序列编号86)的PCR产物。
接着,使用In-Fusion HD cloning kit(产品名、Clontech公司制造),将该PCR产物插入到pACYCDuet-1(产品名、Merck公司制造)的限制酶NdeI和AvrII切断部位间。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。得到作为MaCar(m)表达质粒的“Macar(m)-pACYCDuet”。
由艾阿华诺卡氏菌(Nocardia iowensis)JCM18299株(通过日本文部科学省国家生物资源项目由理研BRC提供)的基因组DNA进行克隆来取得编码艾阿华诺卡氏菌(Nocardia iowensis)的Npt(序列编号24)的多核苷酸。以序列编号90和91的寡核苷酸作为引物进行PCR,得到包含npt基因的编码区域(序列编号89)的PCR产物。接下来,以“MaCar(m)-pACYCDuet”作为模板、以序列编号92和93的寡核苷酸作为引物进行PCR,得到“Macar(m)-pACYCDuet”片段。将PCR产物彼此使用In-Fusion HD cloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。取得作为MaCar(m)、Npt共表达质粒的“Macar(m)-npt-pACYCDuet”。
由大肠杆菌(Eshcherichia coli)W3110株(NBRC12713)基因组DNA进行克隆,取得编码大肠杆菌(Eshcherichia coli)的Ahr(序列编号32)的多核苷酸。以序列编号95和96的寡核苷酸作为引物进行PCR,得到包含ahr基因(序列编号94)的编码区域的PCR产物。接着以“Macar(m)-npt-pACYCDuet”作为模板、以序列编号97和98的寡核苷酸作为引物进行PCR,得到“macar(m)-npt-pACYCDuet”片段。将包含各基因的编码区域的PCR产物与“macar(m)-npt-pACYCDuet”片段使用In-Fusion HD cloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。分别得到作为Ahr、MaCar、Npt共表达质粒的“ahr-macar(m)-npt-pACYCDuet”。
<C6化合物生产菌株的构建>
将所构建的质粒转化到大肠杆菌BL2l(DE3)株感受态细胞(Nippon Gene公司制造)中,涂布至包含氨苄青霉素钠50mg/L、卡那霉素硫酸盐30mg/L、(菌株HDOl的构建中进一步包含氯霉素30mg/L)的LB琼脂培养基(胰蛋白胨10g/L、酵母抽提物5g/L、氯化钠5g/L、琼脂末15g/L),在37℃培养24小时,得到转化体的单个菌落。将所取得的转化体示于表A-3。
[表A-3]
表A-3:转化体
转化体 | 质粒① | 质粒② | 质粒③ |
ADA1 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(Ec)-dcaA(Ab)-pETDuet | - |
ADA2 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(Ct)-pETDuet | - |
ADA3 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(caur)-pETDuet | - |
ADA4 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(kmx)-pETDuet | - |
ADA5 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(pkz)-pETDuet | - |
ADA6 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(mtm)-pETDuet | - |
ADA7 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(ttt)-pETDuet | |
ADA8 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(cthr)-pETDuet | |
ADA9 | paaJ(Ec)*paaH(Ec)-pRSFDuet | paaF(L3)-ter(pan)-pETDuet | - |
ADA10 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(plj)-pETDuet | - |
ADA11 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(pte)-pETDuet | - |
HDO1 | paaJ(Ec)-paaH(Ec)-pRSFDuet | paaF(L3)-ter(mtm)-pETDuet | ahr-macar(m)-npt-pACYCDuet |
<基于重组大肠杆菌培养的己二酸生产试验>
将一白金环的转化体ADA1~11接种到包含氨苄青霉素钠50mg/L、卡那霉素硫酸盐30mg/L的LB液体培养基(胰蛋白胨10g/L、酵母抽提物5g/L、氯化钠5g/L)2mL(14mL容量的圆底试管)中,在37℃进行24小时振荡培养,得到前培养液。将所得到的前培养液10μL接种到包含氨苄青霉素钠50mg/L、卡那霉素硫酸盐30mg/L的MM培养基1mL(96孔深孔板)中,在37℃进行48小时振荡培养。将MM培养基的组成示于表A-4。
[表A-4]
表A-4:MM培养基组成
培养结束后,对培养液中的己二酸浓度进行分析。分析通过使用了三甲基甲硅烷基衍生物化的GC/MS分析来进行。在培养液离心上清40μL(按照作为内标的癸二酸二钠成为最终浓度10mM的方式添加)中添加按照水:甲醇:氯仿=5:2:2(v/v/v)的方式混合而成的提取液360μL,利用涡流混合器充分搅拌。离心分离(16,000×g、5分钟)后,将上清40μL采集到另一微量管中,利用离心蒸发器离心干固1小时。在所得到的干固物中添加甲氧基胺盐酸盐20mg/mL吡啶溶液100μL,在30℃振荡90分钟。添加N-甲基-N-三甲基甲硅烷基三氟乙酰胺50μL,在37℃振荡30分钟。将反应溶液作为GC/MS测定试样,在下述条件下进行分析。
GC/MS分析条件
装置:GCMS-QP-2020NX(岛津制作所制)
柱:熔融二氧化硅毛细管惰性处理管(长度1m、外径0.35mm、内径0.25mm、GLScience公司制造)、InertCap 5MS/NP(长度30m、内径0.25mm、膜厚0.25μm、GL Science公司制造)
试样注入量:1μL
试样导入法:分流(分流比25:1)
气化室温度:230℃
载气:氦
载气线速度:39.0cm/秒
烘箱温度:80℃、保持2分钟→15℃/分钟升温→325℃、保持13分钟
离子化法:电子离子化法(EI)
离子化能量:70eV
离子源温度:230℃
扫描范围:m/z=50~500
将结果示于表A-5。转化体ADA1~ADA11为导入有可进行图1的步骤A、B、C、D的反应的酶基因的表达质粒的转化体。步骤E的反应通过大肠杆菌的内源性酶来进行。
在表达有大肠杆菌来源的PaaJ(Ec)、PaaH(Ec)、PaaF(Ec)、以及贝氏不动杆菌(Acinetobacter baylyi)来源的dcaA(Ab)的转化体ADA1的培养(比较例1)中,生成了0.14g/L的己二酸。在表达有大肠杆菌来源的PaaJ(Ec)、PaaH(Ec)、L3株来源的PaaF(L3)、以及热带念珠菌(Candida tropicalis)的Ter(Ct)的转化体ADA2的培养(比较例2)中,生成了0.33g/L的己二酸。
在表达有大肠杆菌来源的PaaJ(Ec)、PaaH(Ec)、L3株来源的PaaF(L3)、作为催化图1的步骤D的反应的酶的Ter(caur)、Ter(kmx)、Ter(pkz)、Ter(mtm)、Ter(ttt)、Ter(cthr)、Ter(pan)、Ter(plj)、或Ter(pte)的转化体ADA3~ADA11的培养(实施例1~9)中,分别生成己二酸,显示出Ter(caur)、Ter(kmx)、Ter(pkz)、Ter(mtm)、Ter(ttt)、Ter(cthr)、Ter(pan)、Ter(plj)、或Ter(pte)具有进行己二酸生产路径反应的酶活性。
另外,在转化体ADA6(实施例4)、ADA7(实施例5)、ADA8(实施例6)、ADA9(实施例7)和ADA11(实施例9)的培养中,生成了高于比较例的己二酸,显示出通过表达酶Ter(mtm)、Ter(ttt)、Ter(cthr)、Ter(pan)、或Ter(pte)而提高了C6化合物生成量。另外,由这些结果可知,作为实施例示出的微生物中显示出了良好的2,3-脱氢己二酰-CoA还原酶活性。
[表A-5]
表A-5:己二酸生产试验结果
<基于重组大肠杆菌培养的1,6-己二醇和6-羟基己酸生产试验>
将一白金环的转化体HDO1接种到包含氨苄青霉素钠50mg/L、卡那霉素硫酸盐30mg/L、氯霉素30mg/L的LB液体培养基2mL(14mL容量的圆底试管)中,在37℃进行24小时振荡培养,得到前培养液。将所得到的前培养液10μL接种到包含氨苄青霉素钠50mg/L、卡那霉素硫酸盐30mg/L、氯霉素30mg/L的MM培养基1mL(96孔深孔板)中,在37℃进行48小时振荡培养。
培养结束后,对培养液中的1,6-己二醇浓度、6-羟基己酸浓度进行分析。使用GC/MS,在与己二酸浓度分析同样的条件下对6-羟基己酸浓度进行分析。使用高效液相色谱仪Prominence系统(岛津制作所制)在下述条件下进行1,6-己二醇浓度的分析。
高效液相色谱法(HPLC)分析条件
检测器:差示折射率检测器
柱:Shim-Pack Fast-OA(G)、Fast-OA(岛津制作所制)
烘箱温度:40℃
流动相:8mM甲磺酸水溶液
流速:0.6mL/min
注入量:10μL
将结果示于表A-6。转化体HDO1是除了可进行图1的步骤A、B、C、D的反应的酶基因的表达质粒以外还导入有羧酸还原酶、醇脱氢酶的表达质粒的转化体。在转化体HDO1的培养(实施例10)中,生成了6-羟基己酸和1,6-己二醇。
[表A-6]
表A-6:6-羟基己酸和1,6-己二醇的生产试验结果
<基因破坏用质粒的构建>
在大肠杆菌的基因的破坏·插入中,使用pHAK1(于2019年3月18日以受理号NITEP-02919保藏在独立行政法人制品评价技术基础机构生物技术中心专利微生物保藏中心(NPMD)(地址:千叶县木更津市かずさ镰足2-5-8122号室)。国际保藏号:NITE BP-02919)通过同源重组法来进行。pHAK1包含温度敏感性突变型repA基因、卡那霉素抗性基因、枯草杆菌(Bacillus subtilis)来源的左聚糖蔗糖酶基因sacB。左聚糖蔗糖酶基因在蔗糖存在下对宿主微生物产生致死作用。PCR片段的扩增使用PrimeSTAR Max DNA Polymerase(产品名、Takara Bio制)或KOD FX Neo(产品名、东洋纺制)进行,质粒制备使用大肠杆菌HST08株进行。
以大肠杆菌BL21(DE3)株的基因组DNA作为模板,得到包含破坏靶基因的5’同源区域、编码区域、以及3’同源区域的PCR产物。将靶基因与引物序列的组合示于下述表A-7。
[表A-7]
表A-7:用于质粒构建的引物组
基因名 | 序列编号 |
atoB | 99,100 |
sucD | 101,102 |
IdhA | 103,104 |
adhE | 105,106 |
yqhD,dkgA | 107,108 |
yahK | 109,110 |
ahr | 111,112 |
pflB | 113,114 |
接着,使用In-Fusion HD cloning kit(产品名、Clontech公司制造),将该PCR产物插入到使用序列编号115和116的引物扩增得到的pHAK1质粒片段中,进行环状化。转化到大肠杆菌HST08株中,由所得到的转化体提取质粒。
将上述提取的插入有破坏靶基因的5’同源区域、编码区域、以及3’同源区域的DNA片段的pHAK1质粒作为模板,使用下述表A-8中记载的引物进行PCR,除去破坏靶基因的编码区域的部分区域或全部区域,得到内含有5’同源区域和3’同源区域的pHAK1质粒片段。
[表A-8]
表A-8:用于质粒构建的引物组
基因名 | 序列编号 |
atoB | 117,118 |
sucD | 119,120 |
IdhA | 121,122 |
adhE | 123,124 |
yqhD,dkgA | 125,126 |
yahK | 127,128 |
ahr | 129,130 |
pflB | 131,132 |
将所得到的质粒片段通过末端磷酸化、自连接而进行环状化。转化到大肠杆菌HST08株中,由所得到的转化体提取质粒,得到基因破坏用质粒。
<六亚甲基二胺生产路径酶基因插入用质粒的构建>
关于编码大肠杆菌(Eshcherichia coli)的PaaJ(Ec)(序列编号1)的多核苷酸(序列编号33),由大肠杆菌(Eshcherichia coli)W3110株(NBRC12713)基因组DNA进行克隆。关于编码大肠杆菌(Eshcherichia coli)的PaaH(Ec)(序列编号2)的多核苷酸(序列编号38),由大肠杆菌(Eshcherichia coli)W3110株(NBRC12713)的基因组DNA进行克隆来取得。
对于paaJ(Ec)和paaH(Ec)基因,按照作为表达盒插入到pflB基因区域中的方式来设计基因插入用质粒。在内含在先前构建的pflB基因破坏用质粒中的pflB基因编码区域的5’同源区域与3’同源区域之间,插入了以启动子区域(序列编号133)、paaJ(Ec)基因编码区域、连接子序列(序列编号134)、paaH基因编码区域、终止子区域(序列编号135)的顺序配置的表达盒。
对于编码被使用序列编号4所示的核苷酸和序列编号46所示的核苷酸作为引物、以L3株的染色体DNA作为模板的PCR扩增物的碱基序列所编码的L3株的PaaF(L3)的核苷酸,进行用于大肠杆菌表达的碱基序列的最佳化,利用Eurofins Genomics公司的人工基因合成服务取得该核苷酸。对于编码被使用序列编号162所示的核苷酸和序列编号163所示的核苷酸作为引物、以L3株的染色体DNA作为模板的PCR扩增物的碱基序列所编码的L3株的MmgC(L3)的多核苷酸(1155bp),进行用于大肠杆菌表达的碱基序列的最佳化,利用EurofinsGenomics公司的人工基因合成服务取得该核苷酸。
对于paaF(L3)和mmgC(L3)基因的编码区域的最佳化序列,按照作为表达盒插入到yahK基因区域中的方式来设计基因插入用质粒。在内含在先前构建的yahK基因破坏用质粒中的yahK基因编码区域的5’同源区域与3’同源区域之间,插入了以启动子区域(序列编号136)、paaF(L3)基因编码区域、连接子序列(序列编号137)、mmgC(L3)基因编码区域、终止子区域(序列编号138)的顺序配置的表达盒。
对于脓肿分枝杆菌(Mycobacterium abscessus)的羧酸还原酶MaCar(序列编号20),以将氨基酸序列的第283位的色氨酸残基置换成精氨酸残基、将第303位的丙氨酸残基置换成蛋氨酸残基的突变体MaCar(m)的形式使用(序列编号22)。对于编码MaCar(m)的多核苷酸(序列编号86),进行用于大肠杆菌表达的碱基序列的最佳化,利用Eurofins Genomics公司的人工基因合成服务取得该核苷酸。对于编码艾阿华诺卡氏菌(Nocardia iowensis)的Npt(序列编号24)的多核苷酸(序列编号89),由艾阿华诺卡氏菌(Nocardia iowensis)JCM18299株(通过日本文部科学省国家生物资源项目由理研BRC提供)的基因组DNA进行克隆来取得该核苷酸。
对于macar(m)和npt基因,按照作为表达盒插入到ldhA基因区域中的方式来设计基因插入用质粒。在内含在先前构建的ldhA基因破坏用质粒中的ldhA基因编码区域的5’同源区域与3’同源区域之间,插入了以启动子区域(序列编号139)、macar(m)基因编码区域、连接子序列(序列编号140)、npt基因编码区域、终止子区域(序列编号141)的顺序配置的表达盒。
<大肠杆菌修饰株的构建>
通过电穿孔法(参照羊土社基因工程实验笔记田村隆明著)在大肠杆菌BL21(DE3)株中转化用于破坏或插入所期望的基因的质粒后,涂布至含有卡那霉素硫酸盐100mg/L的LB琼脂培养基(胰蛋白胨10g/L、酵母抽提物5g/L、氯化钠5g/L、琼脂末15g/L),在30℃培养一夜,取得单个菌落,得到转化体。将一白金环的本转化体接种到含有卡那霉素硫酸盐100mg/L的LB液体培养基(胰蛋白胨10g/L、酵母抽提物5g/L、氯化钠5g/L)1mL中,在30℃进行振荡培养。将所得到的培养液涂布至含有卡那霉素硫酸盐100mg/L的LB琼脂培养基,在42℃培养一夜,取得菌落。将一白金环所得到的菌落接种到LB液体培养基1mL中,在30℃进行振荡培养。将所得到的培养液涂布至含有蔗糖20%的LB琼脂培养基,在30℃培养2天。对于所得到的菌落,使用表A-9所示的引物组通过菌落直接PCR来确认所期望的基因的破坏或插入。
重复以上的操作,构建出表A-10所示的包含复数个基因破坏和基因插入的大肠杆菌株“No.060”。表中,Δ表示该酶基因缺损,“基因名A::基因名B”表示基因A区域被置换成包含基因B的区域。
[表A-9]
表A-9:破坏·插入目标确认用引物组
基因名 | 序列编号 |
atoB | 142,143 |
sucD | 144,145 |
IdhA | 146,147 |
adhE | 148,149 |
yqhD,dkgA | 150,151 |
yahK | 152,153 |
ahr | 154,155 |
pflB | 156,157 |
[表A-10]
表A-1O:大肠杆菌修饰株
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<六亚甲基二胺生产路径酶基因表达质粒的构建>
在PCR片段的扩增中使用PrimeSTAR Max DNA Polymerase(产品名、Takara Bio制)或者KOD FXNeo(产品名、东洋纺制),在质粒的制备中使用大肠杆菌JM109株。
大肠杆菌(Eshcherichia coli)的YgjG(序列编号27)使用一部分氨基酸序列被置换的突变型YgjG(m)(序列编号158)。关于编码YgjG(m)的多核苷酸,以序列编号160和161的寡核苷酸作为引物进行PCR,得到包含ygjG(m)基因(序列编号159)的编码区域的PCR产物。接着使用In-Fusion HD cloning kit(产品名、Clontech公司制造)将该PCR产物插入到pACYCDuet-1(产品名、Merck公司制造)的限制酶NcoI和HindIII切断部位间。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。得到作为MaCar(m)表达质粒的“ygjG(m)-pACYCDuet”。
<基于重组大肠杆菌培养的六亚甲基二胺生产试验>
将“paaF(L3)-ter(mtm)-pETDuet”和“ygjG(m)-pACYCDuet”通过电穿孔法转化到大肠杆菌修饰株No.060中,在包含氨苄青霉素钠50mg/L、氯霉素30mg/L的LB琼脂培养基上在37℃培养一天,形成菌落,得到转化体HMD1。将一白金环的转化体HMD1的菌落接种到包含氨苄青霉素钠50mg/L、氯霉素30mg/L的LB液体培养基2mL(14mL容量的圆底试管)中,在37℃进行3~5小时振荡培养,得到前培养液。在250mL容量的罐状培养装置(机种名BioJr.8、Biott公司制造)中在包含羧苄西林钠50mg/L、氯霉素30mg/L、IPTG 0.02mM的合成培养基(下表)中添加前培养液0.5mL,进行主培养。培养条件如下:培养温度37℃;培养pH 7.0;pH调整10%(w/v)氨水;搅拌750rpm;通气1vvm。在前培养液接种后经过了22小时的时刻追加添加葡萄糖以使其最终浓度为35g/L,培养至经过41小时为止。
[表A-11]
表A-11:合成培养基组成
培养结束后,将培养液通过离心分离而分离成菌体和上清,对上清中的六亚甲基二胺浓度进行分析。六亚甲基二胺浓度的分析使用离子色谱在下述条件下进行。
离子色谱分析条件
装置:ICS-3000(Dionex公司制造)
检测器:电导率检测器
柱:IonPac CG19(2×50mm)/CS19(2×250mm)(Thermo Scientific公司制造)
烘箱温度:30℃
流动相:8mM甲磺酸水溶液(A)、70mM甲磺酸水溶液(B)
梯度条件:(A:100%、B:0%)-(10min)-(A:0%、B:100%)-(保持1min)
流速:0.28mL/min
注入量:20μL
将结果示于表A-12中。转化体HMD1是除了可进行图1的步骤A、B、C、D的反应的酶基因以外还导入有羧酸还原酶、氨基转移酶基因的转化体。转化体HMD1的培养(实施例11)中生成了六亚甲基二胺。
[表A-12]
表A--12:六亚甲基二胺生产菌株的培养结果
<2>发明B
以下基于实施例说明本发明B,但本发明B并不被这些实施例所限定。
<伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的分离和鉴定>
使用以己二酸作为唯一碳源和能量源的培养基,由日本国内的活性污泥中分离出伯克霍尔德菌属(Burkholderia sp.)LEBP-3株。伯克霍尔德菌属(Burkholderiasp.)LEBP-3株于2020年12月4日(原保藏日)向独立行政法人制品评价技术基础机构专利微生物保藏中心NPMD)(地址:千叶县木更津市かずさ镰足2-5-8122号室)提交了保藏申请(受理号:NITE ABP-03334),以“保藏号:NITE BP-03334”进行了国际保藏。伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的分类性状如下所示。
<LEBP-3株的细菌学的各性质>
培养温度:37℃、
细胞形态:杆菌(0.7×1.7-3.0μm)、
革兰氏染色性:-、
芽孢的有无:-、
运动性:+、
LB琼脂培养基48h培养时的菌落形态:直径1.9~2.5mm、淡黄色、圆形、透镜状、全缘、平滑、不透明、黄油样、
30℃生长:+、
45℃生长:+、
过氧化氢酶反应:+、
氧化酶反应:+、
葡萄糖中的酸/气体产生:+/-、
O/F检测(氧化/发酵):+/-、
硝酸盐还原性:-、
吲哚产生性:-、
葡萄糖酸性化性:-、
脲酶:不具有、
七叶苷分解性:+、
葡萄糖、L-阿拉伯糖、D-甘露糖、N-乙酰基-D-葡萄糖胺、葡萄糖酸、正癸酸、己二酸、苹果酸、柠檬酸和乙酸苯酯:能够同化、
麦芽糖:不能同化。
<基于LEBP-3株的16SrRNA基因序列同源性的系统分析>
通过进行16SrDNA基因的碱基序列分析来进行鉴定。将16SrDNA基因的碱基序列利用PCR扩增,进行序列分析。BLAST同源性检索的数据库使用DB-BA11.0(Techno SurugaLaboratory公司)和国际碱基序列数据库。其结果,LEBP-3株包含在由Burkhlderia属构成的聚类中,但显示出与任一已知种均不同的分子系统学的位置,因此未能进行种名的鉴定。
<基于PCR扩增的酶基因的取得和同源性评价>
实施伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的全基因组草图分析。将LEBP-3株在2mL的LB培养基(胰蛋白胨10g/L、酵母抽提物5g/L、氯化钠10g/L)中实施37℃振荡培养。培养结束后,由培养液回收菌体,使用Nucleo Spin Tissue(产品名、MACHEREY-NAGEL公司制造)提取基因组DNA。实施伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的全基因组草图分析和注释,鉴定出7679个开放阅读框(Open reading frame;ORF)。根据该信息,作为琥珀酰CoA:乙酰CoA酰基转移酶鉴定出paaJ(L3),作为3-羟基己二酰CoA脱氢酶鉴定出paaH(L3),作为3-羟基己二酰CoA脱水酶鉴定出paaF(L3),作为2,3-脱氢己二酰CoA还原酶鉴定出mmgC(L3)。
将PaaJ(L3)序列作为查询序列实施BLAST检索的结果示于表B-1,将PaaH(L3)序列作为查询序列实施BLAST检索的结果示于表B-2,将PaaF(L3)序列作为查询序列实施BLAST检索的结果示于表B-3,将MmgC(L3)序列作为查询序列实施BLAST检索的结果示于表B-4。需要说明的是,序列检索时的数据库指定了“Refseq__Protein”。
[表B-1]
[表B-2]
[表B-3]
[表B-4]
根据上述结果,伯克霍尔德菌属(Burkholderia sp.)LEBP-3株所具有的上述酶被鉴定为新序列。paaJ(L3)基因可以以序列编号164所示的核苷酸和序列编号165所示的核苷酸作为引物进行PCR扩增。paaH(L3)基因可以以序列编号166所示的核苷酸和序列编号167所示的核苷酸作为引物进行PCR扩增。paaF(L3)基因可以以序列编号168所示的核苷酸和序列编号169所示的核苷酸作为引物进行PCR扩增。mmgC(L3)基因可以以序列编号170所示的核苷酸和序列编号171所示的核苷酸作为引物进行PCR扩增。将PCR中使用的引物、各酶和编码酶的碱基序列示于图17~20。
PCR扩增条件的一例如下所示。酶使用PrimeSTAR Max DNA Polymerase(产品名、Takara Bio制)。将热处理98℃10秒、退火55℃15秒、延伸72℃5秒/kb的循环实施30次。作为引物使用的寡核苷酸各添加1μM添加。以液量为25μL实施扩增。
<表达取得酶基因的重组大肠杆菌的构建>
在PCR片段的扩增中使用PrimeSTAR Max DNA Polymerase(产品名、Takara Bio制),在质粒的制备中使用大肠杆菌JM109株。在碱基序列的密码子最佳化中使用GeneArtGeneOptinizer(软件名、Thermo Fisher Scientific公司)或Eurofins Genomics公司的人工基因合成服务。
对于编码大肠杆菌(Eshcherichia coli)的PaaJ(Ec)的多核苷酸,由大肠杆菌(Eshcherichia coli)W3110株(NBRC12713)基因组DNA进行克隆。以序列编号188和189的寡核苷酸(图20A)作为引物进行PCR,得到包含paaJ(Ec)基因的编码区域(序列编号180)(图19A)的PCR扩增物。接下来,以pRSFDuet-1(产品名、Merck公司制造)作为模板、以序列编号190和191所示的核苷酸(图20A)作为引物进行PCR,得到pRSFDuet-1片段。将包含基因编码区域的DNA片段和pRSFDuet-1片段使用In-Fusion HD cloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。得到作为PaaJ(Ec)表达质粒的“prm203”。
对于编码大肠杆菌(Eshcherichia coli)的PaaH(Ec)的多核苷酸,由大肠杆菌(Eshcherichia coli)W3110株(NBRC12713)的基因组DNA进行克隆。以序列编号192和193所示的核苷酸(图20A)作为引物进行PCR,得到包含paaH(Ec)基因的编码区域(序列编号181)(图19A)的PCR扩增物。接下来,以prm203作为模板、以序列编号194和195所示的核苷酸(图20A)作为引物进行PCR,得到prm203片段。将包含各基因编码区域的DNA片段与prm203片段使用In-Fusion HD cloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。得到作为PaaJ(Ec)-PaaH(Ec)表达质粒的“prm69”。
对于编码大肠杆菌(Eshcherichia coli)的PaaF(Ec)的多核苷酸,由大肠杆菌(Eshcherichia coli)W3110株(NBRC12713)基因组DNA进行克隆。以序列编号196和197所示的核苷酸(图20A)作为引物进行PCR,得到包含paaF(Ec)基因的编码区域(序列编号182)(图19A)的PCR扩增物。对于编码L3株的PaaF(L3)的多核苷酸,进行用于大肠杆菌表达的碱基序列的最佳化,利用Eurofins Genomics公司的人工基因合成服务取得该核苷酸。以序列编号198和199所示的核苷酸(图20A)作为引物进行PCR,得到包含paaF(L3)基因的编码区域的PCR扩增物。以pETDuet-1(产品名、Merck公司制造)作为模板、以序列编号204和205所示的核苷酸(图20A)作为引物进行PCR,得到pETDuet-1片段。将包含各基因的编码区域的DNA片段与pETDuet-1片段使用In-Fusion HD cloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。作为PaaF(Ec)表达质粒得到“prm74”、作为PaaF(L3)表达质粒得到“prm194”。
对于编码贝氏不动杆菌(Acinetobacter baylyi)的dcaA(Ab)、褐色嗜热裂孢菌(Thermobifida fusca)的Tfu1647、L3株的MmgC(L3)的多核苷酸,进行用于大肠杆菌表达的碱基序列的最佳化,利用Eurofins Genomics公司的人工基因合成服务取得该核苷酸。对于dcaA(Ab)基因(序列编号185)(图19B),以序列编号206和207所示的核苷酸(图20A)作为引物进行PCR,对于褐色嗜热裂孢菌(Thermobifida fusca)的Tfu1647基因(序列编号186)(图19B),以序列编号208和209所示的核苷酸(图20A)作为引物进行PCR,对于mmgC5(L3)基因,以序列编号210和211(图20A)所示的核苷酸作为引物进行PCR,分别得到包含dcaA(Ab)、Tfu1647、mmgC5(L3)各基因的编码区域的PCR扩增物。以prm74和prm194作为模板、以序列编号212和213所示的核苷酸(图20A)作为引物进行PCR,分别得到prm74片段和prm194片段。将包含各基因的编码区域的DNA片段与prm74片段和prm194片段使用In-Fusion HD cloningkit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。分别得到作为PaaF(Ec)-dcaA(Ab)表达质粒的“prm127”、作为PaaF(Ec)-mmgC(L3)表达质粒的“prm234”、作为PaaF(L3)-mmgC(L3)表达质粒的“prm166”。
对于编码贝氏不动杆菌(Acinetobacter baylyi)的tesB(Ab)的多核苷酸,以序列编号214和215所示的核苷酸(图20A)作为引物由贝氏不动杆菌(Acinetobacter baylyi)的染色体DNA进行PCR,分别得到包含tesB(Ab)基因的编码区域的PCR扩增物。以pCDFDuet-1作为模板、以序列编号216和217所示的核苷酸(图20A)作为引物进行PCR,得到pCDFDuet-1片段。将包含各tesB(Ab)基因编码区域的DNA片段与pCDFDuet-1片段使用In-Fusion HDcloning kit(产品名、Clontech公司制造)进行连接。转化到大肠杆菌JM109株中,由所得到的转化体提取质粒。得到作为TesB(Ab)表达质粒的“prm92”。
如下所述制作大肠杆菌BL21(DE3)株的基因破坏株。编码乳酸脱氢酶的ldh基因、编码乙酰CoA乙酰基转移酶的atoB基因、以及编码琥珀酰CoA合成酶α亚基的sucD基因的破坏通过使用pHAK1(于2019年3月18日以受理号NITE P-02919保藏于独立行政法人制品评价技术基础机构生物技术中心专利微生物保藏中心(NPMD)(地址:千叶县木更津市かずさ镰足2-5-8 122号室)。国际保藏号:NITE BP-02919)的同源重组法进行。pHAK1包含温度敏感性突变型repA基因、卡那霉素抗性基因、枯草杆菌(Bacillus subtilis)来源的左聚糖蔗糖酶基因SacB。左聚糖蔗糖酶基因在蔗糖存在下对宿主微生物产生致死作用。PCR片段的扩增使用PrimeSTAR Max DNA Polymerase(产品名、Takara Bio制)进行,质粒制备使用大肠杆菌HST08株进行。
以大肠杆菌BL21(DE3)株的基因组DNA作为模板,得到包含破坏靶基因的上游区域、编码区域、以及下游区域的PCR扩增物。对于ldh基因周边区域,以序列编号218和219所示的核苷酸(图20B)作为引物进行PCR,对于atoB基因周边区域,以序列编号220和221所示的核苷酸(图20B)作为引物进行PCR,对于sucD基因周边区域,以序列编号222和223所示的核苷酸(图20B)作为引物进行PCR,得到各片段。
接着,使用In-Fusion HD cloning kit(产品名、Clontech公司制造),将该PCR扩增物插入到使用序列编号224和225所示的引物(图20B)扩增得到的pHAK1质粒片段中,进行环状化。
以所得到的插入有破坏靶基因的上游区域、编码区域、以及下游区域的DNA片段的pHAK1质粒作为模板,对于ldh基因周边区域,以序列编号226和227所示的核苷酸(图20B)作为引物进行PCR,对于atoB基因周边区域,以序列编号228和229所示的核苷酸(图20B)作为引物进行PCR,对于sucD基因周边区域,以序列编号230和231所示的核苷酸作为引物(图20B)进行PCR,得到除去了破坏靶基因的编码区域的一部分区域或全部区域的质粒片段。
对所得到的质粒片段通过末端磷酸化、自连接而进行环状化,得到基因破坏用质粒。
通过氯化钙法(参照羊土社基因工程实验笔记田村隆明著)在大肠杆菌BL21(DE3)株中转化用于破坏所期望的基因的质粒后,涂布至含有卡那霉素硫酸盐100mg/L的LB琼脂培养基(胰蛋白胨10g/L、酵母抽提物5g/L、氯化钠5g/L、琼脂末15g/L),在30℃培养一夜,取得单个菌落,得到转化体。将一白金环的该转化体接种到含有卡那霉素硫酸盐100mg/L的LB液体培养基(胰蛋白胨10g/L、酵母抽提物5g/L、氯化钠5g/L)1mL中,在30℃进行振荡培养。将所得到的培养液涂布至含有卡那霉素硫酸盐100mg/L的LB琼脂培养基,在42℃培养一夜。对于所得到的菌落,通过单交换将质粒插入到基因组中。将一白金环的菌落接种到LB液体培养基1mL中,在30℃进行振荡培养。将所得到的培养液涂布至含有蔗糖10%的LB琼脂培养基,培养一夜。对于所得到的菌落,通过菌落直接PCR来确认所期望的基因的破坏。首先构建ldh基因破坏株,以该ldh基因破坏株为基础构建atoB基因破坏株,进一步以ldh、atoB双重破坏株为基础构建sucD基因破坏株。将所制作的ldh、atoB、sucD基因的三重破坏株作为ASL000株。
将所构建的质粒转化到ASL000株中,涂布至包含氨苄青霉素钠50mg/L、卡那霉素硫酸盐30mg/L、链霉素硫酸盐50mg/L的LB琼脂培养基(胰蛋白胨10g/L、酵母抽提物5g/L、氯化钠5g/L、琼脂末15g/L),在37℃培养24小时,得到转化体的单个菌落。将所取得的转化体示于表B-5。
[表B-5]
<基于表达取得酶基因的重组大肠杆菌的己二酸生产试验>
将一白金环的表B-5所示的菌株接种到包含氨苄青霉素钠50mg/L、卡那霉素硫酸盐30mg/L和链霉素硫酸盐50mg/L的LB液体培养基(胰蛋白胨10g/L、酵母抽提物5g/L、氯化钠5g/L)2mL(14mL容量的圆底试管)中,在37℃进行24小时振荡培养,得到前培养液。将上述得到的前培养液10μL接种在包含氨苄青霉素钠50mg/L、卡那霉素硫酸盐30mg/L和链霉素硫酸盐50mg/L的MM培养基1mL(96孔深孔板)中,在37℃进行48小时振荡培养。
将MM培养基的组成示于表B-6。
[表B-6]
MM培养基
培养结束后,对培养液中的己二酸和己二酸类似物的浓度进行分析。分析通过使用了三甲基甲硅烷基衍生物化的GC/MS分析来进行。在培养液离心上清40μL(按照作为内标的癸二酸二钠成为最终浓度10mM的方式添加)中添加按照水:甲醇:氯仿=5:2:2(v/v/v)进行混合而成的提取液360μL,充分混合。离心分离(16,000×g、5分钟)后,将上清100μL采集到另一微量管中,利用离心蒸发器进行1小时离心浓缩。在所得到的干固物中添加甲氧基胺盐酸盐20mg/mL吡啶溶液100μL,在30℃振荡90分钟。添加N-甲基-N-三甲基甲硅烷基三氟乙酰胺50μL,在37℃振荡30分钟。将反应溶液作为GC/MS测定试样,在下述条件下进行分析。
GC/MS分析条件
装置:GCMS-QP-2020NX(岛津制作所制)
柱:熔融二氧化硅毛细管惰性处理管(长度1m、外径0.35mm、内径0.25mm、GLScience公司制造)、InertCap 5MS/NP(长度30m、内径0.25mm、膜厚0.25μm、GL Science公司制造)
试样注入量:1μL
试样导入法:分流(分流比25:1)
气化室温度:230℃
载气:氦
载气线速度:39.0cm/秒
烘箱温度:80℃、保持2分钟→15℃/分钟升温→325℃、保持13分钟
离子化法:电子离子化法(EI)
离子化能量:70eV
离子源温度:230℃
扫描范围:m/z=50~500
注入量:1μL
将结果示于表B-7。表中,“3OA”表示3-氧代己二酸,“LEV”表示乙酰丙酸,“3HA”表示3-羟基己二酸,“23DA”表示2,3-脱氢己二酸,“ADA”表示己二酸。
[表B-7]
在表达了L3株来源的PaaF(L3)和mmgC(L3)这两者的发明株、即ADA500株和ADA496株中,3-氧代己二酸(3-OA)、乙酰丙酸(LEV)、3-羟基己二酸(3HA)、2,3-脱氢己二酸(23DA)和己二酸(ADA)生成得比对照株多,以它们的合计值计分别生成了355mg/L和613mg/L。根据以上的结果显示出,被这些基因所编码的酶具有进行己二酸路径反应的高活性,提高己二酸和己二酸衍生物的生成量。
工业实用性
本发明能够提供例如C6化合物的有效的制造工艺,期待其在工业规模的生产中的应用。
本发明还适合用于由生物物质原料直接进行己二酸类的发酵生产。
序列表
<110> 旭化成株式会社
<120> 重组微生物和C6化合物的制造方法
<130> 210089WO01
<150> JP2021-069632
<151> 2021-04-16
<150> JP2021-057325
<151> 2021-03-30
<160> 251
<170> PatentIn version 3.5
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<213> 大肠杆菌
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Pro Asn Glu Val Ile Val Lys Thr Leu Gly Ser Pro Val Asn Pro Ser
35 40 45
Asp Ile Asn Gln Ile Gln Gly Val Tyr Pro Ser Lys Pro Ala Lys Thr
50 55 60
Thr Gly Phe Gly Thr Thr Glu Pro Ala Ala Pro Cys Gly Asn Glu Gly
65 70 75 80
Leu Phe Glu Val Ile Lys Val Gly Ser Asn Val Ser Ser Leu Glu Ala
85 90 95
Gly Asp Trp Val Ile Pro Ser His Val Asn Phe Gly Thr Trp Arg Thr
100 105 110
His Ala Leu Gly Asn Asp Asp Asp Phe Ile Lys Leu Pro Asn Pro Ala
115 120 125
Gln Ser Lys Ala Asn Gly Lys Pro Asn Gly Leu Thr Ile Asn Gln Gly
130 135 140
Ala Thr Ile Ser Val Asn Pro Leu Thr Ala Tyr Leu Met Leu Thr His
145 150 155 160
Tyr Val Lys Leu Thr Pro Gly Lys Asp Trp Phe Ile Gln Asn Gly Gly
165 170 175
Thr Ser Ala Val Gly Lys Tyr Ala Ser Gln Ile Gly Lys Leu Leu Asn
180 185 190
Phe Asn Ser Ile Ser Val Ile Arg Asp Arg Pro Asn Leu Asp Glu Val
195 200 205
Val Ala Ser Leu Lys Glu Leu Gly Ala Thr Gln Val Ile Thr Glu Asp
210 215 220
Gln Asn Asn Ser Arg Glu Phe Gly Pro Thr Ile Lys Glu Trp Ile Lys
225 230 235 240
Gln Ser Gly Gly Glu Ala Lys Leu Ala Leu Asn Cys Val Gly Gly Lys
245 250 255
Ser Ser Thr Gly Ile Ala Arg Lys Leu Asn Asn Asn Gly Leu Met Leu
260 265 270
Thr Tyr Gly Gly Met Ser Phe Gln Pro Val Thr Ile Pro Thr Ser Leu
275 280 285
Tyr Ile Phe Lys Asn Phe Thr Ser Ala Gly Phe Trp Val Thr Glu Leu
290 295 300
Leu Lys Asn Asn Lys Glu Leu Lys Thr Ser Thr Leu Asn Gln Ile Ile
305 310 315 320
Ala Trp Tyr Glu Glu Gly Lys Leu Thr Asp Ala Lys Ser Ile Glu Thr
325 330 335
Leu Tyr Asp Gly Thr Lys Pro Leu His Glu Leu Tyr Gln Asp Gly Val
340 345 350
Ala Asn Ser Lys Asp Gly Lys Gln Leu Ile Thr Tyr
355 360
<210> 8
<211> 388
<212> PRT
<213> 耳念珠菌
<400> 8
Met Phe Val Pro Thr Val Ala Gly Lys Arg Phe Leu Thr Asn Gln Leu
1 5 10 15
Arg Lys Ser Pro Gln Phe Thr Arg Met Leu Thr Ser His Ala Val Ile
20 25 30
Phe Ser Ser His Gly Glu Pro Lys Asp Val Leu Lys Thr His Thr Tyr
35 40 45
Glu Ile Asp Glu Asn Asn Ile Lys Pro Asn Glu Ile Val Leu Lys Thr
50 55 60
Leu Gly Ala Pro Val Asn Pro Ser Asp Ile Asn Gln Val Gln Gly Val
65 70 75 80
Tyr Pro Ser Gln Pro Glu Lys Thr Thr Gln Leu Gly Thr Ser Glu Pro
85 90 95
Ser Ala Val Gly Gly Asn Glu Gly Leu Phe Glu Ile Ile Lys Ile Gly
100 105 110
Ser Asp Val Lys Gly Phe Gln Val Gly Asp Trp Ala Ile Pro Thr Ser
115 120 125
Val Asn Phe Gly Thr Trp Arg Ser His Ala Leu Cys Glu Glu Gly Lys
130 135 140
Met Met Lys Val Pro Asn Pro Glu Gln Ser Lys Gln Ala Gly Lys Lys
145 150 155 160
Gln Gly Leu Thr Ile Asn Gln Gly Ala Thr Leu Ser Val Asn Pro Leu
165 170 175
Thr Ala Tyr Leu Met Leu Thr His Tyr Val Lys Leu Thr Pro Gly Lys
180 185 190
Asp Trp Phe Ile Gln Asn Gly Gly Asn Ser Ala Val Gly Lys Phe Ala
195 200 205
Ser Gln Ile Gly Lys Leu Leu Gly Ile His Ser Ile Ser Val Val Arg
210 215 220
Asp Arg Pro Glu Ile Glu Glu Leu Lys Lys Glu Leu Lys Glu Val Cys
225 230 235 240
Gly Ala Thr Gln Val Ile Thr Glu Glu Gln Asn Asn Ser Lys Asp Phe
245 250 255
Gly Pro Gln Val Lys Gln Trp Val Lys Glu Thr Gly Gly Glu Ile Lys
260 265 270
Leu Ala Leu Asn Cys Val Gly Gly Lys Ser Ser Ala Gly Val Ala Arg
275 280 285
Lys Leu Ala Pro Asn Gly Leu Met Leu Thr Tyr Gly Gly Met Ser Phe
290 295 300
Gln Pro Val Thr Leu Pro Thr Ser Leu His Ile Phe Lys Asn Val Thr
305 310 315 320
Ser Ala Gly Phe Trp Val Thr Glu Leu Leu Lys Ser Asp Pro Asn Leu
325 330 335
Lys Arg Glu Thr Val Asp Lys Ile Val Glu Trp Tyr Thr Ser Gly Glu
340 345 350
Leu Leu Asp Ala Pro Ser Lys Glu Asn Gln Trp Gly Asp Gln Asp Leu
355 360 365
Ala Gln Val Phe Ile Asp Ala Val Ala Asn Ser Lys Ser Gly Lys Gln
370 375 380
Leu Val Lys Phe
385
<210> 9
<211> 380
<212> PRT
<213> 马克斯克鲁维酵母
<400> 9
Met Ser Ser Phe Leu Ser Lys Arg Phe Ile Ser Thr Thr Gln Arg Ala
1 5 10 15
Met Ser Gln Leu Pro Lys Ala Lys Ser Leu Ile Tyr Ser Ser His Asp
20 25 30
Gln Asp Val Ser Lys Ile Leu Lys Val His Thr Tyr Gln Pro Lys Gly
35 40 45
Ser Ala Glu Ser Ser Ile Leu Leu Lys Thr Leu Ala Phe Pro Ile Asn
50 55 60
Pro Ser Asp Ile Asn Gln Leu Glu Gly Val Tyr Pro Ser Lys Pro Glu
65 70 75 80
Lys Val Leu Asp Tyr Ser Thr Glu Glu Pro Ser Ala Ile Ala Gly Asn
85 90 95
Glu Gly Leu Phe Glu Val Val Ser Val Pro Ser Gly Val Lys Asn Leu
100 105 110
Lys Ala Gly Asp Arg Val Ile Pro Leu Gln Ala Asn Phe Gly Thr Trp
115 120 125
Ser Thr Tyr Arg Thr Cys Glu Ser Glu Asn Asp Leu Ile Lys Ile Glu
130 135 140
Gly Val Asp Leu Tyr Thr Ala Ala Thr Ile Ala Val Asn Gly Cys Thr
145 150 155 160
Ala Tyr Gln Met Val Asn Asp Tyr Ile Glu Trp Asp Pro Ser Gly Asn
165 170 175
Asp Trp Leu Val Gln Asn Ala Gly Thr Ser Ser Val Ser Lys Ile Val
180 185 190
Thr Gln Ile Ala Lys Asp Lys Gly Ile Lys Thr Leu Ser Val Val Arg
195 200 205
Asp Arg Asp Asn Phe Asp Glu Val Ala Glu Lys Leu Glu Lys Lys Tyr
210 215 220
Gly Ala Thr Lys Val Ile Ser Glu Ser Gln Asn Gly Glu Arg Glu Phe
225 230 235 240
Gly Asn Glu Val Leu Pro Lys Ile Leu Gly Pro Asn Ala Gln Val Lys
245 250 255
Leu Ala Leu Asn Ser Val Gly Gly Lys Ser Cys Thr Asn Ile Ala Arg
260 265 270
Lys Leu Ser Pro Asn Gly Leu Met Leu Thr Tyr Gly Gly Met Ser Lys
275 280 285
Gln Pro Val Thr Leu Pro Thr Gly Leu Phe Ile Phe Asn Ser Ile Arg
290 295 300
Ser His Gly Phe Trp Val Thr Ala Asn Ser Lys Arg Asp Pro Glu Asn
305 310 315 320
Lys Arg Lys Thr Val Asp Ala Val Val Lys Leu Tyr Arg Asp Gly Lys
325 330 335
Ile Ile Ser Pro Lys Glu Asp Ile Arg Thr Leu Glu Trp Asp Val Asn
340 345 350
Asn Leu Ser Asp Glu Gly Val Leu Asp Leu Val Asn Arg Gly Ile Ala
355 360 365
Thr Lys Gly Ala Lys Asn Met Val Val Leu Lys Trp
370 375 380
<210> 10
<211> 373
<212> PRT
<213> 库德里阿兹威毕赤酵母
<400> 10
Met Ser Leu Arg Ser Val Phe Ser Val Ser Ser Ile Arg Arg Met Ser
1 5 10 15
Ser Ile Thr Ala Arg Ala Ile Val Tyr Asn Ala His Gly Glu Pro Lys
20 25 30
Asp Thr Leu Lys Cys His Ser Tyr Lys Ile Asp Leu Gln Asn Leu Gln
35 40 45
Pro Asn Glu Val Val Val Gln Thr Leu Ala Ala Pro Leu Asn Pro Ser
50 55 60
Asp Ile Asn Gln Ile Gln Gly Val Tyr Pro Ser Lys Pro Pro Leu Ser
65 70 75 80
Ile Asp Ser Leu Pro Glu Leu Lys Glu Pro Ala Ala Val Thr Gly Asn
85 90 95
Glu Gly Val Phe Lys Val Ile Ala Lys Gly Ser Asn Val Gly Glu Leu
100 105 110
Glu Thr Gly Asp Trp Cys Ile Pro Ser Gly Val Asn Phe Gly Thr Trp
115 120 125
Cys Thr His Arg Leu Thr Thr Asp Lys Asn Leu Leu Lys Met Pro Lys
130 135 140
Thr Ile Ser Leu Asn Gln Ala Ala Thr Ile Ala Val Asn Pro Ser Ser
145 150 155 160
Ala Tyr Gln Met Leu Thr His Phe Val Lys Leu Gln Pro Gly Asp Trp
165 170 175
Phe Ile Gln Asn Gly Ala Asn Ser Gln Val Gly Arg Ala Ala Ile Gln
180 185 190
Ile Gly His Lys Leu Gly Tyr Lys Ser Leu Asn Ile Val Arg Asn Arg
195 200 205
Asp Asn Leu Asp Glu Leu Val Lys Asp Leu Glu Ser Ile Gly Ala Thr
210 215 220
Lys Val Ile Thr Glu Asp Glu Ser Ala Ser Lys Glu Phe Gly Ala Thr
225 230 235 240
Val Lys Glu Trp Leu Glu Gly Lys Glu Ile Lys Leu Gly Leu Asn Cys
245 250 255
Ile Gly Gly Pro Ser Ala Ser Asn Leu Ala Arg Lys Leu Gly His Asp
260 265 270
Ala Thr Ile Leu Thr Tyr Gly Ala Met Ser Lys Lys Pro Leu Ser Leu
275 280 285
Pro Thr Gly Asn Phe Ile Phe Lys Asn Leu Thr Ala Lys Gly Phe Trp
290 295 300
Ile Thr Gly Asn Ile Lys Arg His Pro Glu Leu Arg Ile Glu Thr Ile
305 310 315 320
Asn Ala Val Leu Lys Phe Met Glu Asp Gly Thr Leu Lys Glu Pro Lys
325 330 335
Val Asn Glu Pro Lys Val Lys Leu Ser Thr Thr Thr Asn Glu Ser Phe
340 345 350
Leu Glu Thr Val Leu Ala Ala Leu Glu Asp Ser Gln Lys Gly Lys Gln
355 360 365
Leu Leu Lys Phe Glu
370
<210> 11
<211> 448
<212> PRT
<213> 喜热梭孢壳
<400> 11
Met Ala Ser Val Leu Ser Pro Asn Ala Ala Ala Arg His Leu Leu Arg
1 5 10 15
Pro Ala Thr Arg Ser Thr Pro Pro Arg Leu Leu Pro Ser Ala Ile Leu
20 25 30
His Gln Ser Gly Asn Asn Ser Thr Arg Asn Pro Arg Ile Ser Asp Pro
35 40 45
Gln Ser Ile Ala Gln Arg Leu Gly Arg Arg Tyr Lys Ser Gly Pro Tyr
50 55 60
Gly Tyr Thr Gln Ala Lys Thr Leu Val Phe Ser Arg Phe Gly Glu Pro
65 70 75 80
Ser Asp Val Leu Arg Leu His Thr His Ser Ile Ser Pro Thr Leu Pro
85 90 95
Asp Gly Ala Val Leu Val Arg Ala Leu Ala Ala Pro Val Asn Pro Ala
100 105 110
Asp Val Asn Thr Ile Gln Gly Thr Tyr Gly Ala Arg Pro Ala Phe Ser
115 120 125
Pro Leu Leu Gly Thr Pro Glu Pro Ser Ala Val Pro Gly Asn Glu Gly
130 135 140
Cys Phe Glu Val Val Ala Val Gly Pro Arg Val Gly Gly Gly Leu Arg
145 150 155 160
Lys Gly Asp Trp Val Ile Pro Ala Thr Thr Gly Phe Gly Thr Phe Arg
165 170 175
Thr His Ala Leu Val Glu Asn Ala Asp Arg Ala Leu Leu Arg Val Gly
180 185 190
Gly Asp Lys Gly Thr Ala Gly Leu Thr Ala Lys Gln Val Ala Thr Val
195 200 205
Ser Val Asn Pro Cys Ser Ala Tyr Arg Met Leu Lys Asp Tyr Val Asp
210 215 220
Leu Val Asp Leu Ser Val Lys Ser Phe Ala Arg Gly Asp Gly Ala Thr
225 230 235 240
Gly Gly Ala Trp Phe Leu Gln Asn Gly Ala Asn Ser Gly Val Gly Arg
245 250 255
Ala Ala Ile Gln Leu Gly Arg Leu Trp Gly Leu Arg Ser Ile Asn Val
260 265 270
Val Arg Glu Arg Ala Thr Ala Glu Glu Thr Glu Ala Leu Lys Ser Glu
275 280 285
Leu Arg Glu Leu Gly Ala Thr Val Val Val Thr Glu Ala Glu Phe Leu
290 295 300
Asp Arg Ser Phe Ser Ala Arg Leu Lys Glu Glu Trp Thr Arg Gly Asp
305 310 315 320
Arg Glu Pro Val Met Leu Gly Leu Asn Cys Val Gly Gly Lys Ser Ala
325 330 335
Ser Ala Met Ile Lys Ala Leu Ser Pro Lys Gly Cys Met Val Thr Tyr
340 345 350
Gly Gly Met Ser Arg Gln Ser Phe Pro Phe Pro Thr Gly Pro Gln Ile
355 360 365
Phe Lys Arg Leu Arg Phe Glu Gly Phe Trp Leu Ser Glu Trp Ala Lys
370 375 380
Glu Asn Pro Ala Glu Lys Arg Asn Thr Ile Asn Glu Ile Leu Glu Leu
385 390 395 400
Met Arg Glu Gly Lys Phe Lys Glu Ser Pro Phe Lys Glu Val Glu Trp
405 410 415
Asn Trp Asp Thr Glu Glu Lys Val Leu Lys Asp Ala Ile Gln Gly Thr
420 425 430
Leu Glu Gly Phe Lys Ser Gly Lys Gly Leu Phe Val Phe Gly Asp Thr
435 440 445
<210> 12
<211> 443
<212> PRT
<213> 太瑞斯梭孢壳霉
<400> 12
Met Ala Ser Ala Leu Asn Pro Arg His Leu Pro Leu Arg Leu Ala Leu
1 5 10 15
Arg Pro Arg Leu Pro Leu Leu Pro Ser Thr Ser Ser Thr Thr Thr Thr
20 25 30
Thr Thr Pro Pro Ser Cys Leu Pro Ile Pro Thr Ser Ile Arg Leu Pro
35 40 45
Pro Arg Arg His Lys Ser Gly Pro Tyr Gly Tyr Thr Leu Ala Lys Thr
50 55 60
Leu Val Phe Ser Arg Phe Gly Glu Pro Arg Asp Val Leu Ser Leu His
65 70 75 80
Thr His Ser Ile Ser Pro Ala Leu Pro Asp Gly Ala Ala Leu Leu Arg
85 90 95
Ala Leu Ala Ala Pro Val Asn Pro Ala Asp Val Asn Thr Val Gln Gly
100 105 110
Thr Tyr Gly Ala Lys Pro Ala Phe Glu Arg Leu Leu Gly Thr Pro Glu
115 120 125
Pro Ala Ala Val Pro Gly Asn Glu Gly Cys Phe Glu Val Val Ala Ile
130 135 140
Gly Gly Gly Ala Gly Gly Gly Gly Gly Leu Lys Lys Gly Asp Trp Val
145 150 155 160
Ile Pro Ala Gln Ser Gly Phe Gly Thr Phe Arg Thr His Ala Leu Val
165 170 175
Glu Gly Ala Glu Arg Lys Leu Ile Arg Val Gly Gly Ala Lys Gly Arg
180 185 190
Glu Gly Leu Arg Ala Ala Gln Val Ala Thr Val Ser Val Asn Pro Cys
195 200 205
Ser Ala Tyr Arg Met Leu Arg Asp Tyr Val Asp Leu Val Asp Leu Ser
210 215 220
Val Gln Ser Phe Ala Arg Gly Asp Gly Ala Thr Gly Gly Ala Trp Phe
225 230 235 240
Val Gln Asn Gly Ala Asn Ser Gly Val Gly Arg Ala Ala Ile Gln Leu
245 250 255
Gly Arg Leu Trp Gly Leu Arg Ser Ile Asn Val Val Arg Glu Arg Ala
260 265 270
Thr Pro Glu Glu Thr Ala Ala Leu Lys Arg Glu Leu Ala Glu Leu Gly
275 280 285
Ala Thr Val Val Val Thr Glu Ser Glu Phe Leu Asp Arg Ser Phe Ala
290 295 300
Asp Arg Leu Arg Asp Glu Trp Thr Arg Gly Gly Arg Glu Pro Val Met
305 310 315 320
Leu Gly Leu Asn Cys Val Gly Gly Lys Ser Ala Ala Ala Met Val Lys
325 330 335
Ala Leu Ser Pro Arg Gly Cys Met Val Thr Tyr Gly Gly Met Ser Arg
340 345 350
Gln Ser Phe Pro Phe Pro Thr Gly Gln Gln Ile Phe Lys Arg Leu Arg
355 360 365
Phe Glu Gly Phe Trp Leu Ser Glu Trp Ala Lys Glu Asn Pro Ala Ala
370 375 380
Lys Arg Asp Thr Ile Asn Glu Ile Leu Glu Leu Met Arg Glu Gly Lys
385 390 395 400
Phe Lys Glu Ala Pro Leu Gln Glu Val Glu Trp Asn Trp Asp Thr Glu
405 410 415
Glu Ser Val Leu Lys Asp Thr Ile Gln Gly Thr Leu Glu Gly Phe Arg
420 425 430
Pro Gly Lys Ser Ile Phe Val Phe Lys Asp Thr
435 440
<210> 13
<211> 439
<212> PRT
<213> 嗜热毛壳菌
<400> 13
Met Ala Ser Ile Leu Ser Pro His Ser Ala Leu Arg Leu Gly Gly Ile
1 5 10 15
Ser Thr Leu Ser Arg Gln Thr Leu Leu Arg Thr Ser Arg Val Thr Leu
20 25 30
Thr Gln Leu Pro Lys Thr Ala Pro Gln Leu Pro Gln Arg Arg His Lys
35 40 45
Ser Ala Pro Tyr Gly Tyr Thr Gln Ser Lys Thr Leu Val Phe Pro Arg
50 55 60
Phe Gly Glu Pro Ile Asp Val Leu Ser Leu His Thr His Ser Ile Ser
65 70 75 80
Pro Thr Leu Pro Asp Thr Ala Val Leu Leu Arg Thr Leu Ala Ala Pro
85 90 95
Ile Asn Pro Ala Asp Val Asn Thr Ile Gln Gly Thr Tyr Gly Ala Lys
100 105 110
Pro Thr Phe Ser Asn Leu Leu Gly Thr Ala Glu Pro Ala Ala Val Pro
115 120 125
Gly Asn Glu Gly Val Phe Glu Val Val Ser Val Gly Ser Glu Ile Ala
130 135 140
Lys Arg Gly Val Phe Lys Lys Gly Asp Trp Val Ile Pro Ser Ser Ser
145 150 155 160
Gly Phe Gly Thr Phe Arg Thr His Val Leu Val Glu Glu Ala Glu Gln
165 170 175
Lys Leu Trp Arg Ile Gly Gly Glu Lys Gly Thr Glu Gly Leu Thr Pro
180 185 190
Val Gln Val Ala Thr Val Ser Val Asn Pro Cys Ser Ala Tyr Arg Met
195 200 205
Leu Arg Asp Tyr Val Asp Leu Val Gly Val Ser Val Arg Met Tyr Gln
210 215 220
Glu Gly Gly Ser Asp Val Arg Gly Gly Ala Trp Phe Leu Gln Asn Gly
225 230 235 240
Ala Asn Ser Gly Val Gly Arg Ala Ala Ile Gln Leu Gly Arg Leu Trp
245 250 255
Gly Leu Arg Ser Ile Asn Val Val Arg Glu Arg Ala Thr Ala Glu Glu
260 265 270
Thr Glu Ala Leu Lys Lys Glu Leu Tyr Asp Leu Gly Ala Thr Val Val
275 280 285
Val Thr Glu Ser Glu Phe Leu Asp Arg Ser Phe Thr Gln Arg Leu Asn
290 295 300
Glu Glu Trp Thr Arg Gly Gly Lys Glu Pro Leu Leu Leu Ala Leu Asn
305 310 315 320
Cys Val Gly Gly Lys Ser Ala Gln Gln Ile Val Arg Ala Leu Ser Pro
325 330 335
Lys Gly Thr Met Val Thr Tyr Gly Gly Met Ser Arg Gln Ser Phe Pro
340 345 350
Phe Pro Thr Gly Pro Gln Ile Phe Lys Arg Leu Arg Phe Glu Gly Phe
355 360 365
Trp Leu Ser Glu Trp Ala Lys Glu Asn Pro Ala Glu Lys Lys Lys Cys
370 375 380
Val Asp Glu Ile Ile Glu Leu Met Arg Glu Gly Lys Phe Lys Glu Ala
385 390 395 400
Pro Val Gln Glu Ile Arg Trp Asp Trp Glu Thr Glu Glu Lys Val Leu
405 410 415
Lys Glu Ala Val Gln Gly Thr Leu Glu Gly Phe Arg Ser Gly Lys Gly
420 425 430
Val Phe Ile Phe Gly Asp Thr
435
<210> 14
<211> 422
<212> PRT
<213> 丝状真菌柄孢霉
<400> 14
Met Ala Ala Pro Arg Leu Thr Ser Lys Ala Leu Arg Leu Pro Thr Ser
1 5 10 15
Lys Leu Leu Arg Pro Ala Leu Thr Thr Ala Thr Pro Arg Ala Ala Pro
20 25 30
Thr Leu Thr Gln Lys Arg His Leu Thr Gly Pro Tyr Gly Tyr His Gln
35 40 45
Ser Lys Ala Leu Thr Phe Ser Ser Phe Gly Glu Pro Ile Asp Val Leu
50 55 60
Ser Leu His Thr His Ser Ile Ser Pro Thr Leu Pro Ser Gly Ser Val
65 70 75 80
Leu Val Arg Thr Leu Ala Ala Pro Val Asn Pro Ala Asp Val Asn Thr
85 90 95
Ile Gln Gly Thr Tyr Gly Ser Lys Pro Pro Phe Thr Thr Leu Leu Gly
100 105 110
Thr Ala Gln Pro Ser Ala Val Pro Gly Asn Glu Ala Cys Phe Glu Val
115 120 125
Leu Ser Val Gly Gln Gly Val Lys Gly Leu Glu Lys Gly Asp Trp Val
130 135 140
Ile Pro Ala Lys Thr Gly Phe Gly Thr Phe Arg Thr His Ala Leu Val
145 150 155 160
Glu Glu Ala Glu Gly Lys Leu Met Arg Val Glu Arg Glu Gly Leu Thr
165 170 175
Pro Val Gln Val Ala Thr Val Ser Val Asn Pro Cys Ser Ala Tyr Arg
180 185 190
Met Leu Lys Asp Tyr Val Asp Leu Val Gly Leu Ser Met Arg Trp Tyr
195 200 205
Arg Glu Gly Lys Asp Val Ser Gly Gly Ala Trp Phe Leu Gln Asn Gly
210 215 220
Ala Asn Ser Gly Val Gly Arg Ala Ala Val Gln Phe Gly Arg Leu Trp
225 230 235 240
Gly Leu Arg Ser Ile Asn Val Val Arg Glu Arg Glu Thr Pro Glu Glu
245 250 255
Thr Glu Lys Leu Lys Glu Glu Leu Thr Gly Leu Gly Ala Asn Val Val
260 265 270
Leu Thr Glu Gln Glu Phe Leu Asp Arg Ser Phe Arg Asp Arg Leu Gly
275 280 285
Glu Leu Thr Lys Arg Gly Lys Glu Pro Leu Leu Leu Gly Met Asn Cys
290 295 300
Val Gly Gly Lys Ser Ala Ser Ala Val Val Lys Ala Leu Ser Pro Lys
305 310 315 320
Gly Cys Met Val Thr Tyr Gly Gly Met Ser Arg Gln Ser Phe Pro Phe
325 330 335
Pro Thr Gly Pro Gln Ile Phe Lys Arg Leu Arg Phe Glu Gly Phe Trp
340 345 350
Leu Ser Glu Trp Gly Lys Glu Asn Pro Glu Gly Lys Lys Lys Met Ile
355 360 365
Glu Asp Ile Leu Asn Leu Met Arg Glu Gly Lys Phe Lys Glu Ser Pro
370 375 380
Val Gln Glu Val Glu Trp Asn Trp Glu Thr Glu Glu Lys Thr Leu Lys
385 390 395 400
Glu Ala Val Gln Gly Thr Leu Gly Gly Phe Arg Ser Gly Lys Gly Val
405 410 415
Phe Val Phe Gly Glu Thr
420
<210> 15
<211> 424
<212> PRT
<213> 淡紫拟青霉菌
<400> 15
Met Ala Ser Ser Arg Leu Leu Gly Pro Pro Leu Arg Leu Pro Ser Ser
1 5 10 15
Gly Phe Ser Arg Pro Val Ala Ala Leu Asn Cys Arg Ala Ala Ala Leu
20 25 30
Pro Leu His Leu Pro Arg Thr Ala Val Arg Tyr Arg Ser Gly Pro Tyr
35 40 45
Gly Tyr Thr Gln Ala Lys Ala Leu Val Tyr Ser Lys Asn Gly Glu Pro
50 55 60
Ala Asp Val Leu Lys Leu His Thr His Ser Ile Ser Pro Ser Ile Pro
65 70 75 80
Ser Thr Ser Val Leu Val Arg Cys Leu Ala Ala Pro Ile Asn Pro Ala
85 90 95
Asp Val Asn Thr Ile Gln Gly Thr Tyr Gly Ser Lys Gln Pro Leu Thr
100 105 110
Ser Leu Ile Gly Thr Ser Glu Pro Ser Ala Val Pro Gly Asn Glu Gly
115 120 125
Val Phe Glu Val Leu Ser Ala Gly Ser Pro Ser Ser Glu Leu Lys Lys
130 135 140
Gly Asp Trp Val Ile Pro Ala Ala Ser Gln Ile Gly Thr Trp Arg Thr
145 150 155 160
His Ala Val Phe Gly Ala Asp Glu Leu Leu Lys Val Asp Lys Glu Gly
165 170 175
Leu Thr Pro Thr Gln Val Ser Leu Val Ser Val Asn Pro Cys Thr Ala
180 185 190
Tyr Arg Ile Leu Arg Ser Tyr Gly Pro Ser Ala Gly Val Lys Ala Ala
195 200 205
Leu Gly Met Arg Pro Leu Glu Val Gly Ser Gly Gln Trp Phe Ile Gln
210 215 220
Asn Gly Ala Asn Ser Gly Val Gly Arg Ala Ala Ile Gln Phe Gly Lys
225 230 235 240
Leu Trp Gly Leu Arg Ser Ile Asn Val Ile Arg Asp Arg Asp Thr Val
245 250 255
Glu Ala Thr Asp Ala Leu Lys Gln Glu Leu Arg Asp Leu Gly Ala Asp
260 265 270
Val Val Val Ala Glu Ser Glu Phe Leu Ser Arg Gly Trp Lys Asp Gln
275 280 285
Leu Ala Glu Ile Thr Arg Lys Gly Arg Glu Gln Val Gly Leu Gly Leu
290 295 300
Asn Cys Val Gly Gly Lys Ser Ala Thr Ala Leu Ala Arg Ser Leu Gly
305 310 315 320
Glu Gly Ala Thr Leu Val Ser Tyr Gly Gly Met Ala Lys Gln Pro Val
325 330 335
Ala Leu Pro Val Gly Leu Leu Ile Phe Lys Asp Ile Arg Phe Val Gly
340 345 350
Phe Trp Leu Ser Lys Trp Asn Gln Gln Asp Val Val Gly Arg Arg His
355 360 365
Met Val Ala Asp Ile Leu Gly Leu Met Arg Asp Gly Lys Phe Arg Asp
370 375 380
Ala Pro Val Asp Glu Val Lys Trp Asp Trp Asp Thr Asp Glu Ala Thr
385 390 395 400
Leu Arg Gly Ala Ala Gln Arg Gly Leu Glu Gly Phe Arg Gln Gly Lys
405 410 415
Gly Val Tyr Val Phe Gly Asp Thr
420
<210> 16
<211> 405
<212> PRT
<213> 圆核腔菌
<400> 16
Met Ala Ser Lys Leu Ile Ser Pro Ser Pro Arg Leu Ala Gln Ser Cys
1 5 10 15
Leu Arg Arg Thr Arg Asn Leu Ala Pro Val Thr Ala Arg Phe Pro Gln
20 25 30
Arg Arg Tyr Ile Ser Ala Tyr Gly Tyr Glu Gln Ala Lys Ala Leu Thr
35 40 45
Phe Thr Glu Tyr Gly Asp Pro Ser Ala Val Leu Ser Leu His Ser His
50 55 60
Ser Ile Ser Pro Pro His Ser Asn Tyr Met Thr Leu Arg Phe Leu Ala
65 70 75 80
Ser Pro Ile Asn Pro Ala Asp Ile Asn Gln Ile Gln Gly Val Tyr Pro
85 90 95
Ser Lys Pro Thr Phe Thr Thr Ser Leu Gly Thr Pro Asn Pro Ile Ala
100 105 110
Val Ala Gly Asn Glu Gly Val Ala Glu Ile Ile Ala Leu Gly Glu Gly
115 120 125
Val Lys Lys Glu Gly Phe Lys Lys Gly Asp Trp Val Phe Met Lys Gly
130 135 140
Pro Gly Phe Gly Thr Trp Arg Thr His Ala Ser Ala Thr Thr Asn Asp
145 150 155 160
Val Val Lys Leu Asn Asp Gln Met Arg Glu Gly Ile Thr Ala Ile Gln
165 170 175
Ala Gly Thr Val Ser Ile Asn Pro Cys Thr Ala Tyr Arg Met Leu Arg
180 185 190
Asp Phe Thr Thr Leu Ser Glu Gly Asp Trp Phe Ile Gln Asn Gly Ala
195 200 205
Asn Ser Gly Val Gly Arg Ala Ala Ile Gln Leu Gly Arg Lys Trp Gly
210 215 220
Tyr Lys Ser Ile Asn Ile Ile Arg Ser Arg Glu Asp Lys Asn Lys Glu
225 230 235 240
Glu Ala Met Lys Lys Glu Leu His Asn Leu Gly Ala Asp Val Val Ile
245 250 255
Thr Asp Ala Glu Leu Gln Ala Gln Gly Ile Lys Asp Gln Ala Lys Glu
260 265 270
Trp Thr Asn Gly Gly Arg Ser Pro Ile Arg Leu Ala Leu Asn Cys Val
275 280 285
Asn Gly Lys Ala Ala Thr Ala Met Ala Lys Leu Leu Ser Ser Ser Ala
290 295 300
His Phe Val Thr Tyr Gly Ala Met Ser Lys Gln Pro Leu Thr Ile Pro
305 310 315 320
Ala Ser Met Leu Ile Phe Lys Asp Ile His Phe His Gly Phe Trp Val
325 330 335
Ser Arg Trp Ala Glu Glu His Pro Glu Glu Lys Gln Lys Thr Val Ala
340 345 350
Asp Val Leu Asp Met Thr Arg Lys Gly Glu Phe Lys Asp Met Pro Val
355 360 365
Asp Glu Ile Lys Trp Glu Trp Glu Thr Lys Gly Glu Glu Leu Val Ala
370 375 380
Lys Val Lys Asp Thr Leu Glu Gly Tyr Arg Asp Gly Lys Gly Ile Phe
385 390 395 400
Val Phe Gly Lys Thr
405
<210> 17
<211> 453
<212> PRT
<213> 克氏梭菌
<400> 17
Met Ser Asn Glu Val Ser Ile Lys Glu Leu Ile Glu Lys Ala Lys Val
1 5 10 15
Ala Gln Lys Lys Leu Glu Ala Tyr Ser Gln Glu Gln Val Asp Val Leu
20 25 30
Val Lys Ala Leu Gly Lys Val Val Tyr Asp Asn Ala Glu Met Phe Ala
35 40 45
Lys Glu Ala Val Glu Glu Thr Glu Met Gly Val Tyr Glu Asp Lys Val
50 55 60
Ala Lys Cys His Leu Lys Ser Gly Ala Ile Trp Asn His Ile Lys Asp
65 70 75 80
Lys Lys Thr Val Gly Ile Ile Lys Glu Glu Pro Glu Arg Ala Leu Val
85 90 95
Tyr Val Ala Lys Pro Lys Gly Val Val Ala Ala Thr Thr Pro Ile Thr
100 105 110
Asn Pro Val Val Thr Pro Met Cys Asn Ala Met Ala Ala Ile Lys Gly
115 120 125
Arg Asn Thr Ile Ile Val Ala Pro His Pro Lys Ala Lys Lys Val Ser
130 135 140
Ala His Thr Val Glu Leu Met Asn Ala Glu Leu Lys Lys Leu Gly Ala
145 150 155 160
Pro Glu Asn Ile Ile Gln Ile Val Glu Ala Pro Ser Arg Glu Ala Ala
165 170 175
Lys Glu Leu Met Glu Ser Ala Asp Val Val Ile Ala Thr Gly Gly Ala
180 185 190
Gly Arg Val Lys Ala Ala Tyr Ser Ser Gly Arg Pro Ala Tyr Gly Val
195 200 205
Gly Pro Gly Asn Ser Gln Val Ile Val Asp Lys Gly Tyr Asp Tyr Asn
210 215 220
Lys Ala Ala Gln Asp Ile Ile Thr Gly Arg Lys Tyr Asp Asn Gly Ile
225 230 235 240
Ile Cys Ser Ser Glu Gln Ser Val Ile Ala Pro Ala Glu Asp Tyr Asp
245 250 255
Lys Val Ile Ala Ala Phe Val Glu Asn Gly Ala Phe Tyr Val Glu Asp
260 265 270
Glu Glu Thr Val Glu Lys Phe Arg Ser Thr Leu Phe Lys Asp Gly Lys
275 280 285
Ile Asn Ser Lys Ile Ile Gly Lys Ser Val Gln Ile Ile Ala Asp Leu
290 295 300
Ala Gly Val Lys Val Pro Glu Gly Thr Lys Val Ile Val Leu Lys Gly
305 310 315 320
Lys Gly Ala Gly Glu Lys Asp Val Leu Cys Lys Glu Lys Met Cys Pro
325 330 335
Val Leu Val Ala Leu Lys Tyr Asp Thr Phe Glu Glu Ala Val Glu Ile
340 345 350
Ala Met Ala Asn Tyr Met Tyr Glu Gly Ala Gly His Thr Ala Gly Ile
355 360 365
His Ser Asp Asn Asp Glu Asn Ile Arg Tyr Ala Gly Thr Val Leu Pro
370 375 380
Ile Ser Arg Leu Val Val Asn Gln Pro Ala Thr Thr Ala Gly Gly Ser
385 390 395 400
Phe Asn Asn Gly Phe Asn Pro Thr Thr Thr Leu Gly Cys Gly Ser Trp
405 410 415
Gly Arg Asn Ser Ile Ser Glu Asn Leu Thr Tyr Glu His Leu Ile Asn
420 425 430
Val Ser Arg Ile Gly Tyr Phe Asn Lys Glu Ala Lys Val Pro Ser Tyr
435 440 445
Glu Glu Ile Trp Gly
450
<210> 18
<211> 1174
<212> PRT
<213> 艾阿华诺卡氏菌
<400> 18
Met Ala Val Asp Ser Pro Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln
1 5 10 15
Leu Phe Ala Glu Asp Glu Gln Val Lys Ala Ala Arg Pro Leu Glu Ala
20 25 30
Val Ser Ala Ala Val Ser Ala Pro Gly Met Arg Leu Ala Gln Ile Ala
35 40 45
Ala Thr Val Met Ala Gly Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg
50 55 60
Ala Phe Glu Leu Asn Thr Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg
65 70 75 80
Leu Leu Pro Arg Phe Glu Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg
85 90 95
Val Gly Glu Val Ala Ala Ala Trp His His Asp Pro Glu Asn Pro Leu
100 105 110
Arg Ala Gly Asp Phe Val Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr
115 120 125
Ala Thr Leu Asp Leu Ala Asp Ile His Leu Gly Ala Val Thr Val Pro
130 135 140
Leu Gln Ala Ser Ala Ala Val Ser Gln Leu Ile Ala Ile Leu Thr Glu
145 150 155 160
Thr Ser Pro Arg Leu Leu Ala Ser Thr Pro Glu His Leu Asp Ala Ala
165 170 175
Val Glu Cys Leu Leu Ala Gly Thr Thr Pro Glu Arg Leu Val Val Phe
180 185 190
Asp Tyr His Pro Glu Asp Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala
195 200 205
Arg Arg Arg Leu Ala Asp Ala Gly Ser Ser Val Ile Val Glu Thr Leu
210 215 220
Asp Ala Val Arg Ala Arg Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe
225 230 235 240
Val Pro Asp Thr Asp Asp Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser
245 250 255
Gly Ser Thr Gly Thr Pro Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala
260 265 270
Ala Thr Met Trp Gln Gly Asn Ser Met Leu Gln Gly Asn Ser Gln Arg
275 280 285
Val Gly Ile Asn Leu Asn Tyr Met Pro Met Ser His Ile Ala Gly Arg
290 295 300
Ile Ser Leu Phe Gly Val Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala
305 310 315 320
Ala Lys Ser Asp Met Ser Thr Leu Phe Glu Asp Ile Gly Leu Val Arg
325 330 335
Pro Thr Glu Ile Phe Phe Val Pro Arg Val Cys Asp Met Val Phe Gln
340 345 350
Arg Tyr Gln Ser Glu Leu Asp Arg Arg Ser Val Ala Gly Ala Asp Leu
355 360 365
Asp Thr Leu Asp Arg Glu Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu
370 375 380
Gly Gly Arg Phe Leu Val Ala Val Val Gly Ser Ala Pro Leu Ala Ala
385 390 395 400
Glu Met Lys Thr Phe Met Glu Ser Val Leu Asp Leu Pro Leu His Asp
405 410 415
Gly Tyr Gly Ser Thr Glu Ala Gly Ala Ser Val Leu Leu Asp Asn Gln
420 425 430
Ile Gln Arg Pro Pro Val Leu Asp Tyr Lys Leu Val Asp Val Pro Glu
435 440 445
Leu Gly Tyr Phe Arg Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu
450 455 460
Leu Lys Ala Glu Thr Thr Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val
465 470 475 480
Thr Ala Glu Ile Phe Asp Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile
485 490 495
Val Ala Glu Leu Glu His Asp Arg Leu Val Tyr Val Asp Arg Arg Asn
500 505 510
Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Thr Val Ala His Leu
515 520 525
Glu Ala Val Phe Ala Ser Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr
530 535 540
Gly Ser Ser Glu Arg Ser Tyr Leu Leu Ala Val Ile Val Pro Thr Asp
545 550 555 560
Asp Ala Leu Arg Gly Arg Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala
565 570 575
Glu Ser Ile Gln Arg Ile Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu
580 585 590
Ile Pro Arg Asp Phe Leu Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn
595 600 605
Gly Leu Leu Ser Gly Ile Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu
610 615 620
Arg Tyr Gly Ala Gln Leu Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly
625 630 635 640
Gln Ala Asp Glu Leu Leu Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro
645 650 655
Val Leu Glu Thr Val Ser Arg Ala Ala Lys Ala Met Leu Gly Val Ala
660 665 670
Ser Ala Asp Met Arg Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp
675 680 685
Ser Leu Ser Ala Leu Ser Phe Ser Asn Leu Leu His Glu Ile Phe Gly
690 695 700
Val Glu Val Pro Val Gly Val Val Val Ser Pro Ala Asn Glu Leu Arg
705 710 715 720
Asp Leu Ala Asn Tyr Ile Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg
725 730 735
Pro Thr Phe Thr Ser Val His Gly Gly Gly Ser Glu Ile Arg Ala Ala
740 745 750
Asp Leu Thr Leu Asp Lys Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala
755 760 765
Asp Ser Ile Pro His Ala Pro Val Pro Ala Gln Thr Val Leu Leu Thr
770 775 780
Gly Ala Asn Gly Tyr Leu Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu
785 790 795 800
Arg Leu Asp Lys Thr Gly Gly Thr Leu Ile Cys Val Val Arg Gly Ser
805 810 815
Asp Ala Ala Ala Ala Arg Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly
820 825 830
Asp Pro Gly Leu Leu Glu His Tyr Gln Gln Leu Ala Ala Arg Thr Leu
835 840 845
Glu Val Leu Ala Gly Asp Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp
850 855 860
Ala Thr Trp Gln Arg Leu Ala Glu Thr Val Asp Leu Ile Val His Pro
865 870 875 880
Ala Ala Leu Val Asn His Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro
885 890 895
Asn Val Val Gly Thr Ala Glu Ile Val Arg Leu Ala Ile Thr Ala Arg
900 905 910
Arg Lys Pro Val Thr Tyr Leu Ser Thr Val Gly Val Ala Asp Gln Val
915 920 925
Asp Pro Ala Glu Tyr Gln Glu Asp Ser Asp Val Arg Glu Met Ser Ala
930 935 940
Val Arg Val Val Arg Glu Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys
945 950 955 960
Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys Gly Leu
965 970 975
Pro Val Ala Val Phe Arg Ser Asp Met Ile Leu Ala His Ser Arg Tyr
980 985 990
Ala Gly Gln Leu Asn Val Gln Asp Val Phe Thr Arg Leu Ile Leu Ser
995 1000 1005
Leu Val Ala Thr Gly Ile Ala Pro Tyr Ser Phe Tyr Arg Thr Asp
1010 1015 1020
Ala Asp Gly Asn Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Ala
1025 1030 1035
Asp Phe Thr Ala Ala Ala Ile Thr Ala Leu Gly Ile Gln Ala Thr
1040 1045 1050
Glu Gly Phe Arg Thr Tyr Asp Val Leu Asn Pro Tyr Asp Asp Gly
1055 1060 1065
Ile Ser Leu Asp Glu Phe Val Asp Trp Leu Val Glu Ser Gly His
1070 1075 1080
Pro Ile Gln Arg Ile Thr Asp Tyr Ser Asp Trp Phe His Arg Phe
1085 1090 1095
Glu Thr Ala Ile Arg Ala Leu Pro Glu Lys Gln Arg Gln Ala Ser
1100 1105 1110
Val Leu Pro Leu Leu Asp Ala Tyr Arg Asn Pro Cys Pro Ala Val
1115 1120 1125
Arg Gly Ala Ile Leu Pro Ala Lys Glu Phe Gln Ala Ala Val Gln
1130 1135 1140
Thr Ala Lys Ile Gly Pro Glu Gln Asp Ile Pro His Leu Ser Ala
1145 1150 1155
Pro Leu Ile Asp Lys Tyr Val Ser Asp Leu Glu Leu Leu Gln Leu
1160 1165 1170
Leu
<210> 19
<211> 1174
<212> PRT
<213> 海洋分枝杆菌
<400> 19
Met Ser Pro Ile Thr Arg Glu Glu Arg Leu Glu Arg Arg Ile Gln Asp
1 5 10 15
Leu Tyr Ala Asn Asp Pro Gln Phe Ala Ala Ala Lys Pro Ala Thr Ala
20 25 30
Ile Thr Ala Ala Ile Glu Arg Pro Gly Leu Pro Leu Pro Gln Ile Ile
35 40 45
Glu Thr Val Met Thr Gly Tyr Ala Asp Arg Pro Ala Leu Ala Gln Arg
50 55 60
Ser Val Glu Phe Val Thr Asp Ala Gly Thr Gly His Thr Thr Leu Arg
65 70 75 80
Leu Leu Pro His Phe Glu Thr Ile Ser Tyr Gly Glu Leu Trp Asp Arg
85 90 95
Ile Ser Ala Leu Ala Asp Val Leu Ser Thr Glu Gln Thr Val Lys Pro
100 105 110
Gly Asp Arg Val Cys Leu Leu Gly Phe Asn Ser Val Asp Tyr Ala Thr
115 120 125
Ile Asp Met Thr Leu Ala Arg Leu Gly Ala Val Ala Val Pro Leu Gln
130 135 140
Thr Ser Ala Ala Ile Thr Gln Leu Gln Pro Ile Val Ala Glu Thr Gln
145 150 155 160
Pro Thr Met Ile Ala Ala Ser Val Asp Ala Leu Ala Asp Ala Thr Glu
165 170 175
Leu Ala Leu Ser Gly Gln Thr Ala Thr Arg Val Leu Val Phe Asp His
180 185 190
His Arg Gln Val Asp Ala His Arg Ala Ala Val Glu Ser Ala Arg Glu
195 200 205
Arg Leu Ala Gly Ser Ala Val Val Glu Thr Leu Ala Glu Ala Ile Ala
210 215 220
Arg Gly Asp Val Pro Arg Gly Ala Ser Ala Gly Ser Ala Pro Gly Thr
225 230 235 240
Asp Val Ser Asp Asp Ser Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser
245 250 255
Thr Gly Ala Pro Lys Gly Ala Met Tyr Pro Arg Arg Asn Val Ala Thr
260 265 270
Phe Trp Arg Lys Arg Thr Trp Phe Glu Gly Gly Tyr Glu Pro Ser Ile
275 280 285
Thr Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gln Ile Leu
290 295 300
Tyr Gly Thr Leu Cys Asn Gly Gly Thr Ala Tyr Phe Val Ala Lys Ser
305 310 315 320
Asp Leu Ser Thr Leu Phe Glu Asp Leu Ala Leu Val Arg Pro Thr Glu
325 330 335
Leu Thr Phe Val Pro Arg Val Trp Asp Met Val Phe Asp Glu Phe Gln
340 345 350
Ser Glu Val Asp Arg Arg Leu Val Asp Gly Ala Asp Arg Val Ala Leu
355 360 365
Glu Ala Gln Val Lys Ala Glu Ile Arg Asn Asp Val Leu Gly Gly Arg
370 375 380
Tyr Thr Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser Asp Glu Met Lys
385 390 395 400
Ala Trp Val Glu Glu Leu Leu Asp Met His Leu Val Glu Gly Tyr Gly
405 410 415
Ser Thr Glu Ala Gly Met Ile Leu Ile Asp Gly Ala Ile Arg Arg Pro
420 425 430
Ala Val Leu Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe
435 440 445
Leu Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu Val Lys Thr Asp
450 455 460
Ser Leu Phe Pro Gly Tyr Tyr Gln Arg Ala Glu Val Thr Ala Asp Val
465 470 475 480
Phe Asp Ala Asp Gly Phe Tyr Arg Thr Gly Asp Ile Met Ala Glu Val
485 490 495
Gly Pro Glu Gln Phe Val Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys
500 505 510
Leu Ser Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe
515 520 525
Gly Asp Ser Pro Leu Val Arg Gln Ile Tyr Ile Tyr Gly Asn Ser Ala
530 535 540
Arg Ala Tyr Leu Leu Ala Val Ile Val Pro Thr Gln Glu Ala Leu Asp
545 550 555 560
Ala Val Pro Val Glu Glu Leu Lys Ala Arg Leu Gly Asp Ser Leu Gln
565 570 575
Glu Val Ala Lys Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp
580 585 590
Phe Ile Ile Glu Thr Thr Pro Trp Thr Leu Glu Asn Gly Leu Leu Thr
595 600 605
Gly Ile Arg Lys Leu Ala Arg Pro Gln Leu Lys Lys His Tyr Gly Glu
610 615 620
Leu Leu Glu Gln Ile Tyr Thr Asp Leu Ala His Gly Gln Ala Asp Glu
625 630 635 640
Leu Arg Ser Leu Arg Gln Ser Gly Ala Asp Ala Pro Val Leu Val Thr
645 650 655
Val Cys Arg Ala Ala Ala Ala Leu Leu Gly Gly Ser Ala Ser Asp Val
660 665 670
Gln Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala
675 680 685
Leu Ser Phe Thr Asn Leu Leu His Glu Ile Phe Asp Ile Glu Val Pro
690 695 700
Val Gly Val Ile Val Ser Pro Ala Asn Asp Leu Gln Ala Leu Ala Asp
705 710 715 720
Tyr Val Glu Ala Ala Arg Lys Pro Gly Ser Ser Arg Pro Thr Phe Ala
725 730 735
Ser Val His Gly Ala Ser Asn Gly Gln Val Thr Glu Val His Ala Gly
740 745 750
Asp Leu Ser Leu Asp Lys Phe Ile Asp Ala Ala Thr Leu Ala Glu Ala
755 760 765
Pro Arg Leu Pro Ala Ala Asn Thr Gln Val Arg Thr Val Leu Leu Thr
770 775 780
Gly Ala Thr Gly Phe Leu Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu
785 790 795 800
Arg Met Asp Leu Val Asp Gly Lys Leu Ile Cys Leu Val Arg Ala Lys
805 810 815
Ser Asp Thr Glu Ala Arg Ala Arg Leu Asp Lys Thr Phe Asp Ser Gly
820 825 830
Asp Pro Glu Leu Leu Ala His Tyr Arg Ala Leu Ala Gly Asp His Leu
835 840 845
Glu Val Leu Ala Gly Asp Lys Gly Glu Ala Asp Leu Gly Leu Asp Arg
850 855 860
Gln Thr Trp Gln Arg Leu Ala Asp Thr Val Asp Leu Ile Val Asp Pro
865 870 875 880
Ala Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu Phe Gly Pro
885 890 895
Asn Ala Leu Gly Thr Ala Glu Leu Leu Arg Leu Ala Leu Thr Ser Lys
900 905 910
Ile Lys Pro Tyr Ser Tyr Thr Ser Thr Ile Gly Val Ala Asp Gln Ile
915 920 925
Pro Pro Ser Ala Phe Thr Glu Asp Ala Asp Ile Arg Val Ile Ser Ala
930 935 940
Thr Arg Ala Val Asp Asp Ser Tyr Ala Asn Gly Tyr Ser Asn Ser Lys
945 950 955 960
Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys Gly Leu
965 970 975
Pro Val Ala Val Phe Arg Cys Asp Met Ile Leu Ala Asp Thr Thr Trp
980 985 990
Ala Gly Gln Leu Asn Val Pro Asp Met Phe Thr Arg Met Ile Leu Ser
995 1000 1005
Leu Ala Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Glu Leu Ala
1010 1015 1020
Ala Asp Gly Ala Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Val
1025 1030 1035
Glu Phe Ile Ala Glu Ala Ile Ser Thr Leu Gly Ala Gln Ser Gln
1040 1045 1050
Asp Gly Phe His Thr Tyr His Val Met Asn Pro Tyr Asp Asp Gly
1055 1060 1065
Ile Gly Leu Asp Glu Phe Val Asp Trp Leu Asn Glu Ser Gly Cys
1070 1075 1080
Pro Ile Gln Arg Ile Ala Asp Tyr Gly Asp Trp Leu Gln Arg Phe
1085 1090 1095
Glu Thr Ala Leu Arg Ala Leu Pro Asp Arg Gln Arg His Ser Ser
1100 1105 1110
Leu Leu Pro Leu Leu His Asn Tyr Arg Gln Pro Glu Arg Pro Val
1115 1120 1125
Arg Gly Ser Ile Ala Pro Thr Asp Arg Phe Arg Ala Ala Val Gln
1130 1135 1140
Glu Ala Lys Ile Gly Pro Asp Lys Asp Ile Pro His Val Gly Ala
1145 1150 1155
Pro Ile Ile Val Lys Tyr Val Ser Asp Leu Arg Leu Leu Gly Leu
1160 1165 1170
Leu
<210> 20
<211> 1178
<212> PRT
<213> 脓肿分枝杆菌
<400> 20
Met Thr Glu Thr Ile Ser Thr Ala Ala Val Pro Thr Thr Asp Leu Glu
1 5 10 15
Glu Gln Val Lys Arg Arg Ile Glu Gln Val Val Ser Asn Asp Pro Gln
20 25 30
Leu Ala Ala Leu Leu Pro Glu Asp Ser Val Thr Glu Ala Val Asn Glu
35 40 45
Pro Asp Leu Pro Leu Val Glu Val Ile Arg Arg Leu Leu Glu Gly Tyr
50 55 60
Gly Asp Arg Pro Ala Leu Gly Gln Arg Ala Phe Glu Phe Val Thr Gly
65 70 75 80
Asp Asp Gly Ala Thr Val Ile Ala Leu Lys Pro Glu Tyr Thr Thr Val
85 90 95
Ser Tyr Arg Glu Leu Trp Glu Arg Ala Glu Ala Ile Ala Ala Ala Trp
100 105 110
His Glu Gln Gly Ile Arg Asp Gly Asp Phe Val Ala Gln Leu Gly Phe
115 120 125
Thr Ser Thr Asp Phe Ala Ser Leu Asp Val Ala Gly Leu Arg Leu Gly
130 135 140
Thr Val Ser Val Pro Leu Gln Thr Gly Ala Ser Leu Gln Gln Arg Asn
145 150 155 160
Ala Ile Leu Glu Glu Thr Arg Pro Ala Val Phe Ala Ala Ser Ile Glu
165 170 175
Tyr Leu Asp Ala Ala Val Asp Ser Val Leu Ala Thr Pro Ser Val Arg
180 185 190
Leu Leu Ser Val Phe Asp Tyr His Ala Glu Val Asp Ser Gln Arg Glu
195 200 205
Ala Leu Glu Ala Val Arg Ala Arg Leu Glu Ser Ala Gly Arg Thr Ile
210 215 220
Val Val Glu Ala Leu Ala Glu Ala Leu Ala Arg Gly Arg Asp Leu Pro
225 230 235 240
Ala Ala Pro Leu Pro Ser Ala Asp Pro Asp Ala Leu Arg Leu Leu Ile
245 250 255
Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys Gly Ala Met Tyr Pro Gln
260 265 270
Trp Leu Val Ala Asn Leu Trp Gln Lys Lys Trp Leu Thr Asp Asp Val
275 280 285
Ile Pro Ser Ile Gly Val Asn Phe Met Pro Met Ser His Leu Ala Gly
290 295 300
Arg Leu Thr Leu Met Gly Thr Leu Ser Gly Gly Gly Thr Ala Tyr Tyr
305 310 315 320
Ile Ala Ser Ser Asp Leu Ser Thr Phe Phe Glu Asp Ile Ala Leu Ile
325 330 335
Arg Pro Ser Glu Val Leu Phe Val Pro Arg Val Val Glu Met Val Phe
340 345 350
Gln Arg Phe Gln Ala Glu Leu Asp Arg Ser Leu Ala Pro Gly Glu Ser
355 360 365
Asn Ser Glu Ile Ala Glu Arg Ile Lys Val Arg Ile Arg Glu Gln Asp
370 375 380
Phe Gly Gly Arg Val Leu Ser Ala Gly Ser Gly Ser Ala Pro Leu Ser
385 390 395 400
Pro Glu Met Thr Glu Phe Met Glu Ser Leu Leu Gln Val Pro Leu Arg
405 410 415
Asp Gly Tyr Gly Ser Thr Glu Ala Gly Gly Val Trp Arg Asp Gly Val
420 425 430
Leu Gln Arg Pro Pro Val Thr Asp Tyr Lys Leu Val Asp Val Pro Glu
435 440 445
Leu Gly Tyr Phe Thr Thr Asp Ser Pro His Pro Arg Gly Glu Leu Arg
450 455 460
Leu Lys Ser Glu Thr Met Phe Pro Gly Tyr Tyr Lys Arg Pro Glu Thr
465 470 475 480
Thr Ala Asp Val Phe Asp Asp Glu Gly Tyr Tyr Lys Thr Gly Asp Val
485 490 495
Val Ala Glu Leu Gly Pro Asp His Leu Lys Tyr Leu Asp Arg Val Lys
500 505 510
Asn Val Leu Lys Leu Ala Gln Gly Glu Phe Val Ala Val Ser Lys Leu
515 520 525
Glu Ala Ala Tyr Thr Gly Ser Pro Leu Val Arg Gln Ile Phe Val Tyr
530 535 540
Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val Val Val Pro Thr Pro
545 550 555 560
Glu Val Leu Glu Arg Tyr Ala Asp Ser Pro Asp Ala Leu Lys Pro Leu
565 570 575
Ile Gln Asp Ser Leu Gln Gln Val Ala Lys Asp Ala Glu Leu Gln Ser
580 585 590
Tyr Glu Ile Pro Arg Asp Phe Ile Val Glu Thr Val Pro Phe Thr Val
595 600 605
Glu Ser Gly Leu Leu Ser Asp Ala Arg Lys Leu Leu Arg Pro Lys Leu
610 615 620
Lys Asp His Tyr Gly Glu Arg Leu Glu Ala Leu Tyr Ala Glu Leu Ala
625 630 635 640
Glu Ser Gln Asn Glu Arg Leu Arg Gln Leu Ala Arg Glu Ala Ala Thr
645 650 655
Arg Pro Val Leu Glu Thr Val Thr Asp Ala Ala Ala Ala Leu Leu Gly
660 665 670
Ala Ser Ser Ser Asp Leu Ala Pro Asp Val Arg Phe Ile Asp Leu Gly
675 680 685
Gly Asp Ser Leu Ser Ala Leu Ser Tyr Ser Glu Leu Leu Arg Asp Ile
690 695 700
Phe Glu Val Asp Val Pro Val Gly Val Ile Asn Ser Val Ala Asn Asp
705 710 715 720
Leu Ala Ala Ile Ala Arg His Ile Glu Ala Gln Arg Thr Gly Ala Ala
725 730 735
Thr Gln Pro Thr Phe Ala Ser Val His Gly Lys Asp Ala Thr Val Ile
740 745 750
Thr Ala Gly Glu Leu Thr Leu Asp Lys Phe Leu Asp Glu Ser Leu Leu
755 760 765
Lys Ala Ala Lys Asp Val Gln Pro Ala Thr Ala Asp Val Lys Thr Val
770 775 780
Leu Val Thr Gly Gly Asn Gly Trp Leu Gly Arg Trp Leu Val Leu Asp
785 790 795 800
Trp Leu Glu Arg Leu Ala Pro Asn Gly Gly Lys Val Tyr Ala Leu Ile
805 810 815
Arg Gly Ala Asp Ala Glu Ala Ala Arg Ala Arg Leu Asp Ala Val Tyr
820 825 830
Glu Ser Gly Asp Pro Lys Leu Ser Ala His Tyr Arg Gln Leu Ala Gln
835 840 845
Gln Ser Leu Glu Val Ile Ala Gly Asp Phe Gly Asp Gln Asp Leu Gly
850 855 860
Leu Ser Gln Glu Val Trp Gln Lys Leu Ala Lys Asp Val Asp Leu Ile
865 870 875 880
Val His Ser Gly Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu
885 890 895
Phe Gly Pro Asn Val Ala Gly Thr Ala Glu Ile Ile Lys Leu Ala Ile
900 905 910
Ser Glu Arg Leu Lys Pro Val Thr Tyr Leu Ser Thr Val Gly Ile Ala
915 920 925
Asp Gln Ile Pro Val Thr Glu Phe Glu Glu Asp Ser Asp Val Arg Val
930 935 940
Met Ser Ala Glu Arg Gln Ile Asn Asp Gly Tyr Ala Asn Gly Tyr Gly
945 950 955 960
Asn Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu
965 970 975
Ala Gly Leu Pro Val Arg Val Phe Arg Ser Asp Met Ile Leu Ala His
980 985 990
Ser Asp Tyr His Gly Gln Leu Asn Val Thr Asp Val Phe Thr Arg Ser
995 1000 1005
Ile Gln Ser Leu Leu Leu Thr Gly Val Ala Pro Ala Ser Phe Tyr
1010 1015 1020
Glu Leu Asp Ala Asp Gly Asn Arg Gln Arg Ala His Tyr Asp Gly
1025 1030 1035
Val Pro Gly Asp Phe Thr Ala Ala Ser Ile Thr Ala Ile Gly Gly
1040 1045 1050
Val Asn Val Val Asp Gly Tyr Arg Ser Phe Asp Val Phe Asn Pro
1055 1060 1065
His His Asp Gly Val Ser Met Asp Thr Phe Val Asp Trp Leu Ile
1070 1075 1080
Asp Ala Gly Tyr Lys Ile Ala Arg Ile Asp Asp Tyr Asp Gln Trp
1085 1090 1095
Leu Ala Arg Phe Glu Leu Ala Leu Lys Gly Leu Pro Glu Gln Gln
1100 1105 1110
Arg Gln Gln Ser Val Leu Pro Leu Leu Lys Met Tyr Glu Lys Pro
1115 1120 1125
Gln Pro Ala Ile Asp Gly Ser Ala Leu Pro Thr Ala Glu Phe Ser
1130 1135 1140
Arg Ala Val His Glu Ala Lys Val Gly Asp Ser Gly Glu Ile Pro
1145 1150 1155
His Val Thr Lys Glu Leu Ile Leu Lys Tyr Ala Ser Asp Ile Gln
1160 1165 1170
Leu Leu Gly Leu Val
1175
<210> 21
<211> 1193
<212> PRT
<213> 耻垢分枝杆菌
<400> 21
Met His Gln Leu Thr Val Thr Gly Met Asn Ile Cys Glu Val Gln Arg
1 5 10 15
Leu Phe Pro Arg Met Thr Ser Asp Val His Asp Ala Thr Asp Gly Val
20 25 30
Thr Glu Thr Ala Leu Asp Asp Glu Gln Ser Thr Arg Arg Ile Ala Glu
35 40 45
Leu Tyr Ala Thr Asp Pro Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala
50 55 60
Val Val Asp Ala Ala His Lys Pro Gly Leu Arg Leu Ala Glu Ile Leu
65 70 75 80
Gln Thr Leu Phe Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly Tyr Arg
85 90 95
Ala Arg Glu Leu Ala Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Leu
100 105 110
Leu Pro Arg Phe Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg Val
115 120 125
Gln Ala Val Ala Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr
130 135 140
Pro Gly Asp Ala Val Ala Thr Ile Gly Phe Ala Ser Pro Asp Tyr Leu
145 150 155 160
Thr Leu Asp Leu Val Cys Ala Tyr Leu Gly Leu Val Ser Val Pro Leu
165 170 175
Gln His Asn Ala Pro Val Ser Arg Leu Ala Pro Ile Leu Ala Glu Val
180 185 190
Glu Pro Arg Ile Leu Thr Val Ser Ala Glu Tyr Leu Asp Leu Ala Val
195 200 205
Glu Ser Val Arg Asp Val Asn Ser Val Ser Gln Leu Val Val Phe Asp
210 215 220
His His Pro Glu Val Asp Asp His Arg Asp Ala Leu Ala Arg Ala Arg
225 230 235 240
Glu Gln Leu Ala Gly Lys Gly Ile Ala Val Thr Thr Leu Asp Ala Ile
245 250 255
Ala Asp Glu Gly Ala Gly Leu Pro Ala Glu Pro Ile Tyr Thr Ala Asp
260 265 270
His Asp Gln Arg Leu Ala Met Ile Leu Tyr Thr Ser Gly Ser Thr Gly
275 280 285
Ala Pro Lys Gly Ala Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp
290 295 300
Thr Met Ser Phe Ile Thr Gly Asp Pro Thr Pro Val Ile Asn Val Asn
305 310 315 320
Phe Met Pro Leu Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr Ala
325 330 335
Val Gln Asn Gly Gly Thr Ser Tyr Phe Val Pro Glu Ser Asp Met Ser
340 345 350
Thr Leu Phe Glu Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Gly Leu
355 360 365
Val Pro Arg Val Ala Asp Met Leu Tyr Gln His His Leu Ala Thr Val
370 375 380
Asp Arg Leu Val Thr Gln Gly Ala Asp Glu Leu Thr Ala Glu Lys Gln
385 390 395 400
Ala Gly Ala Glu Leu Arg Glu Gln Val Leu Gly Gly Arg Val Ile Thr
405 410 415
Gly Phe Val Ser Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu
420 425 430
Asp Ile Thr Leu Gly Ala His Ile Val Asp Gly Tyr Gly Leu Thr Glu
435 440 445
Thr Gly Ala Val Thr Arg Asp Gly Val Ile Val Arg Pro Pro Val Ile
450 455 460
Asp Tyr Lys Leu Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr Asp
465 470 475 480
Lys Pro Tyr Pro Arg Gly Glu Leu Leu Val Arg Ser Gln Thr Leu Thr
485 490 495
Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Ser Val Phe Asp Arg
500 505 510
Asp Gly Tyr Tyr His Thr Gly Asp Val Met Ala Glu Thr Ala Pro Asp
515 520 525
His Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ala Gln
530 535 540
Gly Glu Phe Val Ala Val Ala Asn Leu Glu Ala Val Phe Ser Gly Ala
545 550 555 560
Ala Leu Val Arg Gln Ile Phe Val Tyr Gly Asn Ser Glu Arg Ser Phe
565 570 575
Leu Leu Ala Val Val Val Pro Thr Pro Glu Ala Leu Glu Gln Tyr Asp
580 585 590
Pro Ala Ala Leu Lys Ala Ala Leu Ala Asp Ser Leu Gln Arg Thr Ala
595 600 605
Arg Asp Ala Glu Leu Gln Ser Tyr Glu Val Pro Ala Asp Phe Ile Val
610 615 620
Glu Thr Glu Pro Phe Ser Ala Ala Asn Gly Leu Leu Ser Gly Val Gly
625 630 635 640
Lys Leu Leu Arg Pro Asn Leu Lys Asp Arg Tyr Gly Gln Arg Leu Glu
645 650 655
Gln Met Tyr Ala Asp Ile Ala Ala Thr Gln Ala Asn Gln Leu Arg Glu
660 665 670
Leu Arg Arg Ala Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln
675 680 685
Ala Ala Ala Thr Ile Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala
690 695 700
His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Thr Leu Ser
705 710 715 720
Asn Leu Leu Ser Asp Phe Phe Gly Phe Glu Val Pro Val Gly Thr Ile
725 730 735
Val Asn Pro Ala Thr Asn Leu Ala Gln Leu Ala Gln His Ile Glu Ala
740 745 750
Gln Arg Thr Ala Gly Asp Arg Arg Pro Ser Phe Thr Thr Val His Gly
755 760 765
Ala Asp Ala Thr Glu Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe
770 775 780
Ile Asp Ala Glu Thr Leu Arg Ala Ala Pro Gly Leu Pro Lys Val Thr
785 790 795 800
Thr Glu Pro Arg Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly
805 810 815
Arg Phe Leu Thr Leu Gln Trp Leu Glu Arg Leu Ala Pro Val Gly Gly
820 825 830
Thr Leu Ile Thr Ile Val Arg Gly Arg Asp Asp Ala Ala Ala Arg Ala
835 840 845
Arg Leu Thr Gln Ala Tyr Asp Thr Asp Pro Glu Leu Ser Arg Arg Phe
850 855 860
Ala Glu Leu Ala Asp Arg His Leu Arg Val Val Ala Gly Asp Ile Gly
865 870 875 880
Asp Pro Asn Leu Gly Leu Thr Pro Glu Ile Trp His Arg Leu Ala Ala
885 890 895
Glu Val Asp Leu Val Val His Pro Ala Ala Leu Val Asn His Val Leu
900 905 910
Pro Tyr Arg Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala Glu Val
915 920 925
Ile Lys Leu Ala Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr Leu Ser
930 935 940
Thr Val Ser Val Ala Met Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp
945 950 955 960
Ile Arg Thr Val Ser Pro Val Arg Pro Leu Asp Gly Gly Tyr Ala Asn
965 970 975
Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala
980 985 990
His Asp Leu Cys Gly Leu Pro Val Ala Thr Phe Arg Ser Asp Met Ile
995 1000 1005
Leu Ala His Pro Arg Tyr Arg Gly Gln Val Asn Val Pro Asp Met
1010 1015 1020
Phe Thr Arg Leu Leu Leu Ser Leu Leu Ile Thr Gly Val Ala Pro
1025 1030 1035
Arg Ser Phe Tyr Ile Gly Asp Gly Glu Arg Pro Arg Ala His Tyr
1040 1045 1050
Pro Gly Leu Thr Val Asp Phe Val Ala Glu Ala Val Thr Thr Leu
1055 1060 1065
Gly Ala Gln Gln Arg Glu Gly Tyr Val Ser Tyr Asp Val Met Asn
1070 1075 1080
Pro His Asp Asp Gly Ile Ser Leu Asp Val Phe Val Asp Trp Leu
1085 1090 1095
Ile Arg Ala Gly His Pro Ile Asp Arg Val Asp Asp Tyr Asp Asp
1100 1105 1110
Trp Val Arg Arg Phe Glu Thr Ala Leu Thr Ala Leu Pro Glu Lys
1115 1120 1125
Arg Arg Ala Gln Thr Val Leu Pro Leu Leu His Ala Phe Arg Ala
1130 1135 1140
Pro Gln Ala Pro Leu Arg Gly Ala Pro Glu Pro Thr Glu Val Phe
1145 1150 1155
His Ala Ala Val Arg Thr Ala Lys Val Gly Pro Gly Asp Ile Pro
1160 1165 1170
His Leu Asp Glu Ala Leu Ile Asp Lys Tyr Ile Arg Asp Leu Arg
1175 1180 1185
Glu Phe Gly Leu Ile
1190
<210> 22
<211> 1178
<212> PRT
<213> 耻垢分枝杆菌
<400> 22
Met Thr Glu Thr Ile Ser Thr Ala Ala Val Pro Thr Thr Asp Leu Glu
1 5 10 15
Glu Gln Val Lys Arg Arg Ile Glu Gln Val Val Ser Asn Asp Pro Gln
20 25 30
Leu Ala Ala Leu Leu Pro Glu Asp Ser Val Thr Glu Ala Val Asn Glu
35 40 45
Pro Asp Leu Pro Leu Val Glu Val Ile Arg Arg Leu Leu Glu Gly Tyr
50 55 60
Gly Asp Arg Pro Ala Leu Gly Gln Arg Ala Phe Glu Phe Val Thr Gly
65 70 75 80
Asp Asp Gly Ala Thr Val Ile Ala Leu Lys Pro Glu Tyr Thr Thr Val
85 90 95
Ser Tyr Arg Glu Leu Trp Glu Arg Ala Glu Ala Ile Ala Ala Ala Trp
100 105 110
His Glu Gln Gly Ile Arg Asp Gly Asp Phe Val Ala Gln Leu Gly Phe
115 120 125
Thr Ser Thr Asp Phe Ala Ser Leu Asp Val Ala Gly Leu Arg Leu Gly
130 135 140
Thr Val Ser Val Pro Leu Gln Thr Gly Ala Ser Leu Gln Gln Arg Asn
145 150 155 160
Ala Ile Leu Glu Glu Thr Arg Pro Ala Val Phe Ala Ala Ser Ile Glu
165 170 175
Tyr Leu Asp Ala Ala Val Asp Ser Val Leu Ala Thr Pro Ser Val Arg
180 185 190
Leu Leu Ser Val Phe Asp Tyr His Ala Glu Val Asp Ser Gln Arg Glu
195 200 205
Ala Leu Glu Ala Val Arg Ala Arg Leu Glu Ser Ala Gly Arg Thr Ile
210 215 220
Val Val Glu Ala Leu Ala Glu Ala Leu Ala Arg Gly Arg Asp Leu Pro
225 230 235 240
Ala Ala Pro Leu Pro Ser Ala Asp Pro Asp Ala Leu Arg Leu Leu Ile
245 250 255
Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys Gly Ala Met Tyr Pro Gln
260 265 270
Trp Leu Val Ala Asn Leu Trp Gln Lys Lys Arg Leu Thr Asp Asp Val
275 280 285
Ile Pro Ser Ile Gly Val Asn Phe Met Pro Met Ser His Leu Met Gly
290 295 300
Arg Leu Thr Leu Met Gly Thr Leu Ser Gly Gly Gly Thr Ala Tyr Tyr
305 310 315 320
Ile Ala Ser Ser Asp Leu Ser Thr Phe Phe Glu Asp Ile Ala Leu Ile
325 330 335
Arg Pro Ser Glu Val Leu Phe Val Pro Arg Val Val Glu Met Val Phe
340 345 350
Gln Arg Phe Gln Ala Glu Leu Asp Arg Ser Leu Ala Pro Gly Glu Ser
355 360 365
Asn Ser Glu Ile Ala Glu Arg Ile Lys Val Arg Ile Arg Glu Gln Asp
370 375 380
Phe Gly Gly Arg Val Leu Ser Ala Gly Ser Gly Ser Ala Pro Leu Ser
385 390 395 400
Pro Glu Met Thr Glu Phe Met Glu Ser Leu Leu Gln Val Pro Leu Arg
405 410 415
Asp Gly Tyr Gly Ser Thr Glu Ala Gly Gly Val Trp Arg Asp Gly Val
420 425 430
Leu Gln Arg Pro Pro Val Thr Asp Tyr Lys Leu Val Asp Val Pro Glu
435 440 445
Leu Gly Tyr Phe Thr Thr Asp Ser Pro His Pro Arg Gly Glu Leu Arg
450 455 460
Leu Lys Ser Glu Thr Met Phe Pro Gly Tyr Tyr Lys Arg Pro Glu Thr
465 470 475 480
Thr Ala Asp Val Phe Asp Asp Glu Gly Tyr Tyr Lys Thr Gly Asp Val
485 490 495
Val Ala Glu Leu Gly Pro Asp His Leu Lys Tyr Leu Asp Arg Val Lys
500 505 510
Asn Val Leu Lys Leu Ala Gln Gly Glu Phe Val Ala Val Ser Lys Leu
515 520 525
Glu Ala Ala Tyr Thr Gly Ser Pro Leu Val Arg Gln Ile Phe Val Tyr
530 535 540
Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val Val Val Pro Thr Pro
545 550 555 560
Glu Val Leu Glu Arg Tyr Ala Asp Ser Pro Asp Ala Leu Lys Pro Leu
565 570 575
Ile Gln Asp Ser Leu Gln Gln Val Ala Lys Asp Ala Glu Leu Gln Ser
580 585 590
Tyr Glu Ile Pro Arg Asp Phe Ile Val Glu Thr Val Pro Phe Thr Val
595 600 605
Glu Ser Gly Leu Leu Ser Asp Ala Arg Lys Leu Leu Arg Pro Lys Leu
610 615 620
Lys Asp His Tyr Gly Glu Arg Leu Glu Ala Leu Tyr Ala Glu Leu Ala
625 630 635 640
Glu Ser Gln Asn Glu Arg Leu Arg Gln Leu Ala Arg Glu Ala Ala Thr
645 650 655
Arg Pro Val Leu Glu Thr Val Thr Asp Ala Ala Ala Ala Leu Leu Gly
660 665 670
Ala Ser Ser Ser Asp Leu Ala Pro Asp Val Arg Phe Ile Asp Leu Gly
675 680 685
Gly Asp Ser Leu Ser Ala Leu Ser Tyr Ser Glu Leu Leu Arg Asp Ile
690 695 700
Phe Glu Val Asp Val Pro Val Gly Val Ile Asn Ser Val Ala Asn Asp
705 710 715 720
Leu Ala Ala Ile Ala Arg His Ile Glu Ala Gln Arg Thr Gly Ala Ala
725 730 735
Thr Gln Pro Thr Phe Ala Ser Val His Gly Lys Asp Ala Thr Val Ile
740 745 750
Thr Ala Gly Glu Leu Thr Leu Asp Lys Phe Leu Asp Glu Ser Leu Leu
755 760 765
Lys Ala Ala Lys Asp Val Gln Pro Ala Thr Ala Asp Val Lys Thr Val
770 775 780
Leu Val Thr Gly Gly Asn Gly Trp Leu Gly Arg Trp Leu Val Leu Asp
785 790 795 800
Trp Leu Glu Arg Leu Ala Pro Asn Gly Gly Lys Val Tyr Ala Leu Ile
805 810 815
Arg Gly Ala Asp Ala Glu Ala Ala Arg Ala Arg Leu Asp Ala Val Tyr
820 825 830
Glu Ser Gly Asp Pro Lys Leu Ser Ala His Tyr Arg Gln Leu Ala Gln
835 840 845
Gln Ser Leu Glu Val Ile Ala Gly Asp Phe Gly Asp Gln Asp Leu Gly
850 855 860
Leu Ser Gln Glu Val Trp Gln Lys Leu Ala Lys Asp Val Asp Leu Ile
865 870 875 880
Val His Ser Gly Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu
885 890 895
Phe Gly Pro Asn Val Ala Gly Thr Ala Glu Ile Ile Lys Leu Ala Ile
900 905 910
Ser Glu Arg Leu Lys Pro Val Thr Tyr Leu Ser Thr Val Gly Ile Ala
915 920 925
Asp Gln Ile Pro Val Thr Glu Phe Glu Glu Asp Ser Asp Val Arg Val
930 935 940
Met Ser Ala Glu Arg Gln Ile Asn Asp Gly Tyr Ala Asn Gly Tyr Gly
945 950 955 960
Asn Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu
965 970 975
Ala Gly Leu Pro Val Arg Val Phe Arg Ser Asp Met Ile Leu Ala His
980 985 990
Ser Asp Tyr His Gly Gln Leu Asn Val Thr Asp Val Phe Thr Arg Ser
995 1000 1005
Ile Gln Ser Leu Leu Leu Thr Gly Val Ala Pro Ala Ser Phe Tyr
1010 1015 1020
Glu Leu Asp Ala Asp Gly Asn Arg Gln Arg Ala His Tyr Asp Gly
1025 1030 1035
Val Pro Gly Asp Phe Thr Ala Ala Ser Ile Thr Ala Ile Gly Gly
1040 1045 1050
Val Asn Val Val Asp Gly Tyr Arg Ser Phe Asp Val Phe Asn Pro
1055 1060 1065
His His Asp Gly Val Ser Met Asp Thr Phe Val Asp Trp Leu Ile
1070 1075 1080
Asp Ala Gly Tyr Lys Ile Ala Arg Ile Asp Asp Tyr Asp Gln Trp
1085 1090 1095
Leu Ala Arg Phe Glu Leu Ala Leu Lys Gly Leu Pro Glu Gln Gln
1100 1105 1110
Arg Gln Gln Ser Val Leu Pro Leu Leu Lys Met Tyr Glu Lys Pro
1115 1120 1125
Gln Pro Ala Ile Asp Gly Ser Ala Leu Pro Thr Ala Glu Phe Ser
1130 1135 1140
Arg Ala Val His Glu Ala Lys Val Gly Asp Ser Gly Glu Ile Pro
1145 1150 1155
His Val Thr Lys Glu Leu Ile Leu Lys Tyr Ala Ser Asp Ile Gln
1160 1165 1170
Leu Leu Gly Leu Val
1175
<210> 23
<211> 224
<212> PRT
<213> 枯草杆菌
<400> 23
Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg Pro Leu Ser Gln Glu Glu
1 5 10 15
Asn Glu Arg Phe Met Thr Phe Ile Ser Pro Glu Lys Arg Glu Lys Cys
20 25 30
Arg Arg Phe Tyr His Lys Glu Asp Ala His Arg Thr Leu Leu Gly Asp
35 40 45
Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr Gln Leu Asp Lys Ser
50 55 60
Asp Ile Arg Phe Ser Thr Gln Glu Tyr Gly Lys Pro Cys Ile Pro Asp
65 70 75 80
Leu Pro Asp Ala His Phe Asn Ile Ser His Ser Gly Arg Trp Val Ile
85 90 95
Gly Ala Phe Asp Ser Gln Pro Ile Gly Ile Asp Ile Glu Lys Thr Lys
100 105 110
Pro Ile Ser Leu Glu Ile Ala Lys Arg Phe Phe Ser Lys Thr Glu Tyr
115 120 125
Ser Asp Leu Leu Ala Lys Asp Lys Asp Glu Gln Thr Asp Tyr Phe Tyr
130 135 140
His Leu Trp Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys Gly
145 150 155 160
Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu His Gln Asp Gly
165 170 175
Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro Cys Tyr Ile Lys
180 185 190
Thr Tyr Glu Val Asp Pro Gly Tyr Lys Met Ala Val Cys Ala Ala His
195 200 205
Pro Asp Phe Pro Glu Asp Ile Thr Met Val Ser Tyr Glu Glu Leu Leu
210 215 220
<210> 24
<211> 222
<212> PRT
<213> 艾阿华诺卡氏菌
<400> 24
Met Ile Glu Thr Ile Leu Pro Ala Gly Val Glu Ser Ala Glu Leu Leu
1 5 10 15
Glu Tyr Pro Glu Asp Leu Lys Ala His Pro Ala Glu Glu His Leu Ile
20 25 30
Ala Lys Ser Val Glu Lys Arg Arg Arg Asp Phe Ile Gly Ala Arg His
35 40 45
Cys Ala Arg Leu Ala Leu Ala Glu Leu Gly Glu Pro Pro Val Ala Ile
50 55 60
Gly Lys Gly Glu Arg Gly Ala Pro Ile Trp Pro Arg Gly Val Val Gly
65 70 75 80
Ser Leu Thr His Cys Asp Gly Tyr Arg Ala Ala Ala Val Ala His Lys
85 90 95
Met Arg Phe Arg Ser Ile Gly Ile Asp Ala Glu Pro His Ala Thr Leu
100 105 110
Pro Glu Gly Val Leu Asp Ser Val Ser Leu Pro Pro Glu Arg Glu Trp
115 120 125
Leu Lys Thr Thr Asp Ser Ala Leu His Leu Asp Arg Leu Leu Phe Cys
130 135 140
Ala Lys Glu Ala Thr Tyr Lys Ala Trp Trp Pro Leu Thr Ala Arg Trp
145 150 155 160
Leu Gly Phe Glu Glu Ala His Ile Thr Phe Glu Ile Glu Asp Gly Ser
165 170 175
Ala Asp Ser Gly Asn Gly Thr Phe His Ser Glu Leu Leu Val Pro Gly
180 185 190
Gln Thr Asn Asp Gly Gly Thr Pro Leu Leu Ser Phe Asp Gly Arg Trp
195 200 205
Leu Ile Ala Asp Gly Phe Ile Leu Thr Ala Ile Ala Tyr Ala
210 215 220
<210> 25
<211> 272
<212> PRT
<213> 酿酒酵母
<400> 25
Met Val Lys Thr Thr Glu Val Val Ser Glu Val Ser Lys Val Ala Gly
1 5 10 15
Val Arg Pro Trp Ala Gly Ile Phe Val Val Glu Ile Gln Glu Asp Ile
20 25 30
Leu Ala Asp Glu Phe Thr Phe Glu Ala Leu Met Arg Thr Leu Pro Leu
35 40 45
Ala Ser Gln Ala Arg Ile Leu Asn Lys Lys Ser Phe His Asp Arg Cys
50 55 60
Ser Asn Leu Cys Ser Gln Leu Leu Gln Leu Phe Gly Cys Ser Ile Val
65 70 75 80
Thr Gly Leu Asn Phe Gln Glu Leu Lys Phe Asp Lys Gly Ser Phe Gly
85 90 95
Lys Pro Phe Leu Asp Asn Asn Arg Phe Leu Pro Phe Ser Met Thr Ile
100 105 110
Gly Glu Gln Tyr Val Ala Met Phe Leu Val Lys Cys Val Ser Thr Asp
115 120 125
Glu Tyr Gln Asp Val Gly Ile Asp Ile Ala Ser Pro Cys Asn Tyr Gly
130 135 140
Gly Arg Glu Glu Leu Glu Leu Phe Lys Glu Val Phe Ser Glu Arg Glu
145 150 155 160
Phe Asn Gly Leu Leu Lys Ala Ser Asp Pro Cys Thr Ile Phe Thr Tyr
165 170 175
Leu Trp Ser Leu Lys Glu Ser Tyr Thr Lys Phe Thr Gly Thr Gly Leu
180 185 190
Asn Thr Asp Leu Ser Leu Ile Asp Phe Gly Ala Ile Ser Phe Phe Pro
195 200 205
Ala Glu Gly Ala Ser Met Cys Ile Thr Leu Asp Glu Val Pro Leu Ile
210 215 220
Phe His Ser Gln Trp Phe Asn Asn Glu Ile Val Thr Ile Cys Met Pro
225 230 235 240
Lys Ser Ile Ser Asp Lys Ile Asn Thr Asn Arg Pro Lys Leu Tyr Asn
245 250 255
Ile Ser Leu Ser Thr Leu Ile Asp Tyr Phe Ile Glu Asn Asp Gly Leu
260 265 270
<210> 26
<211> 209
<212> PRT
<213> 大肠杆菌
<400> 26
Met Val Asp Met Lys Thr Thr His Thr Ser Leu Pro Phe Ala Gly His
1 5 10 15
Thr Leu His Phe Val Glu Phe Asp Pro Ala Asn Phe Cys Glu Gln Asp
20 25 30
Leu Leu Trp Leu Pro His Tyr Ala Gln Leu Gln His Ala Gly Arg Lys
35 40 45
Arg Lys Thr Glu His Leu Ala Gly Arg Ile Ala Ala Val Tyr Ala Leu
50 55 60
Arg Glu Tyr Gly Tyr Lys Cys Val Pro Ala Ile Gly Glu Leu Arg Gln
65 70 75 80
Pro Val Trp Pro Ala Glu Val Tyr Gly Ser Ile Ser His Cys Gly Thr
85 90 95
Thr Ala Leu Ala Val Val Ser Arg Gln Pro Ile Gly Ile Asp Ile Glu
100 105 110
Glu Ile Phe Ser Val Gln Thr Ala Arg Glu Leu Thr Asp Asn Ile Ile
115 120 125
Thr Pro Ala Glu His Glu Arg Leu Ala Asp Cys Gly Leu Ala Phe Ser
130 135 140
Leu Ala Leu Thr Leu Ala Phe Ser Ala Lys Glu Ser Ala Phe Lys Ala
145 150 155 160
Ser Glu Ile Gln Thr Asp Ala Gly Phe Leu Asp Tyr Gln Ile Ile Ser
165 170 175
Trp Asn Lys Gln Gln Val Ile Ile His Arg Glu Asn Glu Met Phe Ala
180 185 190
Val His Trp Gln Ile Lys Glu Lys Ile Val Ile Thr Leu Cys Gln His
195 200 205
Asp
<210> 27
<211> 468
<212> PRT
<213> 大肠杆菌
<400> 27
Met Ile Arg Glu Pro Pro Glu His Ile Leu Asn Arg Leu Pro Ser Ser
1 5 10 15
Ala Ser Ala Leu Ala Cys Ser Ala His Ala Leu Asn Leu Ile Glu Lys
20 25 30
Arg Thr Leu Asp His Glu Glu Met Lys Ala Leu Asn Arg Glu Val Ile
35 40 45
Glu Tyr Phe Lys Glu His Val Asn Pro Gly Phe Leu Glu Tyr Arg Lys
50 55 60
Ser Val Thr Ala Gly Gly Asp Tyr Gly Ala Val Glu Trp Gln Ala Gly
65 70 75 80
Ser Leu Asn Thr Leu Val Asp Thr Gln Gly Gln Glu Phe Ile Asp Cys
85 90 95
Leu Gly Gly Phe Gly Ile Phe Asn Val Gly His Arg Asn Pro Val Val
100 105 110
Val Ser Ala Val Gln Asn Gln Leu Ala Lys Gln Pro Leu His Ser Gln
115 120 125
Glu Leu Leu Asp Pro Leu Arg Ala Met Leu Ala Lys Thr Leu Ala Ala
130 135 140
Leu Thr Pro Gly Lys Leu Lys Tyr Ser Phe Phe Cys Asn Ser Gly Thr
145 150 155 160
Glu Ser Val Glu Ala Ala Leu Lys Leu Ala Lys Ala Tyr Gln Ser Pro
165 170 175
Arg Gly Lys Phe Thr Phe Ile Ala Thr Ser Gly Ala Phe His Gly Lys
180 185 190
Ser Leu Gly Ala Leu Ser Ala Thr Ala Lys Ser Thr Phe Arg Lys Pro
195 200 205
Phe Met Pro Leu Leu Pro Gly Phe Arg His Val Pro Phe Gly Asn Ile
210 215 220
Glu Ala Met Arg Thr Ala Leu Asn Glu Cys Lys Lys Thr Gly Asp Asp
225 230 235 240
Val Ala Ala Val Ile Leu Glu Pro Ile Gln Gly Glu Gly Gly Val Ile
245 250 255
Leu Pro Pro Pro Gly Tyr Leu Thr Ala Val Arg Lys Leu Cys Asp Glu
260 265 270
Phe Gly Ala Leu Met Ile Leu Asp Glu Val Gln Thr Gly Met Gly Arg
275 280 285
Thr Gly Lys Met Phe Ala Cys Glu His Glu Asn Val Gln Pro Asp Ile
290 295 300
Leu Cys Leu Ala Lys Ala Leu Gly Gly Gly Val Met Pro Ile Gly Ala
305 310 315 320
Thr Ile Ala Thr Glu Glu Val Phe Ser Val Leu Phe Asp Asn Pro Phe
325 330 335
Leu His Thr Thr Thr Phe Gly Gly Asn Pro Leu Ala Cys Ala Ala Ala
340 345 350
Leu Ala Thr Ile Asn Val Leu Leu Glu Gln Asn Leu Pro Ala Gln Ala
355 360 365
Glu Gln Lys Gly Asp Met Leu Leu Asp Gly Phe Arg Gln Leu Ala Arg
370 375 380
Glu Tyr Pro Asp Leu Val Gln Glu Ala Arg Gly Lys Gly Met Leu Met
385 390 395 400
Ala Ile Glu Phe Val Asp Asn Glu Ile Gly Tyr Asn Phe Ala Ser Glu
405 410 415
Met Phe Arg Gln Arg Val Leu Val Ala Gly Thr Leu Asn Asn Ala Lys
420 425 430
Thr Ile Arg Ile Glu Pro Pro Leu Thr Leu Thr Ile Glu Gln Cys Glu
435 440 445
Leu Val Ile Lys Ala Ala Arg Lys Ala Leu Ala Ala Met Arg Val Ser
450 455 460
Val Glu Glu Ala
465
<210> 28
<211> 456
<212> PRT
<213> 绿脓杆菌
<400> 28
Met Asn Ser Gln Ile Thr Asn Ala Lys Thr Arg Glu Trp Gln Ala Leu
1 5 10 15
Ser Arg Asp His His Leu Pro Pro Phe Thr Asp Tyr Lys Gln Leu Asn
20 25 30
Glu Lys Gly Ala Arg Ile Ile Thr Lys Ala Glu Gly Val Tyr Ile Trp
35 40 45
Asp Ser Glu Gly Asn Lys Ile Leu Asp Ala Met Ala Gly Leu Trp Cys
50 55 60
Val Asn Val Gly Tyr Gly Arg Glu Glu Leu Val Gln Ala Ala Thr Arg
65 70 75 80
Gln Met Arg Glu Leu Pro Phe Tyr Asn Leu Phe Phe Gln Thr Ala His
85 90 95
Pro Pro Val Val Glu Leu Ala Lys Ala Ile Ala Asp Val Ala Pro Glu
100 105 110
Gly Met Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Ala Asn Asp
115 120 125
Thr Val Leu Arg Met Val Arg His Tyr Trp Ala Thr Lys Gly Gln Pro
130 135 140
Gln Lys Lys Val Val Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr
145 150 155 160
Val Ala Gly Val Ser Leu Gly Gly Met Lys Ala Leu His Glu Gln Gly
165 170 175
Asp Phe Pro Ile Pro Gly Ile Val His Ile Ala Gln Pro Tyr Trp Tyr
180 185 190
Gly Glu Gly Gly Asp Met Ser Pro Asp Glu Phe Gly Val Trp Ala Ala
195 200 205
Glu Gln Leu Glu Lys Lys Ile Leu Glu Val Gly Glu Glu Asn Val Ala
210 215 220
Ala Phe Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro
225 230 235 240
Pro Asp Thr Tyr Trp Pro Lys Ile Arg Glu Ile Leu Ala Lys Tyr Asp
245 250 255
Ile Leu Phe Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly
260 265 270
Glu Trp Phe Gly Ser Gln Tyr Tyr Gly Asn Ala Pro Asp Leu Met Pro
275 280 285
Ile Ala Lys Gly Leu Thr Ser Gly Tyr Ile Pro Met Gly Gly Val Val
290 295 300
Val Arg Asp Glu Ile Val Glu Val Leu Asn Gln Gly Gly Glu Phe Tyr
305 310 315 320
His Gly Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Ile Arg Ile Leu Arg Glu Glu Lys Ile Ile Glu Lys Val Lys
340 345 350
Ala Glu Thr Ala Pro Tyr Leu Gln Lys Arg Trp Gln Glu Leu Ala Asp
355 360 365
His Pro Leu Val Gly Glu Ala Arg Gly Val Gly Met Val Ala Ala Leu
370 375 380
Glu Leu Val Lys Asn Lys Lys Thr Arg Glu Arg Phe Thr Asp Lys Gly
385 390 395 400
Val Gly Met Leu Cys Arg Glu His Cys Phe Arg Asn Gly Leu Ile Met
405 410 415
Arg Ala Val Gly Asp Thr Met Ile Ile Ser Pro Pro Leu Val Ile Asp
420 425 430
Pro Ser Gln Ile Asp Glu Leu Ile Thr Leu Ala Arg Lys Cys Leu Asp
435 440 445
Gln Thr Ala Ala Ala Val Leu Ala
450 455
<210> 29
<211> 426
<212> PRT
<213> 大肠杆菌
<400> 29
Met Asn Ser Asn Lys Glu Leu Met Gln Arg Arg Ser Gln Ala Ile Pro
1 5 10 15
Arg Gly Val Gly Gln Ile His Pro Ile Phe Ala Asp Arg Ala Glu Asn
20 25 30
Cys Arg Val Trp Asp Val Glu Gly Arg Glu Tyr Leu Asp Phe Ala Gly
35 40 45
Gly Ile Ala Val Leu Asn Thr Gly His Leu His Pro Lys Val Val Ala
50 55 60
Ala Val Glu Ala Gln Leu Lys Lys Leu Ser His Thr Cys Phe Gln Val
65 70 75 80
Leu Ala Tyr Glu Pro Tyr Leu Glu Leu Cys Glu Ile Met Asn Gln Lys
85 90 95
Val Pro Gly Asp Phe Ala Lys Lys Thr Leu Leu Val Thr Thr Gly Ser
100 105 110
Glu Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala Ala Thr Lys Arg
115 120 125
Ser Gly Thr Ile Ala Phe Ser Gly Ala Tyr His Gly Arg Thr His Tyr
130 135 140
Thr Leu Ala Leu Thr Gly Lys Val Asn Pro Tyr Ser Ala Gly Met Gly
145 150 155 160
Leu Met Pro Gly His Val Tyr Arg Ala Leu Tyr Pro Cys Pro Leu His
165 170 175
Gly Ile Ser Glu Asp Asp Ala Ile Ala Ser Ile His Arg Ile Phe Lys
180 185 190
Asn Asp Ala Ala Pro Glu Asp Ile Ala Ala Ile Val Ile Glu Pro Val
195 200 205
Gln Gly Glu Gly Gly Phe Tyr Ala Ser Ser Pro Ala Phe Met Gln Arg
210 215 220
Leu Arg Ala Leu Cys Asp Glu His Gly Ile Met Leu Ile Ala Asp Glu
225 230 235 240
Val Gln Ser Gly Ala Gly Arg Thr Gly Thr Leu Phe Ala Met Glu Gln
245 250 255
Met Gly Val Ala Pro Asp Leu Thr Thr Phe Ala Lys Ser Ile Ala Gly
260 265 270
Gly Phe Pro Leu Ala Gly Val Thr Gly Arg Ala Glu Val Met Asp Ala
275 280 285
Val Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Asn Pro Ile Ala
290 295 300
Cys Val Ala Ala Leu Glu Val Leu Lys Val Phe Glu Gln Glu Asn Leu
305 310 315 320
Leu Gln Lys Ala Asn Asp Leu Gly Gln Lys Leu Lys Asp Gly Leu Leu
325 330 335
Ala Ile Ala Glu Lys His Pro Glu Ile Gly Asp Val Arg Gly Leu Gly
340 345 350
Ala Met Ile Ala Ile Glu Leu Phe Glu Asp Gly Asp His Asn Lys Pro
355 360 365
Asp Ala Lys Leu Thr Ala Glu Ile Val Ala Arg Ala Arg Asp Lys Gly
370 375 380
Leu Ile Leu Leu Ser Cys Gly Pro Tyr Tyr Asn Val Leu Arg Ile Leu
385 390 395 400
Val Pro Leu Thr Ile Glu Asp Ala Gln Ile Arg Gln Gly Leu Glu Ile
405 410 415
Ile Ser Gln Cys Phe Asp Glu Ala Lys Gln
420 425
<210> 30
<211> 421
<212> PRT
<213> 大肠杆菌
<400> 30
Met Ser Asn Asn Glu Phe His Gln Arg Arg Leu Ser Ala Thr Pro Arg
1 5 10 15
Gly Val Gly Val Met Cys Asn Phe Phe Ala Gln Ser Ala Glu Asn Ala
20 25 30
Thr Leu Lys Asp Val Glu Gly Asn Glu Tyr Ile Asp Phe Ala Ala Gly
35 40 45
Ile Ala Val Leu Asn Thr Gly His Arg His Pro Asp Leu Val Ala Ala
50 55 60
Val Glu Gln Gln Leu Gln Gln Phe Thr His Thr Ala Tyr Gln Ile Val
65 70 75 80
Pro Tyr Glu Ser Tyr Val Thr Leu Ala Glu Lys Ile Asn Ala Leu Ala
85 90 95
Pro Val Ser Gly Gln Ala Lys Thr Ala Phe Phe Thr Thr Gly Ala Glu
100 105 110
Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala His Thr Gly Arg Pro
115 120 125
Gly Val Ile Ala Phe Ser Gly Gly Phe His Gly Arg Thr Tyr Met Thr
130 135 140
Met Ala Leu Thr Gly Lys Val Ala Pro Tyr Lys Ile Gly Phe Gly Pro
145 150 155 160
Phe Pro Gly Ser Val Tyr His Val Pro Tyr Pro Ser Asp Leu His Gly
165 170 175
Ile Ser Thr Gln Asp Ser Leu Asp Ala Ile Glu Arg Leu Phe Lys Ser
180 185 190
Asp Ile Glu Ala Lys Gln Val Ala Ala Ile Ile Phe Glu Pro Val Gln
195 200 205
Gly Glu Gly Gly Phe Asn Val Ala Pro Lys Glu Leu Val Ala Ala Ile
210 215 220
Arg Arg Leu Cys Asp Glu His Gly Ile Val Met Ile Ala Asp Glu Val
225 230 235 240
Gln Ser Gly Phe Ala Arg Thr Gly Lys Leu Phe Ala Met Asp His Tyr
245 250 255
Ala Asp Lys Pro Asp Leu Met Thr Met Ala Lys Ser Leu Ala Gly Gly
260 265 270
Met Pro Leu Ser Gly Val Val Gly Asn Ala Asn Ile Met Asp Ala Pro
275 280 285
Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Asn Pro Leu Ala Val
290 295 300
Ala Ala Ala His Ala Val Leu Asn Ile Ile Asp Lys Glu Ser Leu Cys
305 310 315 320
Glu Arg Ala Asn Gln Leu Gly Gln Arg Leu Lys Asn Thr Leu Ile Asp
325 330 335
Ala Lys Glu Ser Val Pro Ala Ile Ala Ala Val Arg Gly Leu Gly Ser
340 345 350
Met Ile Ala Val Glu Phe Asn Asp Pro Gln Thr Gly Glu Pro Ser Ala
355 360 365
Ala Ile Ala Gln Lys Ile Gln Gln Arg Ala Leu Ala Gln Gly Leu Leu
370 375 380
Leu Leu Thr Cys Gly Ala Tyr Gly Asn Val Ile Arg Phe Leu Tyr Pro
385 390 395 400
Leu Thr Ile Pro Asp Ala Gln Phe Asp Ala Ala Met Lys Ile Leu Gln
405 410 415
Asp Ala Leu Ser Asp
420
<210> 31
<211> 464
<212> PRT
<213> 帕氏鲁杰氏菌
<400> 31
Met Ala Thr Ile Thr Asn His Met Pro Thr Ala Glu Leu Gln Ala Leu
1 5 10 15
Asp Ala Ala His His Leu His Pro Phe Ser Ala Asn Asn Ala Leu Gly
20 25 30
Glu Glu Gly Thr Arg Val Ile Thr Arg Ala Arg Gly Val Trp Leu Asn
35 40 45
Asp Ser Glu Gly Glu Glu Ile Leu Asp Ala Met Ala Gly Leu Trp Cys
50 55 60
Val Asn Ile Gly Tyr Gly Arg Asp Glu Leu Ala Glu Val Ala Ala Arg
65 70 75 80
Gln Met Arg Glu Leu Pro Tyr Tyr Asn Thr Phe Phe Lys Thr Thr His
85 90 95
Val Pro Ala Ile Ala Leu Ala Gln Lys Leu Ala Glu Leu Ala Pro Gly
100 105 110
Asp Leu Asn His Val Phe Phe Ala Gly Gly Gly Ser Glu Ala Asn Asp
115 120 125
Thr Asn Ile Arg Met Val Arg Thr Tyr Trp Gln Asn Lys Gly Gln Pro
130 135 140
Glu Lys Thr Val Ile Ile Ser Arg Lys Asn Ala Tyr His Gly Ser Thr
145 150 155 160
Val Ala Ser Ser Ala Leu Gly Gly Met Ala Gly Met His Ala Gln Ser
165 170 175
Gly Leu Ile Pro Asp Val His His Ile Asn Gln Pro Asn Trp Trp Ala
180 185 190
Glu Gly Gly Asp Met Asp Pro Glu Glu Phe Gly Leu Ala Arg Ala Arg
195 200 205
Glu Leu Glu Glu Ala Ile Leu Glu Leu Gly Glu Asn Arg Val Ala Ala
210 215 220
Phe Ile Ala Glu Pro Val Gln Gly Ala Gly Gly Val Ile Val Ala Pro
225 230 235 240
Asp Ser Tyr Trp Pro Glu Ile Gln Arg Ile Cys Asp Lys Tyr Asp Ile
245 250 255
Leu Leu Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn
260 265 270
Trp Phe Gly Thr Gln Thr Met Gly Ile Arg Pro His Ile Met Thr Ile
275 280 285
Ala Lys Gly Leu Ser Ser Gly Tyr Ala Pro Ile Gly Gly Ser Ile Val
290 295 300
Cys Asp Glu Val Ala His Val Ile Gly Lys Asp Glu Phe Asn His Gly
305 310 315 320
Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn
325 330 335
Leu Arg Ile Leu Glu Glu Glu Asn Ile Leu Asp His Val Arg Asn Val
340 345 350
Ala Ala Pro Tyr Leu Lys Glu Lys Trp Glu Ala Leu Thr Asp His Pro
355 360 365
Leu Val Gly Glu Ala Lys Ile Val Gly Met Met Ala Ser Ile Ala Leu
370 375 380
Thr Pro Asn Lys Ala Ser Arg Ala Lys Phe Ala Ser Glu Pro Gly Thr
385 390 395 400
Ile Gly Tyr Ile Cys Arg Glu Arg Cys Phe Ala Asn Asn Leu Ile Met
405 410 415
Arg His Val Gly Asp Arg Met Ile Ile Ser Pro Pro Leu Val Ile Thr
420 425 430
Pro Ala Glu Ile Asp Glu Met Phe Val Arg Ile Arg Lys Ser Leu Asp
435 440 445
Glu Ala Gln Ala Glu Ile Glu Lys Gln Gly Leu Met Lys Ser Ala Ala
450 455 460
<210> 32
<211> 339
<212> PRT
<213> 大肠杆菌
<400> 32
Met Ser Met Ile Lys Ser Tyr Ala Ala Lys Glu Ala Gly Gly Glu Leu
1 5 10 15
Glu Val Tyr Glu Tyr Asp Pro Gly Glu Leu Arg Pro Gln Asp Val Glu
20 25 30
Val Gln Val Asp Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Ile
35 40 45
Asp Asn Glu Trp Gly Phe Ser Gln Tyr Pro Leu Val Ala Gly His Glu
50 55 60
Val Ile Gly Arg Val Val Ala Leu Gly Ser Ala Ala Gln Asp Lys Gly
65 70 75 80
Leu Gln Val Gly Gln Arg Val Gly Ile Gly Trp Thr Ala Arg Ser Cys
85 90 95
Gly His Cys Asp Ala Cys Ile Ser Gly Asn Gln Ile Asn Cys Glu Gln
100 105 110
Gly Ala Val Pro Thr Ile Met Asn Arg Gly Gly Phe Ala Glu Lys Leu
115 120 125
Arg Ala Asp Trp Gln Trp Val Ile Pro Leu Pro Glu Asn Ile Asp Ile
130 135 140
Glu Ser Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val Phe Lys Pro
145 150 155 160
Leu Leu Met His His Ile Thr Ala Thr Ser Arg Val Gly Val Ile Gly
165 170 175
Ile Gly Gly Leu Gly His Ile Ala Ile Lys Leu Leu His Ala Met Gly
180 185 190
Cys Glu Val Thr Ala Phe Ser Ser Asn Pro Ala Lys Glu Gln Glu Val
195 200 205
Leu Ala Met Gly Ala Asp Lys Val Val Asn Ser Arg Asp Pro Gln Ala
210 215 220
Leu Lys Ala Leu Ala Gly Gln Phe Asp Leu Ile Ile Asn Thr Val Asn
225 230 235 240
Val Ser Leu Asp Trp Gln Pro Tyr Phe Glu Ala Leu Thr Tyr Gly Gly
245 250 255
Asn Phe His Thr Val Gly Ala Val Leu Thr Pro Leu Ser Val Pro Ala
260 265 270
Phe Thr Leu Ile Ala Gly Asp Arg Ser Val Ser Gly Ser Ala Thr Gly
275 280 285
Thr Pro Tyr Glu Leu Arg Lys Leu Met Arg Phe Ala Ala Arg Ser Lys
290 295 300
Val Ala Pro Thr Thr Glu Leu Phe Pro Met Ser Lys Ile Asn Asp Ala
305 310 315 320
Ile Gln His Val Arg Asp Gly Lys Ala Arg Tyr Arg Val Val Leu Lys
325 330 335
Ala Asp Phe
<210> 33
<211> 1206
<212> DNA
<213> 大肠杆菌
<400> 33
atgcgtgaag cctttatttg tgacggaatt cgtacgccaa ttggtcgcta cggcggggca 60
ttatcaagtg ttcgggctga tgatctggct gctatccctt tgcgggaact gctggtgcga 120
aacccgcgtc tcgatgcgga gtgtatcgat gatgtgatcc tcggctgtgc taatcaggcg 180
ggagaagata accgtaacgt agcccggatg gcgactttac tggcggggct gccgcagagt 240
gtttccggca caaccattaa ccgcttgtgt ggttccgggc tggacgcact ggggtttgcc 300
gcacgggcga ttaaagcggg cgatggcgat ttgctgatcg ccggtggcgt ggagtcaatg 360
tcacgggcac cgtttgttat gggcaaggca gccagtgcat tttctcgtca ggctgagatg 420
ttcgatacca ctattggctg gcgatttgtg aacccgctca tggctcagca atttggaact 480
gacagcatgc cggaaacggc agagaatgta gctgaactgt taaaaatctc acgagaagat 540
caagatagtt ttgcgctacg cagtcagcaa cgtacggcaa aagcgcaatc ctcaggcatt 600
ctggctgagg agattgttcc ggttgtgttg aaaaacaaga aaggtgttgt aacagaaata 660
caacatgatg agcatctgcg cccggaaacg acgctggaac agttacgtgg gttaaaagca 720
ccatttcgtg ccaatggggt gattaccgca ggcaatgctt ccggggtgaa tgacggagcc 780
gctgcgttga ttattgccag tgaacagatg gcagcagcgc aaggactgac accgcgggcg 840
cgtatcgtag ccatggcaac cgccggggtg gaaccgcgcc tgatggggct tggtccggtg 900
cctgcaactc gccgggtgct ggaacgcgca gggctgagta ttcacgatat ggacgtgatt 960
gaactgaacg aagcgttcgc ggcccaggcg ttgggtgtac tacgcgaatt ggggctgcct 1020
gatgatgccc cacatgttaa ccccaacgga ggcgctatcg ccttaggcca tccgttggga 1080
atgagtggtg cccgcctggc actggctgcc agccatgagc tgcatcggcg taacggtcgt 1140
tacgcattgt gcaccatgtg catcggtgtc ggtcagggca tcgccatgat tctggagcgt 1200
gtttga 1206
<210> 34
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> paaJ(Ec) pRSFDuet cloning fw
<400> 34
ataaggagat ataccatgcg tgaagccttt atttg 35
<210> 35
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> paaJ(Ec) pRSFDuet cloning rv
<400> 35
tgcggccgca agctttcaaa cacgctccag aatca 35
<210> 36
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> pRSFDuet MCS1 Backbone Fw
<400> 36
aagcttgcgg ccgcataatg cttaa 25
<210> 37
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> pRSFDuet MCS1 Backbone Rv
<400> 37
catggtatat ctccttatta 20
<210> 38
<211> 1428
<212> DNA
<213> 大肠杆菌
<400> 38
atgatgataa atgtgcaaac tgtggcagtg attgggagcg gcaccatggg ggcaggcatt 60
gctgaagttg ctgccagtca tggacaccag gttttactgt atgacatttc tgctgaagcg 120
ctgacccgcg caatcgacgg gatacacgcg cggctaaatt cacgcgtgac gcggggaaaa 180
ctgactgctg aaacctgtga acgcacattg aaacgcctga tcccggtgac cgatattcac 240
gcgctggcag ctgcggacct ggtcattgaa gcggcgtctg aacgtctgga agtcaaaaaa 300
gcgctctttg cacagctggc ggaagtttgc ccgccacaaa cgctattgac cactaacact 360
tcgtcaatct ctataaccgc gattgctgcg gagataaaaa atcctgaacg tgttgcgggg 420
ctgcattttt ttaacccggc accggtgatg aagttggtgg aggtggtcag tgggctggca 480
acggcggcgg aagttgttga gcagttgtgt gaactaacgt tgagttgggg taagcagcct 540
gtgcgctgtc attcgactcc tggatttatc gttaaccgtg ttgcgcgtcc ttattattcc 600
gaggcctggc gggcactgga agagcaggtt gctgcaccag aagtgattga cgctgcactt 660
cgcgatggcg ctggtttccc gatggggccg ctggaattaa ccgatctgat tggtcaggac 720
gtcaattttg ctgtcacctg ttcggtgttt aacgctttct ggcaggagcg tcgtttttta 780
ccttcgctgg tgcaacagga actggtgatt ggtggacggt tgggcaagaa aagtgggctg 840
ggcgtgtacg actggcgcgc ggaacgtgag gcagttgttg gcctggaagc ggtaagcgac 900
agttttagcc caatgaaagt agaaaagaaa agtgacggtg tcacggaaat tgacgatgtt 960
ttattgattg agacacaagg cgagacggca caggcgctgg caatacgact ggcacgcccg 1020
gtggtagtga tcgataaaat ggcgggcaag gtggtgacca ttgctgctgc agcggtgaac 1080
ccggactcag cgacccgcaa ggccatttat tacctgcaac agcagggcaa aacagtgctg 1140
caaattgcag attacccagg aatgctgatt tggcgaacgg tagcaatgat catcaatgaa 1200
gcccttgatg cgcttcaaaa aggcgtggcc tctgaacagg atatcgatac cgccatgcgt 1260
cttggggtga attatccata tggcccactt gcctggggag cgcaacttgg ctggcagcga 1320
atattaaggc tccttgaaaa tctacagcat cactatggcg aagaacgcta tcgcccatgt 1380
tcattgctgc gccaacgggc gcttctggag agcggttatg agtcataa 1428
<210> 39
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> paaH(Ec) pRSFDuet cloning fw
<400> 39
ggagatatac atatgatgat aaatgtgcaa actgt 35
<210> 40
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> paaH(Ec) pRSFDuet cloning rv
<400> 40
agactcgagg gtaccttatg actcataacc gctct 35
<210> 41
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> pRSFDuet MCS2 Backbone Fw
<400> 41
ggtaccctcg agtctggtaa agaaa 25
<210> 42
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> pRSFDuet MCS2 Backbone Rv
<400> 42
catatgtata tctccttctt 20
<210> 43
<211> 768
<212> DNA
<213> 大肠杆菌
<400> 43
atgagcgaac tgatcgtcag ccgtcagcaa cgagtattgt tgctgaccct taaccgtccc 60
gccgcacgta atgcgctaaa taatgccctg ctgatgcaac tggtaaatga actggaagct 120
gcggctaccg ataccagcat ttcggtctgt gtgattaccg gtaatgcacg cttttttgcc 180
gctggggccg atctcaacga aatggcagaa aaagatctcg cggccacctt aaacgataca 240
cgtccgcagc tatgggcgcg attgcaggcc tttaataaac cacttatcgc agccgtcaat 300
ggttacgcgc ttggggcggg ttgcgaactg gcattgttgt gcgatgtggt ggttgccgga 360
gagaacgcgc gttttgggtt gccggaaatc actctcggca tcatgcctgg cgcaggcgga 420
acgcaacgtt taatccgtag tgtcggtaaa tcgttagcca gcaaaatggt gctgagcgga 480
gaaagtatca ccgctcagca agcacagcag gccgggctgg ttagcgacgt cttccccagc 540
gatttaaccc tcgaatacgc cttacagctg gcatcgaaaa tggcacgtca ctcgccgctg 600
gccttacaag cggcaaagca agcgctgcgc cagtcgcagg aagtggcttt gcaagccgga 660
cttgcccagg agcgacagtt attcaccttg ctggcggcaa cagaagatcg tcatgaaggc 720
atctccgctt tcttacaaaa acgcacgccc gactttaaag gacgctaa 768
<210> 44
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> paaF(Ec) pETDuet cloning fw
<400> 44
aggagatata ccatgagcga actgatcgtc agccg 35
<210> 45
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> paaF(Ec) pETDuet cloning rv
<400> 45
tgcggccgca agcttttagc gtcctttaaa gtcgg 35
<210> 46
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 46
ttaacgtcct gcataggtgg gttt 24
<210> 47
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> paaF(L3)opt pETDuet cloning fw
<400> 47
agaaggagat ataccatgca gaccattgaa aatga 35
<210> 48
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> paaF(L3)opt pETDuet cloning rv
<400> 48
tgcggccgca agcttttaac gaccggcata ggtcg 35
<210> 49
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> pETDuet MCS1 Backbone fw
<400> 49
aagcttgcgg ccgcataatg cttaa 25
<210> 50
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> pETDuet MCS1 Backbone rv
<400> 50
catggtatat ctccttctta aagtt 25
<210> 51
<211> 1155
<212> DNA
<213> 贝氏不动杆菌
<400> 51
atgattcgtg atgaaggtat gctgcaacag ctgctgagca ccattcgtga ttttgttaaa 60
aatgaactga ttccgcgtga acatgaagtg gcagaaaaag attgtattcc cgaagatatt 120
attcagcaga tgcgtgaact gggtctgttt ggtctgacca ttccggaaga atatggtggt 180
ctgggtatta ccatggaaga agaagttaac gttgcatttg aactgggcca gaccagtccg 240
gcatttcgta gcctgattgg caccaataat ggtattggta gcagcggtct gattattgat 300
ggcaccgaag aacagaaaca gaaatatctg cctcgttatg caagcggtga aattattggt 360
agtttttgtc tgaccgaacc ggaagcaggt agtgatgcag caagcctgaa aaccaccgca 420
gttaaagatg gtgatttcta tattctgaac ggcaccaaac gctttattac caatgcaccg 480
catgcagcaa cctttaccgt tatggcacgt accaatccgg caattaaagg tgccggtggt 540
attagcgcat ttctggttga agcaaataca ccgggtatta cgctgggtaa aattgatcag 600
aaaatgggtc agaaaggtag ccatacctgt gatgtgattt ttgaaaattg tcgtgttccg 660
gcaagcgcac tgattggtgg tgttgaaggt gttggcttta aaaccgcaat gaaagttctg 720
gataaaggtc gtctgcatat tggtgcatat agcgttggtg ttgcagaacg tatgctgaat 780
gatgcactgc attatgccgt tgaacgtaaa cagtttggtc agccgattgc aaattttcag 840
ctgattcagg caatgctggc agatagcaaa gcagaaattt atgcagccaa atgcatggtt 900
ctggatgcag cacgtcgtcg tgatgagggt cagaatatta gcaccgaagc aagctgtgca 960
aaaatgtttg caaccgaaat gtgtggtcgt gttgccgatc gttgtgttca gattcatggt 1020
ggtgcaggtt atattagcga atacagcatt gaacgcttct atcgtgatgt gcgtctgttt 1080
cgcctgtatg aaggtacaac ccaggtgcag cagattatta tcgccaaaaa catgatcaaa 1140
gaggtgacca gctaa 1155
<210> 52
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> dcaA(Ab)opt pETDuet cloning fw
<400> 52
gaaggagata tacatatgat tcgtgatgaa ggtat 35
<210> 53
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> dcaA(Ab)opt pETDuet cloning rv
<400> 53
tggcagcagc ctaggttagc tggtcacctc tttga 35
<210> 54
<211> 1095
<212> DNA
<213> 热带念珠菌
<400> 54
atgattaccg cacaggcagt tctgtatacc cagcatggtg aaccgaaaga tgtgctgttt 60
acccagagct ttgaaatcga tgatgataat ctggcaccga acgaagttat tgttaaaacc 120
ctgggtagtc cggttaatcc gagcgatatt aatcagattc agggtgttta tccgagcaaa 180
ccggcaaaaa ccaccggttt tggcaccacc gaaccggcag caccgtgtgg taatgaaggt 240
ctgtttgaag ttattaaagt gggcagcaat gttagcagcc tggaagccgg tgattgggtt 300
attccgagcc atgtgaattt tggtacatgg cgtacccatg cactgggcaa tgatgatgat 360
tttatcaaac tgccgaatcc ggcacagagc aaagcaaatg gtaaaccgaa tggcctgacc 420
attaatcagg gtgcaaccat tagcgttaat ccgctgaccg catatctgat gctgacccat 480
tatgttaaac tgacaccggg taaagattgg tttattcaga atggtggcac cagcgcagtt 540
ggtaaatatg caagccagat tggtaaactg ctgaacttta atagcattag cgtgattcgt 600
gatcgtccga atctggatga agttgttgca agcctgaaag aactgggtgc gacccaggtt 660
attaccgaag atcagaataa tagccgtgaa tttggtccga ccattaaaga atggattaaa 720
cagagcggtg gtgaagcaaa actggcactg aattgtgttg gtggtaaaag cagcaccggt 780
attgcacgta aactgaataa taacggtctg atgttaacct atggtggcat gagctttcag 840
ccggttacca ttccgaccag cctgtatatc tttaaaaact ttaccagtgc cggtttttgg 900
gttaccgagc tgctgaaaaa taacaaagaa ctgaaaacca gcacgctgaa ccagattatt 960
gcatggtatg aagagggtaa actgaccgat gcaaaaagca ttgaaaccct gtatgatggc 1020
accaaaccgc tgcatgaact gtatcaggat ggtgttgcaa atagcaaaga tggcaaacag 1080
ctgatcacct actaa 1095
<210> 55
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(Ct)opt N22AAcut pETDuet cloning fw
<400> 55
gaaggagata tacatatgat taccgcacag gcagt 35
<210> 56
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(Ct)opt N22AAcut pETDuet cloning rv
<400> 56
tggcagcagc ctaggttagt aggtgatcag ctgtt 35
<210> 57
<211> 1167
<212> DNA
<213> 耳念珠菌
<400> 57
atgtttgttc caaccgttgc tgggaaacgc tttctgacca atcagctgcg caaaagtccg 60
cagttcaccc gtatgctgac ttcacacgcg gtgattttca gctctcatgg ggaacccaaa 120
gacgttctga aaacgcatac gtacgaaatc gatgagaaca acatcaaacc caacgagatt 180
gtcctgaaaa ccttgggtgc tccggtcaat ccatcggaca ttaaccaggt gcagggtgta 240
tatcctagcc aaccggagaa aacgacccaa ctgggaactt cggaaccgtc agccgttggt 300
ggcaatgaag ggctctttga gatcatcaaa attggcagtg atgtgaaagg ctttcaagtt 360
ggtgattggg ccattccgac ctcggtcaac tttggtacgt ggcgtagtca tgcgctgtgc 420
gaagaaggca aaatgatgaa agtcccgaac ccggaacagt ctaaacaggc cggtaagaaa 480
cagggcttaa ccatcaatca aggtgcgacc ttaagcgtga atccgcttac agcctacctt 540
atgctcacac actacgtgaa actcacaccg ggtaaggact ggtttatcca gaacggtggc 600
aatagcgcag tcggaaaatt tgcgagccaa attgggaaat tactggggat tcatagcatt 660
tccgtggttc gcgatcgtcc tgaaattgaa gaactgaaga aagagctgaa ggaagtatgt 720
ggtgcgactc aggtaatcac cgaagagcag aacaactcca aggacttcgg ccctcaggtg 780
aagcagtggg tgaaagaaac tggcggcgag atcaaactgg cgctgaattg cgtaggtggc 840
aaaagcagtg caggcgttgc acggaaactc gctcccaacg gactgatgct gacatatggc 900
ggcatgtcat ttcaaccggt aaccttgcca acgagcttgc acattttcaa gaatgtgacg 960
tctgcgggct tctgggtgac ggaactgctg aaatctgatc cgaacctgaa acgcgaaacc 1020
gttgacaaaa ttgtcgagtg gtatacctca ggcgaattac ttgatgcgcc atcgaaagaa 1080
aatcagtggg gtgatcaaga tctggcacaa gtgttcattg atgccgtcgc caactccaaa 1140
tccggaaaac agttggtgaa gttttaa 1167
<210> 58
<211> 1143
<212> DNA
<213> 马克斯克鲁维酵母
<400> 58
atgagttcat ttctcagcaa acgcttcatt agcacgacgc aacgtgccat gagccaattg 60
ccgaaagcca aatcgctgat ctactcatcc catgatcagg acgtgagtaa aatcctgaaa 120
gtgcatactt accagccgaa agggtcagcg gaaagctcga tcctgctcaa aacgttagcc 180
tttccgatca atccttccga catcaaccaa ctggaagggg tatatccgag taagccggaa 240
aaggtgttgg attactcaac ggaagaaccg agcgctattg ctggcaatga gggtctgttt 300
gaggttgtat cggtacctag cggtgtcaag aatctgaaag ctggtgaccg tgtcattcca 360
cttcaggcga actttggcac ctggtctacg tatcgcacat gcgaaagcga gaatgacttg 420
atcaaaatcg aaggcgtaga tctctatacc gcagcaacca ttgcggtgaa tggctgtacc 480
gcgtaccaaa tggtgaatga ctacattgag tgggatccta gtggcaacga ttggctggtt 540
cagaacgcgg gtacaagttc tgtatccaaa attgtgaccc agatcgctaa agacaagggc 600
atcaaaaccc tgtcggtggt tcgtgatcgc gataactttg acgaagttgc ggagaaactg 660
gaaaagaaat atggtgcgac caaagtgatt tctgaaagcc agaacggaga acgtgagttc 720
gggaatgaag tcttaccgaa gattcttggt cccaatgccc aggttaaact cgcgttaaac 780
tctgtgggcg gtaaaagctg cactaacatt gcccgcaaac tgagtccaaa cggattaatg 840
ctgacctatg gcggcatgtc caaacagccg gttacactgc caactggcct gtttatcttc 900
aatagcattc ggtcgcacgg tttctgggtt accgcaaatt cgaaacgcga tcccgaaaac 960
aaacggaaaa ctgtggatgc cgtggtcaaa ctgtatcgcg atggcaagat tatttccccg 1020
aaagaggaca ttcgtaccct ggaatgggat gtcaacaatc tgtctgatga aggtgtcctt 1080
gatttggtca accgcggaat tgcaacgaaa ggggcaaaga acatggttgt gctgaaatgg 1140
taa 1143
<210> 59
<211> 1122
<212> DNA
<213> 库德里阿兹威毕赤酵母
<400> 59
atgagtctgc gttccgtgtt tagcgtgagt tccattcgtc ggatgtcctc tatcacggct 60
cgcgcaatcg tctataacgc gcatggcgaa ccgaaagata ccctgaaatg ccacagctac 120
aagatcgacc ttcagaattt acagcctaac gaggttgtcg tacaaactct cgcagcgcca 180
ctgaacccga gtgatatcaa ccagattcag ggtgtttacc cgtctaagcc tccgctgagc 240
attgactcac tgccagaact gaaagaaccg gccgctgtta ctggcaatga gggtgtgttc 300
aaagtgattg ccaaagggtc gaatgtagga gaactggaaa cgggagactg gtgtattccg 360
tctggcgtaa actttggtac ctggtgtacg caccgcttaa ccacggataa gaatctgctg 420
aaaatgccga aaaccatctc gttaaaccag gcagcaacca ttgccgtcaa cccctcatcg 480
gcatatcaga tgctgacgca ctttgtgaaa ctgcaaccag gtgattggtt tatccagaat 540
ggcgccaatt cccaagttgg tcgtgcagcg attcagattg gccataaact ggggtacaaa 600
tcgttgaaca ttgttcgcaa ccgtgacaac ttggatgagc tggtgaagga cctggaatcg 660
attggggcga ctaaagtgat tacagaagat gaaagcgcga gcaaagagtt tggcgctacg 720
gttaaagagt ggcttgaggg caaagagatc aaactcggtc tgaattgcat tggtggtccg 780
tctgcgtcaa acttagcccg caaattgggc catgatgcta ccatcctgac gtatggcgcg 840
atgagcaaga aaccgctctc attgccgact gggaatttca tctttaagaa tctcaccgcc 900
aaaggatttt ggattacagg taacatcaaa cgccatccag aactgcgcat cgaaaccatt 960
aatgcggtcc tgaaattcat ggaagatggc acccttaagg aacctaaagt gaacgaaccc 1020
aaagtcaaac tgagtaccac aacaaatgaa agcttccttg aaaccgtgct ggccgcgttg 1080
gaagatagcc aaaagggcaa acaactgtta aaattcgaat aa 1122
<210> 60
<211> 1347
<212> DNA
<213> 喜热梭孢壳
<400> 60
atggcgtcag tcttatcacc aaacgcagca gcacgtcatc tgctgcgtcc tgctacccgg 60
tctacaccgc ctcgtttact gccgtccgca atcttacacc aaagcggcaa taactccacg 120
cgcaatccgc gcattagcga tccgcagtca attgctcaac ggttaggtcg tcgctacaaa 180
tcgggcccgt acggctatac gcaggccaaa acgctggtgt tttcgcgttt cggagaaccc 240
tccgatgttc tgcgcctgca tacgcatagc atcagtccga cactgccgga tggtgcggtt 300
ctggtgcggg cactggcagc tccagtaaac ccggcggatg tcaacactat ccaaggcacc 360
tatggtgcgc gtccagcttt cagcccctta ctcggcactc ccgaacctag tgcggttcca 420
ggcaacgaag gttgctttga agttgtcgca gttggtccac gtgtcggcgg cggtctgcgt 480
aaaggggatt gggtgattcc ggccaccacc ggctttggca cctttcgcac acacgcgttg 540
gtggagaatg ccgatcgcgc tctgcttcgc gtaggaggtg acaaaggtac cgcgggtctg 600
accgccaaac aggtggccac ggtaagcgtc aatccgtgtt cggcctatcg tatgctcaag 660
gactacgtcg atctggtgga cttgagtgtg aagtcctttg ctcgcggtga tggggccact 720
ggcggggcgt ggtttctcca gaatggcgcg aatagcggag ttggtcgcgc ggcgattcag 780
ctgggccgcc tctggggtct gcgctcgatt aacgtggttc gtgaacgcgc taccgccgaa 840
gaaaccgaag cattgaaaag cgaactgcgt gagcttggtg cgacggttgt tgtgaccgaa 900
gcggaattcc ttgatcgctc tttctcggcc cgcttgaaag aagagtggac acgcggcgat 960
cgcgaaccgg tgatgctggg tttgaactgt gtaggcggga aaagcgcgag cgccatgatc 1020
aaggccctgt ctccgaaagg ttgcatggtg acttatggcg ggatgagtcg tcagtcattc 1080
ccctttccga ctggtcctca gatctttaaa cgcctgcgtt ttgagggatt ctggctgagt 1140
gaatgggcca aagagaatcc ggcggagaaa cgcaacacca ttaatgagat tctggagctg 1200
atgcgggaag gcaaattcaa agagtctccg ttcaaagaag tggaatggaa ctgggacacg 1260
gaagaaaagg tactgaaaga cgcaattcaa ggcacccttg aagggttcaa gtccgggaaa 1320
ggattatttg tctttggcga cacgtaa 1347
<210> 61
<211> 1332
<212> DNA
<213> 太瑞斯梭孢壳霉
<400> 61
atggccagcg ccttgaatcc tcgccatctg ccgctgcgct tagccttacg tcctcgcttg 60
ccactgctgc catccacgag ctctaccacc acaaccacaa ccccaccctc gtgtctgccg 120
attccgacct ctattcgtct gcctccccgt cgtcacaaat cagggccgta tggttacacg 180
cttgcgaaaa ccttagtgtt tagtcgcttt ggtgaacccc gcgatgtact gtcgcttcat 240
acccacagca ttagccccgc actgccggat ggggcggctc tgttacgcgc tcttgcagcg 300
ccggtcaatc cggcggacgt aaacaccgtc caagggacct atggcgccaa accggcgttt 360
gagcgtctgc tgggtactcc ggaaccggct gcagtaccgg gcaatgaggg ttgcttcgaa 420
gtcgttgcca ttggtggagg tgccggtggt ggcggtggtc ttaagaaagg tgactgggtt 480
attcctgcac agagtggctt cggcaccttc cgcacacatg cgttggtgga aggcgcagaa 540
cgcaaactga ttcgtgtggg aggcgccaaa gggcgtgaag gtctgcgtgc tgcgcaagtt 600
gcgacggtgt ctgtgaatcc atgtagcgca tatcggatgc tccgtgatta cgtagactta 660
gtggacctga gcgtgcagtc atttgctcgc ggtgatggcg caaccggcgg cgcgtggttc 720
gtgcagaacg gagccaacag tggcgttggt cgtgcggcta ttcaactggg acgtctgtgg 780
ggcttgcgtt cgatcaacgt ggttcgggaa cgtgcgactc cggaagaaac cgcggcctta 840
aaacgcgaac tcgccgagtt gggtgcaacg gtagtcgtta cggaatccga attcctcgat 900
cgctcgtttg ctgaccggct gcgcgatgag tggactcgcg gcggccgcga accggtgatg 960
ctcggtctga actgcgttgg cgggaaatct gccgcagcca tggtcaaagc gctgagtcca 1020
cgcggatgca tggtcacata cggcggtatg agccgccagt ccttcccgtt tccgactggc 1080
cagcagatct ttaagcgcct gcgctttgag ggcttttggc tgtcagagtg ggcgaaagag 1140
aatcctgcgg cgaaacggga taccatcaac gagattctgg aattgatgcg tgaaggcaaa 1200
ttcaaggaag caccgctcca agaagttgaa tggaactggg atacggaaga atccgtgctg 1260
aaagacacga tccaggggac tctggagggg tttcgcccag gcaaaagcat cttcgtcttt 1320
aaggatacgt aa 1332
<210> 62
<211> 1320
<212> DNA
<213> 嗜热毛壳菌
<400> 62
atggctagca tcctgtcccc tcattctgct ctccgtttgg gaggaatctc cacgctgtca 60
cgtcagaccc tgctgcgtac ctctcgtgtg accttaaccc agttaccgaa aaccgctccg 120
cagttaccgc aacgtcggca taaaagtgcg ccttacggct atacccagtc caaaacgctg 180
gtgtttccgc gtttcgggga gccgattgac gttctgtcgt tacacactca ctcgattagc 240
ccgacccttc ccgatactgc ggtgctgctt cgcacgctgg cggcgcccat taatcctgcg 300
gacgtcaaca cgattcaggg gacctatggc gcaaagccga cgttcagcaa cttactgggc 360
accgcagagc ctgcagcagt tccggggaat gaaggtgtat ttgaggtcgt ttcggttggc 420
tcggaaattg caaaacgtgg cgtgttcaag aaaggtgatt gggtcattcc gagcagtagc 480
ggctttggca cttttcgcac ccatgtactg gttgaagaag cggagcagaa actgtggcgc 540
attggcggcg agaaaggcac cgaaggttta acgccagtac aggtcgcaac ggtttcggtg 600
aatccgtgtt ctgcgtatcg catgctgcgt gactatgtgg atctggtggg cgtgagcgta 660
cgcatgtacc aggaaggtgg gtcagatgtc cgtggtggag cgtggttcct gcaaaatggc 720
gccaactcag gtgttggacg cgctgccatt caactcgggc gtttgtgggg tttgcgctca 780
atcaacgtgg tacgggagcg cgcgactgcc gaagaaacag aagccctgaa gaaagagctg 840
tacgatcttg gggccactgt ggtggtgacc gaatccgaat tcctggatcg cagttttaca 900
cagcgcctga atgaggaatg gacacgggga ggtaaagagc cattgctgct tgccctgaac 960
tgcgtgggtg gcaaaagtgc ccaacagatt gttcgcgctc tgtctccgaa aggcactatg 1020
gtcacgtatg gcggtatgag ccgccaaagc tttccgtttc cgacgggtcc acagatcttt 1080
aaacgtctgc gctttgaagg tttctggctc tccgaatggg ccaaagaaaa ccccgcagaa 1140
aagaagaaat gcgtagatga gatcatcgaa ctgatgcgcg aaggcaaatt caaagaagcg 1200
ccagtccagg aaattcgctg ggattgggaa accgaagaaa aggtcttgaa agaggcggtt 1260
caaggcacac tcgaaggctt tcgcagtggt aaaggtgtgt ttatcttcgg tgacacctaa 1320
<210> 63
<211> 1269
<212> DNA
<213> 丝状真菌柄孢霉
<400> 63
atggcagctc cgcgtctgac ctctaaagcc ttacgcttac cgacgagcaa actcttgcgt 60
ccagcgctga caactgctac gcctcgcgca gccccaacct tgacccagaa acgtcatctg 120
actggtccat atggctacca ccagagcaaa gcgctgacct tctccagctt tggcgaaccg 180
attgacgtgc tctcactgca tacgcactcg attagtccga cattaccctc aggtagtgtc 240
ttagtgcgta ctctcgcggc accggtaaac ccggcagacg ttaacaccat tcaagggacc 300
tacggatcca agccgccctt tacgacgctg ttgggtacgg cccaaccgtc ggcggttccg 360
ggcaatgaag cgtgctttga ggtgctgtct gtgggccaag gggtgaaagg gctcgaaaag 420
ggcgattggg tcattccagc caagactggg ttcggcacct ttcggactca tgcccttgtg 480
gaagaagctg aggggaaact gatgcgtgta gaacgcgaag gcttgacgcc ggttcaggta 540
gccaccgtta gcgtcaaccc ttgttcggcg tatcgcatgc tgaaagacta cgtggatctg 600
gttggcctgt ccatgcgctg gtatcgcgaa ggtaaagatg tgagcggtgg cgcctggttc 660
ctgcagaatg gcgcgaactc tggagttggc cgtgcggccg tgcaatttgg ccgtttgtgg 720
ggtttacgca gcatcaacgt tgtacgcgaa cgtgaaacac ctgaagaaac cgaaaagtta 780
aaagaagaac tgacgggttt gggtgcgaac gtcgtcctga ccgagcagga atttcttgat 840
cgctcttttc gcgatcggct gggtgagctg acgaaacgcg gtaaagaacc cctgctgctt 900
ggtatgaatt gtgtgggcgg aaaatccgca tcagcggtag tcaaagctct ctcgccaaaa 960
ggctgcatgg tcacctatgg cggtatgagt cgtcagagct tcccgtttcc tactggtccg 1020
cagatcttca agcgtctgcg cttcgaaggc ttctggcttt cagagtgggg taaagagaat 1080
ccggaaggga agaagaaaat gatcgaagat attctgaacc tgatgcgcga agggaaattc 1140
aaagaaagcc cggttcaaga ggtggagtgg aattgggaaa ccgaggagaa aaccctgaaa 1200
gaggcagttc agggaacact gggtggcttt cgcagtggca aaggcgtgtt tgtgtttgga 1260
gaaacctaa 1269
<210> 64
<211> 1275
<212> DNA
<213> 淡紫拟青霉菌
<400> 64
atggcgagta gtcgcctgct cggaccacca ctgcgcctcc caagctcggg attcagccgc 60
ccggttgctg ccctgaattg tcgtgcggca gcactgccct tgcatctgcc acgtacagcg 120
gtccggtatc gttcggggcc ttacggttac actcaggcga aggcccttgt gtacagcaag 180
aacggcgaac cggcagatgt gctgaagtta cacacgcaca gtatttcccc ttcaattccg 240
tccacctccg ttctggtacg ctgcctggcc gctccgatta acccggctga tgtcaacacg 300
attcagggga cctatggcag caaacaaccg ctcacgagct taattgggac cagtgaaccc 360
tccgcagttc cgggcaatga aggcgtcttt gaagttctct cagccggttc cccgagcagc 420
gagctgaaga agggagattg ggtgatccct gccgcatctc agattggcac atggcgcacc 480
catgcggtgt ttggggctga cgagctcttg aaagtcgaca aagagggtct gacgccgact 540
caagtttctc ttgtgtcggt taacccgtgt acggcgtatc gcatcctgcg gagctatggt 600
ccctcagctg gcgtgaaagc ggcgttaggc atgcgcccgt tagaggtggg tagtggacag 660
tggtttatcc agaatggcgc gaattctggc gttggccgtg cagccattca gtttggcaaa 720
ctgtggggct tgcgctcgat caacgtcatt cgcgaccgtg acaccgtcga agccacagac 780
gcactgaaac aggagttgcg ggatttgggc gcggatgtcg tagttgctga atctgagttc 840
ctgtctcgtg gttggaaaga ccaactggcg gaaattaccc gcaaagggcg tgaacaggta 900
gggcttggcc tgaattgcgt gggtggcaaa tcagccacgg cacttgcgcg tagcctgggt 960
gaaggtgcca ctctggtaag ctatggtggt atggcgaaac aaccggtggc gttacctgtc 1020
ggtctgttga tcttcaaaga tatccgcttc gttggctttt ggctgtcgaa atggaaccag 1080
caggatgtgg tgggtcgtcg ccatatggta gcagacatcc tgggactgat gcgtgacggt 1140
aaattccgcg atgccccggt ggatgaagtg aaatgggatt gggataccga tgaagcgacc 1200
ctgcgtgggg ctgcccaacg cggcttagaa ggttttcgcc aaggtaaagg cgtatacgtg 1260
tttggcgata cctaa 1275
<210> 65
<211> 1218
<212> DNA
<213> 圆核腔菌
<400> 65
atggcgtcta aactgatctc gcctagtccg cgcctcgcac agagctgttt acgccgcact 60
cgtaatttgg ccccggtgac tgcgcgcttt ccccaacgcc gctacatctc tgcctatggg 120
tatgaacagg caaaagctct gacgtttacc gaatacgggg atccatctgc ggtgctgagc 180
ttgcacagcc actccatctc gccgcctcat agcaactata tgaccctgcg cttcctggcg 240
agcccgatca atccagcaga cattaaccaa attcagggcg tttatccgtc caaacccact 300
tttacgacgt cgctgggtac tccgaaccca attgccgtcg ctggcaatga aggcgtggcg 360
gaaattattg cgctgggtga aggtgttaag aaagaaggct tcaagaaagg cgattgggtc 420
tttatgaaag gcccgggctt tggcacatgg cgtacccatg ccagtgcaac taccaacgat 480
gtcgtcaagc tgaatgacca gatgcgggaa ggtatcacgg ccattcaagc gggcaccgtt 540
tcaattaatc cgtgcaccgc ttatcgtatg cttcgggatt ttaccaccct gagcgaagga 600
gattggttta tccagaatgg cgccaattca ggcgtgggtc gtgcagcaat tcagctgggt 660
cgcaaatggg gatacaaatc cattaacatt attcgcagtc gtgaggataa gaacaaagaa 720
gaggcgatga agaaagagct gcataacctc ggggccgatg tggtaattac ggatgcagag 780
ttacaagcgc aagggatcaa agaccaggcc aaagagtgga caaacggtgg acgtagccct 840
attcgccttg cgttaaactg cgtgaatggc aaagctgcta ccgcgatggc gaaactgctg 900
tcgagttcag cccatttcgt aacctatggt gcgatgtcca agcagccgtt gaccatccca 960
gccagcatgc ttattttcaa agatatccac tttcatggtt tctgggtgtc tcgctgggca 1020
gaagaacacc cggaagagaa acagaaaacg gttgcggatg tcctcgacat gacacgtaag 1080
ggagagttca aagacatgcc ggttgatgaa atcaaatggg aatgggaaac aaaaggggag 1140
gaactggttg ctaaagtgaa agacacgtta gaaggttacc gtgacggtaa aggcatcttc 1200
gtatttggca aaacctaa 1218
<210> 66
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(caur) pETDuet cloning fw
<400> 66
gaaggagata tacatatgtt tgttccaacc gttgc 35
<210> 67
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(caur) pETDuet cloning rv
<400> 67
tggcagcagc ctaggttaaa acttcaccaa ctgtt 35
<210> 68
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(kmx) pETDuet cloning fw
<400> 68
gaaggagata tacatatgag ttcatttctc agcaa 35
<210> 69
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(kmx) pETDuet rv
<400> 69
tggcagcagc ctaggttacc atttcagcac aacca 35
<210> 70
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(pkz) pETDuet cloning fw
<400> 70
gaaggagata tacatatgag tctgcgttcc gtgtt 35
<210> 71
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(pkz) pETDuet cloning rv
<400> 71
tggcagcagc ctaggttatt cgaattttaa cagtt 35
<210> 72
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(mtm) pETDuet cloning fw
<400> 72
gaaggagata tacatatggc gtcagtctta tcacc 35
<210> 73
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(mtm) pETDuet cloning rv
<400> 73
tggcagcagc ctaggttacg tgtcgccaaa gacaa 35
<210> 74
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(ttt) pETDuet cloning fw
<400> 74
gaaggagata tacatatggc cagcgccttg aatcc 35
<210> 75
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(ttt) pETDuet Ecloning rv
<400> 75
tggcagcagc ctaggttacg tatccttaaa gacga 35
<210> 76
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(cthr) pETDuet cloning fw
<400> 76
gaaggagata tacatatggc tagcatcctg tcccc 35
<210> 77
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(cthr) pETDuet cloning rv
<400> 77
tggcagcagc ctaggttagg tgtcaccgaa gataa 35
<210> 78
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(pan) pETDuet cloning fw
<400> 78
gaaggagata tacatatggc agctccgcgt ctgac 35
<210> 79
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(pan) pETDuet cloning rv
<400> 79
tggcagcagc ctaggttagg tttctccaaa cacaa 35
<210> 80
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(plj) pETDuet cloning fw
<400> 80
gaaggagata tacatatggc gagtagtcgc ctgct 35
<210> 81
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(plj) pETDuet cloning rv
<400> 81
tggcagcagc ctaggttagg tatcgccaaa cacgt 35
<210> 82
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(pte) pETDuet Ecloning fw
<400> 82
gaaggagata tacatatggc gtctaaactg atctc 35
<210> 83
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ter(pte) pETDuet cloning rv
<400> 83
tggcagcagc ctaggttagg ttttgccaaa tacga 35
<210> 84
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> pETDuet MCS2 Backbone Fw
<400> 84
cctaggctgc tgccaccgct gagca 25
<210> 85
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> pETDuet MCS2 Backbone Rv
<400> 85
catatgtata tctccttctt atact 25
<210> 86
<211> 3537
<212> DNA
<213> 人工序列
<220>
<223> Mab4714opt W283RA303A
<400> 86
atgaccgaaa ccattagcac cgcagcagtt ccgaccaccg atctggaaga acaggttaaa 60
cgtcgtattg aacaggttgt tagcaatgat ccgcagctgg cagccctgct gccggaagat 120
agcgttaccg aagcagttaa tgaaccggat ctgccgctgg ttgaagttat tcgtcgtctg 180
ctggaaggtt atggtgatcg tccggcactg ggtcagcgtg catttgaatt tgttaccggt 240
gatgatggtg caaccgttat tgcactgaaa ccggaataca ccaccgttag ctatcgtgaa 300
ctgtgggaac gtgccgaagc aattgcagca gcatggcatg aacagggtat tcgtgatggt 360
gattttgttg cacagctggg ttttaccagc accgattttg caagcctgga tgttgcaggt 420
ctgcgtctgg gtacagttag cgttccgctg cagaccggtg ccagcctgca gcagcgtaat 480
gcaatcctgg aagaaacccg tccggcagtt tttgcagcaa gcattgaata tctggatgca 540
gcagttgata gcgttctggc aaccccgagc gtgcgtctgc tgagcgtttt tgattatcat 600
gccgaagtgg atagccagcg tgaagcactg gaagcagttc gtgcacgtct ggaaagcgca 660
ggtcgtacca ttgttgttga agccctggcc gaagcgctgg cacgtggtcg tgatctgcca 720
gcagcaccgc tgccgagtgc cgatccggat gcactgcgcc tgctgattta taccagcggt 780
agcaccggta caccgaaagg tgccatgtat ccgcagtggc tggttgcaaa tctgtggcag 840
aaaaaaaggc tgaccgatga tgttattccg agcattggtg tgaattttat gccgatgagc 900
catctgatgg gtcgtctgac cctgatgggc accctgagcg gtggtggcac cgcctattat 960
atcgcaagca gcgatctgag cacctttttt gaagatattg ccctgattcg tccgagcgaa 1020
gttctgtttg ttccgcgtgt tgtggaaatg gtttttcagc gttttcaggc agaactggat 1080
cgtagcctgg ctccgggtga aagcaatagc gaaattgcag aacgtattaa agtgcgtatt 1140
cgcgaacagg attttggtgg tcgtgtgctg agcgcaggta gcggtagtgc accgctgagt 1200
ccggaaatga ccgaatttat ggaaagcctg ctgcaagtgc cgctgcgtga tggttatggt 1260
agtaccgaag ccggtggtgt ttggcgtgat ggcgttctgc agcgtccgcc tgttaccgat 1320
tataaactgg ttgatgtgcc ggaactgggt tattttacca ccgatagtcc gcatccgcgt 1380
ggtgaactgc gtctgaaaag cgaaaccatg tttccgggtt attacaaacg tccggaaacc 1440
accgcagatg tttttgatga tgaaggctat tacaaaacgg gtgatgtggt tgctgaactg 1500
ggtcctgatc atctgaaata cctggatcgt gttaaaaacg ttctgaaact ggcacagggt 1560
gaatttgtgg cagttagcaa actggaagcc gcatataccg gtagtccgct ggttcgtcag 1620
atttttgttt atggtaatag cgaacgtagc tttctgctgg ccgttgttgt tccgacaccg 1680
gaagttctgg aacgttatgc agatagtccg gatgcgctga aaccgctgat tcaggatagt 1740
ctgcagcagg ttgcaaaaga tgcagaactg cagagctatg aaattccgcg tgattttatt 1800
gttgaaaccg ttccgtttac cgttgaaagc ggactgctga gtgatgcacg taaactgtta 1860
cgtccgaaac tgaaagatca ttatggtgaa cgcctggaag ccctgtatgc cgaactggca 1920
gaaagccaga atgaacgtct gcgtcagctg gcacgcgaag cagcaacacg tccggttctg 1980
gaaaccgtta ccgatgccgc agccgcactg ctgggtgcaa gcagctccga tctggcacca 2040
gatgttcgtt ttattgattt aggtggtgat agcctgagcg cactgagcta tagcgagctg 2100
ctgcgcgata tttttgaagt tgatgttccg gttggtgtga ttaatagcgt tgcaaatgat 2160
ctggcagcaa ttgcccgtca tattgaagca cagcgtacag gtgcagcaac ccagccgacc 2220
tttgcaagcg ttcatggtaa agatgccacc gttattaccg caggcgaact gaccctggat 2280
aaatttctgg atgaaagtct gctgaaagca gccaaagatg ttcagcctgc gacagcagat 2340
gttaaaaccg tgctggtgac cggtggtaat ggctggttag gtcgttggct ggttctggat 2400
tggctggaac gtctggcacc gaatggtggt aaagtttatg cactgattcg tggtgcagat 2460
gcggaagcag cacgcgcacg cctggatgcc gtttatgaaa gcggtgatcc taaactgagt 2520
gcacattatc gtcaactggc ccagcagagc ctggaagtta ttgcaggcga ttttggcgat 2580
caggatctgg gtctgagcca agaagtttgg cagaaactgg cgaaagatgt tgatctgatt 2640
gttcatagcg gtgccctggt taatcatgtt ctgccgtata gccagctgtt tggtccgaat 2700
gttgccggta cagcagaaat tatcaaactg gcaattagcg aacgcctgaa acctgttacc 2760
tatctgagta ccgttggtat tgcagatcag attccggtta ccgaatttga agaggatagt 2820
gatgttcgcg ttatgagcgc agaacgtcag attaatgatg gctatgcaaa tggctatggc 2880
aatagcaaat gggctggtga agttctgctg cgtgaagccc atgatttagc cggtctgccg 2940
gttcgtgttt ttcgtagcga tatgattctg gcacatagcg attatcacgg tcagctgaat 3000
gtgaccgatg tttttacccg tagcattcag agtctgctgc tgacaggtgt tgcaccggca 3060
agcttttatg aactggatgc ggatggtaat cgccagcgtg cccattatga tggtgttcca 3120
ggtgatttta ccgcagccag cattaccgca attggtggtg ttaatgttgt ggatggttat 3180
cgcagctttg atgtgtttaa tccgcatcac gatggtgtta gcatggatac ctttgttgat 3240
tggctgattg atgccggtta caaaattgca cgcatcgatg attatgatca gtggttagca 3300
cgttttgaac tggccctgaa aggcctgcct gaacagcagc gtcagcagag cgttctgcca 3360
ctgctgaaaa tgtatgaaaa accgcagcct gcaattgatg gtagcgcact gccgaccgca 3420
gaatttagcc gtgcagttca tgaagcaaaa gtgggtgata gtggtgaaat cccgcatgtt 3480
accaaagaac tgattctgaa atatgccagc gatattcagc tgctgggttt agtttaa 3537
<210> 87
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> pACYCDuet Mab4714opt cloning fw
<400> 87
gaaggagata tacatatgac cgaaaccatt agcac 35
<210> 88
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> pACYCDuet Mab4714opt cloning rv
<400> 88
gtggcagcag cctagttaaa ctaaacccag cagct 35
<210> 89
<211> 669
<212> DNA
<213> 艾阿华诺卡氏菌
<400> 89
atgatcgaga caattttgcc tgctggtgtc gagtcggctg agctgctgga gtatccggag 60
gacctgaagg cgcatccggc ggaggagcat ctcatcgcga agtcggtgga gaagcggcgc 120
cgggacttca tcggggccag gcattgtgcc cggctggcgc tggctgagct cggcgagccg 180
ccggtggcga tcggcaaagg ggagcggggt gcgccgatct ggccgcgcgg cgtcgtcggc 240
agcctcaccc attgcgacgg atatcgggcc gcggcggtgg cgcacaagat gcgcttccgt 300
tcgatcggca tcgatgccga gccgcacgcg acgctgcccg aaggcgtgct ggattcggtc 360
agcctgccgc cggagcggga gtggttgaag accaccgatt ccgcactgca cctggaccgt 420
ttactgttct gcgccaagga agccacctac aaggcgtggt ggccgctgac cgcgcgctgg 480
ctcggcttcg aggaagcgca catcaccttc gagatcgaag acggctccgc cgattccggc 540
aacggcacct ttcacagcga gctgctggtg ccgggacaga cgaatgacgg tgggacgccg 600
ctgctttcgt tcgacggccg gtggctgatc gccgacgggt tcatcctcac cgcgatcgcg 660
tacgcctga 669
<210> 90
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> Npt cloning fw
<400> 90
tttaaggagt tcgatatgat cgagacaatt ttgcc 35
<210> 91
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> Npt rv
<400> 91
ggtggcagca gcctagtcag gcgtacgcga tcgcg 35
<210> 92
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> Mab4714opt_pACYCDuet Npt insertion backbone Fw
<400> 92
ctaggctgct gccaccgctg 20
<210> 93
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> Mab4714opt_pACYCDuet Npt insertion backbone Rv
<400> 93
atcgaactcc ttaaatttat ttaaactaaa cccagcagct gaata 45
<210> 94
<211> 1020
<212> DNA
<213> 大肠杆菌
<400> 94
atgtcgatga taaaaagcta tgccgcaaaa gaagcgggcg gcgaactgga agtttatgag 60
tacgatcccg gtgagctgag gccacaagat gttgaagtgc aggtggatta ctgcgggatc 120
tgccattccg atctgtcgat gatcgataac gaatggggat tttcacaata tccgctggtt 180
gccgggcatg aggtgattgg gcgcgtggtg gcactcggga gcgccgcgca ggataaaggt 240
ttgcaggtcg gtcagcgtgt cgggattggc tggacggcgc gtagctgtgg tcactgcgac 300
gcctgtatta gcggtaatca gatcaactgc gagcaaggtg cggtgccgac gattatgaat 360
cgcggtggct ttgccgagaa gttgcgtgcg gactggcaat gggtgattcc actgccagaa 420
aatattgata tcgagtccgc cgggccgctg ttgtgcggcg gtatcacggt ctttaaacca 480
ctgttgatgc accatatcac tgctaccagc cgcgttgggg taattggtat tggcgggctg 540
gggcatatcg ctataaaact tctgcacgca atgggatgcg aggtgacagc ctttagttct 600
aatccggcga aagagcagga agtgctggcg atgggtgccg ataaagtggt gaatagccgc 660
gatccgcagg cactgaaagc actggcgggg cagtttgatc tcattatcaa caccgtcaac 720
gtcagcctcg actggcagcc ctattttgag gcgctgacct atggcggtaa tttccatacg 780
gtcggtgcgg ttctcacgcc gctgtctgtt ccggccttta cgttaattgc gggcgatcgc 840
agcgtctctg gttctgctac cggcacgcct tatgagctgc gtaagctgat gcgttttgcc 900
gcccgcagca aggttgcgcc gaccaccgaa ctgttcccga tgtcgaaaat taacgacgcc 960
atccagcatg tgcgcgacgg taaggcgcgt taccgcgtgg tgttgaaagc cgatttttga 1020
<210> 95
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> Ahr cloning fw
<400> 95
ataaggagat ataccatgtc gatgataaaa agcta 35
<210> 96
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> Ahr cloning rv
<400> 96
atgcggccgc aagcttcaaa aatcggcttt caaca 35
<210> 97
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> pACYCDuet MCS1 Backbone Fw
<400> 97
catggtatat ctccttatta aagttaaaca aaatt 35
<210> 98
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> pACYCDuet MCS1 Backbone Rv
<400> 98
agcttgcggc cgcataatgc ttaagtcgaa 30
<210> 99
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> atoB-del-f1
<400> 99
ttgttgaata aatcggcact cggtatcgct tacct 35
<210> 100
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> atoB-del-r1
<400> 100
tatcgcatgc agatcggctg gtggtcaacg gtgcc 35
<210> 101
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> sucD-del-f1
<400> 101
ttgttgaata aatcggagcg cgacctggcg ttgat 35
<210> 102
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> sucD-del-r1
<400> 102
tatcgcatgc agatcacgat atttttcagt taacc 35
<210> 103
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ldhA-del-f1
<400> 103
ttgttgaata aatcgtttat cgatattgat ccagg 35
<210> 104
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ldhA-del-r1
<400> 104
tatcgcatgc agatctgtgt gcattaccca acggc 35
<210> 105
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> adhE-del-f1
<400> 105
ttgttgaata aatcgctgcc gctgtctgat aactg 35
<210> 106
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> adhE-del-r1
<400> 106
tatcgcatgc agatctcgat atacccgtac tttgt 35
<210> 107
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> dkgA-HA-flag-f
<400> 107
ttgttgaata aatcgtctgt ttgccgagaa tacgc 35
<210> 108
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> dkgA-HA-flag-r
<400> 108
tatcgcatgc agatccgacc attttccgcc accat 35
<210> 109
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> yahK-del-f1
<400> 109
ttgttgaata aatcgatatt cgtcctaacg aacag 35
<210> 110
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> yahK-del-r1
<400> 110
tatcgcatgc agatcttttt gattttcaag tatgt 35
<210> 111
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ahr-del-f1
<400> 111
ttgttgaata aatcgctcat aacggtactg caaac 35
<210> 112
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> ahr-del-r1
<400> 112
tatcgcatgc agatctcgca gcaggtaaga tgatt 35
<210> 113
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> pflB-del-f1
<400> 113
ttgttgaata aatcgatcac ctggggtcag ttggc 35
<210> 114
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> pflB-del-r1
<400> 114
tatcgcatgc agatccgtcg ttcatctgtt tgaga 35
<210> 115
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> pHAK-v-fw
<400> 115
gatctgcatg cgatatctct agaacgcgta agc 33
<210> 116
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> pHAK-v-rev3
<400> 116
cgatttattc aacaaagccg ccgtcccg 28
<210> 117
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> atoB-del-f2-2
<400> 117
cgatggtgat tgaacggttg 20
<210> 118
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> atoB-del-r2-2
<400> 118
gcactgacga tgacacaatt 20
<210> 119
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> sucD-del-f2-2
<400> 119
gaaattcgct gctctggaag 20
<210> 120
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> sucD-del-r2-2
<400> 120
cctggcagat aaccttggtg ttt 23
<210> 121
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> ldhA-del-f2-2
<400> 121
tggtttaatc ttgccgctcc 20
<210> 122
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> ldhA-del-r2-2
<400> 122
gtgctataaa cggcgagttt ca 22
<210> 123
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> adhE-del-f2
<400> 123
tcagtagcgc tgtctggcaa tataaa 26
<210> 124
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> adhE-del-r2
<400> 124
aatgctctcc tgataatgtt aaactttttt 30
<210> 125
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> yqhD-del-f2
<400> 125
catgcaaatt ctcccggtgg cggta 25
<210> 126
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> yqhD-del-r2
<400> 126
ccaatatgag ggcagagaac gatc 24
<210> 127
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> yahK-del-f2-2
<400> 127
tcgcacacta acagactgaa 20
<210> 128
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> yahK-del-r2
<400> 128
tgtgtttact cctgattagc 20
<210> 129
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> ahr-del-f2-2
<400> 129
cgtggtgttg aaagccgatt attg 24
<210> 130
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> ahr-del-r2-2
<400> 130
cataaacttc cagttctccg ccc 23
<210> 131
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> pflB-del-f2
<400> 131
ttagatttga ctgaaatcgt acagtaaaaa gc 32
<210> 132
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> pflB-del-r2
<400> 132
gtaacaccta ccttcttaag tggattt 27
<210> 133
<211> 186
<212> DNA
<213> 人工序列
<220>
<223> 启动子区域
<400> 133
gcagctgctg cagctcgaga gatctctcaa gcccaaagga agagtgaggc gagtcagtcg 60
cgtaatgctt aggcacagga ttgatttgtc gcaatgattg acacgattcc gcttgacgct 120
gcgtaaggtt tgtgtataat tacaggcaac cttttattca ctaacaaata gctggtggaa 180
gatatc 186
<210> 134
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 连接子区域
<400> 134
agaaggagat atacat 16
<210> 135
<211> 162
<212> DNA
<213> 人工序列
<220>
<223> 终止子区域
<400> 135
ggtaccctcg agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac 60
tcgtctacta gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca 120
taaccccttg gggcctctaa acgggtcttg aggggttttt tg 162
<210> 136
<211> 186
<212> DNA
<213> 人工序列
<220>
<223> 启动子区域
<400> 136
gcagctgctg cagctcgaga gatctctcaa gcccaaagga agagtgaggc gagtcagtcg 60
cgtaatgctt aggcacagga ttgatttgtc gcaatgattg acacgattcc gcttgacgct 120
gcgtaaggtt tgtgtataat tacaggcaac cttttattca ctaacaaata gctggtggaa 180
gatatc 186
<210> 137
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 连接子区域
<400> 137
aagcttgcgg ccgcagaagg agatatacat 30
<210> 138
<211> 76
<212> DNA
<213> 人工序列
<220>
<223> 终止子区域
<400> 138
ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 60
cttgaggggt tttttg 76
<210> 139
<211> 186
<212> DNA
<213> 人工序列
<220>
<223> 启动子区域
<400> 139
gcagctgctg cagctcgaga gatctctcaa gcccaaagga agagtgaggc gagtcagtcg 60
cgtaatgctt aggcacagga ttgatttgtc gcaatgattg acacgattcc gcttgacgct 120
gcgtaaggtt tgtgtataat tacaggcaac cttttattca ctaacaaata gctggtggaa 180
gatatc 186
<210> 140
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 连接子区域
<400> 140
ataaatttaa ggagttcgat 20
<210> 141
<211> 76
<212> DNA
<213> 人工序列
<220>
<223> 终止子区域
<400> 141
ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 60
cttgaggggt tttttg 76
<210> 142
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> atoB check Fw2
<400> 142
aagcagctac cgaaagatgc ccc 23
<210> 143
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> atoB check Rv2
<400> 143
ggcagatgat caaaatatct cagcgcagat 30
<210> 144
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> sucD-check-f
<400> 144
gggtctggaa ggtaaactgg 20
<210> 145
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> sucD-check-r
<400> 145
cgtcaaagaa gagcgggtca att 23
<210> 146
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> ldhA-check-f
<400> 146
agaggttaat gcgagagaga g 21
<210> 147
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> ldhA-check-r
<400> 147
ctggatggta cggcgattgg 20
<210> 148
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> adhE-check-f
<400> 148
tcgacatcgc tatcgtcacc accagg 26
<210> 149
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> adhE-check-r
<400> 149
cgcgtatagg tttgtcatcg cctgca 26
<210> 150
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> dkgA-check-Fw
<400> 150
tattctcaat ccgtttcagc acgcg 25
<210> 151
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> dkgA-check-Rv
<400> 151
ggattattgg ccgtcttgaa ggtg 24
<210> 152
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> yahK-check-f2
<400> 152
agctgaccat cagtcacgcc tttgc 25
<210> 153
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> yahK-check-r2
<400> 153
cgtccagaac agagagcaat aacatcac 28
<210> 154
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> ahr-check-f
<400> 154
tccgctagtg tgatttcagg 20
<210> 155
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> ahr-check-r
<400> 155
gaaattatta tgccgccagg cgt 23
<210> 156
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> pflB-check-f
<400> 156
ctgattcttt gtgttgtctg cggag 25
<210> 157
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> pflB-check-r
<400> 157
cagatattta gcgaactcca gcgtg 25
<210> 158
<211> 468
<212> PRT
<213> 人工序列
<220>
<223> YgjG(m)
<400> 158
Met Ile Arg Glu Pro Pro Glu His Ile Leu Asn Arg Leu Pro Ser Ser
1 5 10 15
Ala Ser Ala Leu Ala Cys Ser Ala His Ala Leu Asn Leu Ile Glu Lys
20 25 30
Arg Thr Leu Asp His Lys Glu Met Lys Ala Leu Asn Arg Glu Val Ile
35 40 45
Glu Tyr Tyr Lys Glu His Val Asn Pro Gly Phe Leu Glu Tyr Arg Lys
50 55 60
Ser Val Thr Ala Gly Gly Asp Tyr Gly Ala Val Glu Trp Gln Ala Gly
65 70 75 80
Ser Leu Asn Thr Leu Val Asp Thr Gln Gly Gln Glu Phe Ile Asp Cys
85 90 95
Leu Gly Gly Phe Gly Ile Phe Asn Val Gly His Arg Asn Pro Val Val
100 105 110
Val Ser Ala Val Gln Asn Gln Leu Ala Lys Gln Pro Leu His Ser Gln
115 120 125
Glu Leu Leu Asp Pro Leu Arg Ala Met Leu Ala Lys Thr Leu Ala Ala
130 135 140
Leu Thr Pro Gly Lys Leu Lys Tyr Ser Phe Phe Cys Asn Ser Gly Thr
145 150 155 160
Glu Ser Val Glu Ala Ala Leu Lys Leu Ala Lys Ala Tyr Gln Ser Pro
165 170 175
Arg Gly Lys Phe Thr Phe Ile Ala Thr Ser Gly Ala Phe His Gly Lys
180 185 190
Ser Leu Gly Ala Leu Ser Ala Thr Ala Lys Ser Thr Phe Arg Lys Pro
195 200 205
Phe Met Pro Leu Leu Pro Gly Phe Arg His Val Pro Phe Gly Asn Ile
210 215 220
Glu Ala Met Arg Arg Ala Leu Asn Glu Cys Lys Lys Thr Gly Asp Asp
225 230 235 240
Val Ala Ala Val Ile Leu Glu Pro Ile Gln Gly Glu Gly Gly Val Ile
245 250 255
Leu Pro Pro Pro Gly Tyr Leu Thr Ala Val Arg Lys Leu Cys Asp Glu
260 265 270
Phe Gly Ala Leu Met Ile Leu Asp Glu Val Gln Thr Gly Met Gly Arg
275 280 285
Thr Gly Lys Met Phe Ala Cys Glu His Glu Asn Val Gln Pro Asp Ile
290 295 300
Leu Cys Leu Ala Lys Ala Leu Gly Gly Gly Val Met Pro Ile Gly Ala
305 310 315 320
Thr Ile Ala Thr Glu Glu Val Phe Ser Val Leu Phe Asp Asn Pro Phe
325 330 335
Leu His Thr Thr Thr Phe Gly Gly Asn Pro Leu Ala Cys Ala Ala Ala
340 345 350
Leu Ala Thr Ile Asn Val Leu Leu Glu Gln Asn Leu Pro Ala Gln Ala
355 360 365
Glu Gln Lys Gly Asp Met Leu Leu Asp Gly Phe Arg Gln Leu Ala Arg
370 375 380
Glu Tyr Pro Asp Leu Val Gln Glu Ala Arg Gly Lys Gly Met Leu Met
385 390 395 400
Ala Ile Glu Phe Val Asp Asn Glu Ile Gly Tyr Asn Phe Ala Ser Glu
405 410 415
Met Phe Arg Gln Arg Val Leu Val Ala Gly Thr Leu Asn Asn Ala Lys
420 425 430
Thr Ile Arg Ile Glu Pro Pro Leu Thr Leu Thr Ile Glu Gln Cys Glu
435 440 445
Leu Val Ile Lys Ala Ala Arg Lys Ala Leu Ala Ala Met Arg Val Ser
450 455 460
Val Glu Glu Ala
465
<210> 159
<211> 1407
<212> DNA
<213> 人工序列
<220>
<223> ygjG (m)
<400> 159
atgatacgcg agcctccgga gcatattttg aacaggttac cttcgagcgc atcggcttta 60
gcgtgcagcg cccacgccct gaatctcatt gagaagcgaa cgctggatca taaggagatg 120
aaagcactta accgagaggt gattgaatac tacaaagagc atgtcaatcc ggggttttta 180
gagtatcgca aatctgttac cgccggcggg gattacggag ccgtagagtg gcaagcggga 240
agtttaaata cgcttgtcga cacccagggc caggagttta tcgactgcct gggaggtttt 300
ggaattttca acgtggggca ccgtaatcca gttgtggttt ccgccgtaca gaatcaactt 360
gcgaaacaac cgctgcacag ccaggaactg ctcgatccgt tacgggcgat gttggcgaaa 420
acccttgctg cgctaacgcc cggtaaactg aaatacagct tcttctgtaa tagcggcacc 480
gagtccgttg aagcagcgct gaagctggcg aaagcttacc agtcaccgcg cggcaagttt 540
acttttattg ccaccagcgg cgcgttccac ggtaaatcac ttggcgcgct gtcggccacg 600
gcgaaatcga ccttccgcaa accgtttatg ccgttactgc cgggcttccg tcatgtgccg 660
tttggcaata tcgaagccat gcgcagggct cttaacgagt gcaaaaaaac cggtgatgat 720
gtggctgcgg tgatcctcga accgattcag ggtgaaggtg gcgtaattct gccgccgccg 780
ggctatctca ccgccgtacg taagctatgc gatgagttcg gcgcactgat gatcctcgat 840
gaagtacaaa cgggcatggg gcgcacgggc aagatgttcg cctgcgagca tgagaacgta 900
cagccggata tcctctgcct tgccaaagcg ctcggcggcg gcgtgatgcc gattggcgcg 960
accatcgcca ctgaagaggt gttttcagtt ctgttcgaca acccattcct gcataccacc 1020
acctttggcg gcaacccgct ggcctgtgcg gcggcgctgg cgaccatcaa tgtgttgctg 1080
gagcagaact taccggctca ggctgagcaa aaaggcgata tgttgctgga cggtttccgt 1140
caactggcgc gggaatatcc cgatctggta caggaagcgc gtggtaaagg gatgttgatg 1200
gcgattgagt ttgttgataa cgaaatcggc tataactttg ccagcgagat gttccgccag 1260
cgcgtactgg tggccggaac gctcaataac gccaaaacga tccgcattga accgccactg 1320
acactgacca ttgaacagtg tgaactggtg atcaaagcgg cgcgtaaggc gctggcggcc 1380
atgcgagtaa gtgtcgaaga agcgtaa 1407
<210> 160
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 160
ataaggagat ataccatgat acgcgagcct ccgga 35
<210> 161
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 161
atgcggccgc aagctttacg cttcttcgac actta 35
<210> 162
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 162
atgattcgcg accaggaaac 20
<210> 163
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 163
tcaaacttgg acgtcccgaa t 21
<210> 164
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物 for paaJ
<400> 164
atgcttgacg cctatctcta tgctg 25
<210> 165
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物 for paaJ
<400> 165
ttacatgcgt tcgatgacta cgg 23
<210> 166
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 引物 for paaH
<400> 166
atgaacactg acatccagac cc 22
<210> 167
<211> 19
<212> DNA
<213> 人工序列
<220>
<223> 引物 for paaH
<400> 167
tcagttgtca ggggtcagc 19
<210> 168
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 引物 for paaF
<400> 168
atgcaaacca tcgaaaacga ag 22
<210> 169
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物 for paaF
<400> 169
ttaacgtcct gcataggtgg gttt 24
<210> 170
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物 for mmgC
<400> 170
atgattcgcg accaggaaac 20
<210> 171
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> mmgC的引物
<400> 171
tcaaacttgg acgtcccgaa t 21
<210> 172
<211> 401
<212> PRT
<213> 大肠杆菌
<400> 172
Met Arg Glu Ala Phe Ile Cys Asp Gly Ile Arg Thr Pro Ile Gly Arg
1 5 10 15
Tyr Gly Gly Ala Leu Ser Ser Val Arg Ala Asp Asp Leu Ala Ala Ile
20 25 30
Pro Leu Arg Glu Leu Leu Val Arg Asn Pro Arg Leu Asp Ala Glu Cys
35 40 45
Ile Asp Asp Val Ile Leu Gly Cys Ala Asn Gln Ala Gly Glu Asp Asn
50 55 60
Arg Asn Val Ala Arg Met Ala Thr Leu Leu Ala Gly Leu Pro Gln Ser
65 70 75 80
Val Ser Gly Thr Thr Ile Asn Arg Leu Cys Gly Ser Gly Leu Asp Ala
85 90 95
Leu Gly Phe Ala Ala Arg Ala Ile Lys Ala Gly Asp Gly Asp Leu Leu
100 105 110
Ile Ala Gly Gly Val Glu Ser Met Ser Arg Ala Pro Phe Val Met Gly
115 120 125
Lys Ala Ala Ser Ala Phe Ser Arg Gln Ala Glu Met Phe Asp Thr Thr
130 135 140
Ile Gly Trp Arg Phe Val Asn Pro Leu Met Ala Gln Gln Phe Gly Thr
145 150 155 160
Asp Ser Met Pro Glu Thr Ala Glu Asn Val Ala Glu Leu Leu Lys Ile
165 170 175
Ser Arg Glu Asp Gln Asp Ser Phe Ala Leu Arg Ser Gln Gln Arg Thr
180 185 190
Ala Lys Ala Gln Ser Ser Gly Ile Leu Ala Glu Glu Ile Val Pro Val
195 200 205
Val Leu Lys Asn Lys Lys Gly Val Val Thr Glu Ile Gln His Asp Glu
210 215 220
His Leu Arg Pro Glu Thr Thr Leu Glu Gln Leu Arg Gly Leu Lys Ala
225 230 235 240
Pro Phe Arg Ala Asn Gly Val Ile Thr Ala Gly Asn Ala Ser Gly Val
245 250 255
Asn Asp Gly Ala Ala Ala Leu Ile Ile Ala Ser Glu Gln Met Ala Ala
260 265 270
Ala Gln Gly Leu Thr Pro Arg Ala Arg Ile Val Ala Met Ala Thr Ala
275 280 285
Gly Val Glu Pro Arg Leu Met Gly Leu Gly Pro Val Pro Ala Thr Arg
290 295 300
Arg Val Leu Glu Arg Ala Gly Leu Ser Ile His Asp Met Asp Val Ile
305 310 315 320
Glu Leu Asn Glu Ala Phe Ala Ala Gln Ala Leu Gly Val Leu Arg Glu
325 330 335
Leu Gly Leu Pro Asp Asp Ala Pro His Val Asn Pro Asn Gly Gly Ala
340 345 350
Ile Ala Leu Gly His Pro Leu Gly Met Ser Gly Ala Arg Leu Ala Leu
355 360 365
Ala Ala Ser His Glu Leu His Arg Arg Asn Gly Arg Tyr Ala Leu Cys
370 375 380
Thr Met Cys Ile Gly Val Gly Gln Gly Ile Ala Met Ile Leu Glu Arg
385 390 395 400
Val
<210> 173
<211> 475
<212> PRT
<213> 大肠杆菌
<400> 173
Met Met Ile Asn Val Gln Thr Val Ala Val Ile Gly Ser Gly Thr Met
1 5 10 15
Gly Ala Gly Ile Ala Glu Val Ala Ala Ser His Gly His Gln Val Leu
20 25 30
Leu Tyr Asp Ile Ser Ala Glu Ala Leu Thr Arg Ala Ile Asp Gly Ile
35 40 45
His Ala Arg Leu Asn Ser Arg Val Thr Arg Gly Lys Leu Thr Ala Glu
50 55 60
Thr Cys Glu Arg Thr Leu Lys Arg Leu Ile Pro Val Thr Asp Ile His
65 70 75 80
Ala Leu Ala Ala Ala Asp Leu Val Ile Glu Ala Ala Ser Glu Arg Leu
85 90 95
Glu Val Lys Lys Ala Leu Phe Ala Gln Leu Ala Glu Val Cys Pro Pro
100 105 110
Gln Thr Leu Leu Thr Thr Asn Thr Ser Ser Ile Ser Ile Thr Ala Ile
115 120 125
Ala Ala Glu Ile Lys Asn Pro Glu Arg Val Ala Gly Leu His Phe Phe
130 135 140
Asn Pro Ala Pro Val Met Lys Leu Val Glu Val Val Ser Gly Leu Ala
145 150 155 160
Thr Ala Ala Glu Val Val Glu Gln Leu Cys Glu Leu Thr Leu Ser Trp
165 170 175
Gly Lys Gln Pro Val Arg Cys His Ser Thr Pro Gly Phe Ile Val Asn
180 185 190
Arg Val Ala Arg Pro Tyr Tyr Ser Glu Ala Trp Arg Ala Leu Glu Glu
195 200 205
Gln Val Ala Ala Pro Glu Val Ile Asp Ala Ala Leu Arg Asp Gly Ala
210 215 220
Gly Phe Pro Met Gly Pro Leu Glu Leu Thr Asp Leu Ile Gly Gln Asp
225 230 235 240
Val Asn Phe Ala Val Thr Cys Ser Val Phe Asn Ala Phe Trp Gln Glu
245 250 255
Arg Arg Phe Leu Pro Ser Leu Val Gln Gln Glu Leu Val Ile Gly Gly
260 265 270
Arg Leu Gly Lys Lys Ser Gly Leu Gly Val Tyr Asp Trp Arg Ala Glu
275 280 285
Arg Glu Ala Val Val Gly Leu Glu Ala Val Ser Asp Ser Phe Ser Pro
290 295 300
Met Lys Val Glu Lys Lys Ser Asp Gly Val Thr Glu Ile Asp Asp Val
305 310 315 320
Leu Leu Ile Glu Thr Gln Gly Glu Thr Ala Gln Ala Leu Ala Ile Arg
325 330 335
Leu Ala Arg Pro Val Val Val Ile Asp Lys Met Ala Gly Lys Val Val
340 345 350
Thr Ile Ala Ala Ala Ala Val Asn Pro Asp Ser Ala Thr Arg Lys Ala
355 360 365
Ile Tyr Tyr Leu Gln Gln Gln Gly Lys Thr Val Leu Gln Ile Ala Asp
370 375 380
Tyr Pro Gly Met Leu Ile Trp Arg Thr Val Ala Met Ile Ile Asn Glu
385 390 395 400
Ala Leu Asp Ala Leu Gln Lys Gly Val Ala Ser Glu Gln Asp Ile Asp
405 410 415
Thr Ala Met Arg Leu Gly Val Asn Tyr Pro Tyr Gly Pro Leu Val Trp
420 425 430
Gly Ala Gln Leu Gly Trp Gln Arg Ile Leu Arg Leu Leu Glu Asn Leu
435 440 445
Gln His His Tyr Gly Glu Glu Arg Tyr Arg Pro Cys Ser Leu Leu Arg
450 455 460
Gln Arg Ala Leu Leu Glu Ser Gly Tyr Glu Ser
465 470 475
<210> 174
<211> 255
<212> PRT
<213> 大肠杆菌
<400> 174
Met Ser Glu Leu Ile Val Ser Arg Gln Gln Arg Val Leu Leu Leu Thr
1 5 10 15
Leu Asn Arg Pro Ala Ala Arg Asn Ala Leu Asn Asn Ala Leu Leu Met
20 25 30
Gln Leu Val Asn Glu Leu Glu Ala Ala Ala Thr Asp Thr Ser Ile Ser
35 40 45
Val Cys Val Ile Thr Gly Asn Ala Arg Phe Phe Ala Ala Gly Ala Asp
50 55 60
Leu Asn Glu Met Ala Glu Lys Asp Leu Ala Ala Thr Leu Asn Asp Thr
65 70 75 80
Arg Pro Gln Leu Trp Ala Arg Leu Gln Ala Phe Asn Lys Pro Leu Ile
85 90 95
Ala Ala Val Asn Gly Tyr Ala Leu Gly Ala Gly Cys Glu Leu Ala Leu
100 105 110
Leu Cys Asp Val Val Val Ala Gly Glu Asn Ala Arg Phe Gly Leu Pro
115 120 125
Glu Ile Thr Leu Gly Ile Met Pro Gly Ala Gly Gly Thr Gln Arg Leu
130 135 140
Ile Arg Ser Val Gly Lys Ser Leu Ala Ser Lys Met Val Leu Ser Gly
145 150 155 160
Glu Ser Ile Thr Ala Gln Gln Ala Gln Gln Ala Gly Leu Val Ser Asp
165 170 175
Val Phe Pro Ser Asp Leu Thr Leu Glu Tyr Ala Leu Gln Leu Ala Ser
180 185 190
Lys Met Ala Arg His Ser Pro Leu Ala Leu Gln Ala Ala Lys Gln Ala
195 200 205
Leu Arg Gln Ser Gln Glu Val Ala Leu Gln Ala Gly Leu Ala Gln Glu
210 215 220
Arg Gln Leu Phe Thr Leu Leu Ala Ala Thr Glu Asp Arg His Glu Gly
225 230 235 240
Ile Ser Ala Phe Leu Gln Lys Arg Thr Pro Asp Phe Lys Gly Arg
245 250 255
<210> 175
<211> 259
<212> PRT
<213> 未知
<220>
<223> crt(Ck)
<400> 175
Met Glu Phe Lys Asn Ile Ile Leu Glu Lys Asp Gly Asn Val Ala Ser
1 5 10 15
Ile Thr Leu Asn Arg Pro Lys Ala Leu Asn Ala Leu Asn Ala Ala Thr
20 25 30
Leu Lys Glu Ile Asp Ala Ala Ile Asn Asp Ile Ala Glu Asp Asp Asn
35 40 45
Val Tyr Ala Val Ile Ile Thr Gly Ser Gly Lys Ala Phe Val Ala Gly
50 55 60
Ala Asp Ile Ala Glu Met Lys Asp Leu Thr Ala Val Glu Gly Arg Lys
65 70 75 80
Phe Ser Val Leu Gly Asn Lys Ile Phe Arg Lys Leu Glu Asn Leu Glu
85 90 95
Lys Pro Val Ile Ala Ala Ile Asn Gly Phe Ala Leu Gly Gly Gly Cys
100 105 110
Glu Leu Ser Leu Ser Cys Asp Ile Arg Ile Ala Ser Ser Lys Ala Lys
115 120 125
Phe Gly Gln Pro Glu Val Gly Leu Gly Ile Thr Pro Gly Phe Gly Gly
130 135 140
Thr Gln Arg Leu Ala Arg Ala Ile Gly Val Gly Met Ala Lys Glu Leu
145 150 155 160
Ile Tyr Thr Gly Lys Val Ile Asn Ala Glu Glu Ala Leu Arg Ile Gly
165 170 175
Leu Val Asn Lys Val Val Glu Pro Asp Lys Leu Leu Glu Glu Ala Lys
180 185 190
Ala Leu Val Asp Ala Ile Ile Val Asn Ala Pro Ile Ala Val Arg Met
195 200 205
Cys Lys Ala Ala Ile Asn Gln Gly Leu Gln Cys Asp Ile Asp Thr Gly
210 215 220
Val Ala Tyr Glu Ala Glu Val Phe Gly Glu Cys Phe Ala Thr Glu Asp
225 230 235 240
Arg Val Glu Gly Met Thr Ala Phe Val Glu Lys Arg Asp Lys Ala Phe
245 250 255
Lys Asn Lys
<210> 176
<211> 276
<212> PRT
<213> 未知
<220>
<223> ch(Pp)
<400> 176
Met Ser Lys Tyr Glu Gly Arg Trp Thr Thr Val Lys Val Glu Leu Glu
1 5 10 15
Ala Gly Ile Ala Trp Val Thr Leu Asn Arg Pro Glu Lys Arg Asn Ala
20 25 30
Met Ser Pro Thr Leu Asn Arg Glu Met Val Asp Val Leu Glu Thr Leu
35 40 45
Glu Gln Asp Ala Asp Ala Gly Val Leu Val Leu Thr Gly Ala Gly Glu
50 55 60
Ser Trp Thr Ala Gly Met Asp Leu Lys Glu Tyr Phe Arg Glu Val Asp
65 70 75 80
Ala Gly Pro Glu Ile Leu Gln Glu Lys Ile Arg Arg Glu Ala Ser Gln
85 90 95
Trp Gln Trp Lys Leu Leu Arg Leu Tyr Ala Lys Pro Thr Ile Ala Met
100 105 110
Val Asn Gly Trp Cys Phe Gly Gly Gly Phe Ser Pro Leu Val Ala Cys
115 120 125
Asp Leu Ala Ile Cys Ala Asn Glu Ala Thr Phe Gly Leu Ser Glu Ile
130 135 140
Asn Trp Gly Ile Pro Pro Gly Asn Leu Val Ser Lys Ala Met Ala Asp
145 150 155 160
Thr Val Gly His Arg Gln Ser Leu Tyr Tyr Ile Met Thr Gly Lys Thr
165 170 175
Phe Asp Gly Arg Lys Ala Ala Glu Met Gly Leu Val Asn Asp Ser Val
180 185 190
Pro Leu Ala Glu Leu Arg Glu Thr Thr Arg Glu Leu Ala Leu Asn Leu
195 200 205
Leu Glu Lys Asn Pro Val Val Leu Arg Ala Ala Lys Asn Gly Phe Lys
210 215 220
Arg Cys Arg Glu Leu Thr Trp Glu Gln Asn Glu Asp Tyr Leu Tyr Ala
225 230 235 240
Lys Leu Asp Gln Ser Arg Leu Leu Asp Thr Thr Gly Gly Arg Glu Gln
245 250 255
Gly Met Lys Gln Phe Leu Asp Asp Lys Ser Ile Lys Pro Gly Leu Gln
260 265 270
Ala Tyr Lys Arg
275
<210> 177
<211> 384
<212> PRT
<213> 未知
<220>
<223> dcaA(Ab)
<400> 177
Met Ile Arg Asp Glu Gly Met Leu Gln Gln Leu Leu Ser Thr Ile Arg
1 5 10 15
Asp Phe Val Lys Asn Glu Leu Ile Pro Arg Glu His Glu Val Ala Glu
20 25 30
Lys Asp Cys Ile Pro Glu Asp Ile Ile Gln Gln Met Arg Glu Leu Gly
35 40 45
Leu Phe Gly Leu Thr Ile Pro Glu Glu Tyr Gly Gly Leu Gly Ile Thr
50 55 60
Met Glu Glu Glu Val Asn Val Ala Phe Glu Leu Gly Gln Thr Ser Pro
65 70 75 80
Ala Phe Arg Ser Leu Ile Gly Thr Asn Asn Gly Ile Gly Ser Ser Gly
85 90 95
Leu Ile Ile Asp Gly Thr Glu Glu Gln Lys Gln Lys Tyr Leu Pro Arg
100 105 110
Tyr Ala Ser Gly Glu Ile Ile Gly Ser Phe Cys Leu Thr Glu Pro Glu
115 120 125
Ala Gly Ser Asp Ala Ala Ser Leu Lys Thr Thr Ala Val Lys Asp Gly
130 135 140
Asp Phe Tyr Ile Leu Asn Gly Thr Lys Arg Phe Ile Thr Asn Ala Pro
145 150 155 160
His Ala Ala Thr Phe Thr Val Met Ala Arg Thr Asn Pro Ala Ile Lys
165 170 175
Gly Ala Gly Gly Ile Ser Ala Phe Leu Val Glu Ala Asn Thr Pro Gly
180 185 190
Ile Thr Leu Gly Lys Ile Asp Gln Lys Met Gly Gln Lys Gly Ser His
195 200 205
Thr Cys Asp Val Ile Phe Glu Asn Cys Arg Val Pro Ala Ser Ala Leu
210 215 220
Ile Gly Gly Val Glu Gly Val Gly Phe Lys Thr Ala Met Lys Val Leu
225 230 235 240
Asp Lys Gly Arg Leu His Ile Gly Ala Tyr Ser Val Gly Val Ala Glu
245 250 255
Arg Met Leu Asn Asp Ala Leu His Tyr Ala Val Glu Arg Lys Gln Phe
260 265 270
Gly Gln Pro Ile Ala Asn Phe Gln Leu Ile Gln Ala Met Leu Ala Asp
275 280 285
Ser Lys Ala Glu Ile Tyr Ala Ala Lys Cys Met Val Leu Asp Ala Ala
290 295 300
Arg Arg Arg Asp Glu Gly Gln Asn Ile Ser Thr Glu Ala Ser Cys Ala
305 310 315 320
Lys Met Phe Ala Thr Glu Met Cys Gly Arg Val Ala Asp Arg Cys Val
325 330 335
Gln Ile His Gly Gly Ala Gly Tyr Ile Ser Glu Tyr Ser Ile Glu Arg
340 345 350
Phe Tyr Arg Asp Val Arg Leu Phe Arg Leu Tyr Glu Gly Thr Thr Gln
355 360 365
Val Gln Gln Ile Ile Ile Ala Lys Asn Met Ile Lys Glu Val Thr Ser
370 375 380
<210> 178
<211> 385
<212> PRT
<213> 未知
<220>
<223> Tfu1647
<400> 178
Met Ser Asp Phe Asp Leu Tyr Arg Pro Thr Glu Glu His Glu Ala Leu
1 5 10 15
Arg Glu Ala Ile Arg Ser Val Ala Glu Asp Lys Ile Ala Pro His Ala
20 25 30
Ala Asp Val Asp Glu Gln Ser Arg Phe Pro Gln Glu Ala Tyr Glu Ala
35 40 45
Leu Arg Ala Ser Asp Phe His Ala Pro His Val Ala Glu Glu Tyr Gly
50 55 60
Gly Val Gly Ala Asp Ala Leu Ala Thr Cys Ile Val Ile Glu Glu Ile
65 70 75 80
Ala Arg Val Cys Ala Ser Ser Ser Leu Ile Pro Ala Val Asn Lys Leu
85 90 95
Gly Ser Met Pro Leu Ile Leu Ser Gly Ser Asp Glu Val Lys Gln Arg
100 105 110
Tyr Leu Pro Glu Leu Ala Ser Gly Glu Ala Met Phe Ser Tyr Gly Leu
115 120 125
Ser Glu Arg Glu Ala Gly Ser Asp Thr Ala Ser Met Arg Thr Arg Ala
130 135 140
Val Arg Asp Gly Asp Asp Trp Ile Leu Asn Gly Gln Lys Ser Trp Ile
145 150 155 160
Thr Asn Ala Gly Ile Ser Lys Tyr Tyr Thr Val Met Ala Val Thr Asp
165 170 175
Pro Asp Gly Pro Arg Gly Arg Asn Ile Ser Ala Phe Val Val His Ile
180 185 190
Asp Asp Pro Gly Phe Ser Phe Gly Glu Pro Glu Arg Lys Leu Gly Ile
195 200 205
Lys Gly Ser Pro Thr Arg Glu Leu Ile Phe Asp Asn Val Arg Ile Pro
210 215 220
Gly Asp Arg Leu Val Gly Lys Val Gly Glu Gly Leu Arg Thr Ala Leu
225 230 235 240
Arg Thr Leu Asp His Thr Arg Val Thr Ile Gly Ala Gln Ala Val Gly
245 250 255
Ile Ala Gln Gly Ala Leu Asp Tyr Ala Leu Gly Tyr Val Lys Glu Arg
260 265 270
Lys Gln Phe Gly Lys Ala Ile Ala Asp Phe Gln Gly Ile Gln Phe Met
275 280 285
Leu Ala Asp Met Ala Met Lys Leu Glu Ala Ala Arg Gln Met Val Tyr
290 295 300
Val Ala Ala Ala Lys Ser Glu Arg Asp Asp Ala Asp Leu Ser Phe Tyr
305 310 315 320
Gly Ala Ala Ala Lys Cys Phe Ala Ser Asp Val Ala Met Glu Ile Thr
325 330 335
Thr Asp Ala Val Gln Leu Leu Gly Gly Tyr Gly Tyr Thr Arg Asp Tyr
340 345 350
Pro Val Glu Arg Met Met Arg Asp Ala Lys Ile Thr Gln Ile Tyr Glu
355 360 365
Gly Thr Asn Gln Ile Gln Arg Val Val Met Ala Arg Gln Leu Leu Lys
370 375 380
Lys
385
<210> 179
<211> 290
<212> PRT
<213> 未知
<220>
<223> tesB(Ab)
<400> 179
Met Asn Thr Leu Thr Gln Glu Leu Val Glu Leu Leu Ser Leu Glu Lys
1 5 10 15
Leu Glu Glu Asn Leu Tyr Arg Gly Met Ser Arg Asn Leu Val Gly Lys
20 25 30
Arg Val Phe Gly Gly Gln Val Leu Gly Gln Ala Leu Arg Ala Ala Ser
35 40 45
Tyr Thr Thr Asp Arg Pro Ala His Ser Leu His Ala Tyr Phe Leu Tyr
50 55 60
Gly Gly Asp Val Asn Ala Pro Ile Ile Tyr Glu Val Asp Pro Leu Arg
65 70 75 80
Asp Gly Lys Ser Phe Val Ser Arg Gln Val Arg Ala Ile Gln His Gly
85 90 95
Arg Thr Ile Phe Ser Ala Met Val Ser Phe Ala Ser Pro Glu Glu Gly
100 105 110
Leu Asn Tyr Gln Asn Asp Met Pro Asp Tyr Pro Ala Pro Glu Gln Leu
115 120 125
Lys Ser Glu Ala Glu Leu Lys Leu Gly Leu Ile Asp Phe Val Pro Glu
130 135 140
Asn Val Arg Ala Ser Phe Met Arg Glu Arg His Ile Glu Ile Arg Pro
145 150 155 160
Val Glu Pro Val Asn Pro Phe Gln Pro Gln Pro Gln Ala Pro Thr Asn
165 170 175
Ala His Tyr Ile Arg Thr His Asp Lys Ile Gly Lys Ala Phe Asp Gln
180 185 190
Ile Ala Leu His Gln Ser Ile Val Ala Phe Tyr Ser Asp Phe Thr Leu
195 200 205
Met Thr Thr Ala Leu Lys Pro His Gly Leu Ser Tyr Leu Ser Pro Ser
210 215 220
Leu Gln Cys Ala Ser Ile Asp His Thr Ile Tyr Phe His Arg Pro Leu
225 230 235 240
Arg Ala Asp Glu Trp Met Leu Tyr Asp Met Asp Ala Thr Val Ser Ala
245 250 255
Gly Ser Arg Gly Leu Asn Phe Gly Arg Met Trp Gln Asn Gly Leu Leu
260 265 270
Val Cys Ser Thr Val Gln Glu Gly Leu Met Arg Leu Arg Glu Ile Glu
275 280 285
Thr Gln
290
<210> 180
<211> 1206
<212> DNA
<213> 大肠杆菌
<400> 180
atgcgtgaag cctttatttg tgacggaatt cgtacgccaa ttggtcgcta cggcggggca 60
ttatcaagtg ttcgggctga tgatctggct gctatccctt tgcgggaact gctggtgcga 120
aacccgcgtc tcgatgcgga gtgtatcgat gatgtgatcc tcggctgtgc taatcaggcg 180
ggagaagata accgtaacgt agcccggatg gcgactttac tggcggggct gccgcagagt 240
gtttccggca caaccattaa ccgcttgtgt ggttccgggc tggacgcact ggggtttgcc 300
gcacgggcga ttaaagcggg cgatggcgat ttgctgatcg ccggtggcgt ggagtcaatg 360
tcacgggcac cgtttgttat gggcaaggca gccagtgcat tttctcgtca ggctgagatg 420
ttcgatacca ctattggctg gcgatttgtg aacccgctca tggctcagca atttggaact 480
gacagcatgc cggaaacggc agagaatgta gctgaactgt taaaaatctc acgagaagat 540
caagatagtt ttgcgctacg cagtcagcaa cgtacggcaa aagcgcaatc ctcaggcatt 600
ctggctgagg agattgttcc ggttgtgttg aaaaacaaga aaggtgttgt aacagaaata 660
caacatgatg agcatctgcg cccggaaacg acgctggaac agttacgtgg gttaaaagca 720
ccatttcgtg ccaatggggt gattaccgca ggcaatgctt ccggggtgaa tgacggagcc 780
gctgcgttga ttattgccag tgaacagatg gcagcagcgc aaggactgac accgcgggcg 840
cgtatcgtag ccatggcaac cgccggggtg gaaccgcgcc tgatggggct tggtccggtg 900
cctgcaactc gccgggtgct ggaacgcgca gggctgagta ttcacgatat ggacgtgatt 960
gaactgaacg aagcgttcgc ggcccaggcg ttgggtgtac tacgcgaatt ggggctgcct 1020
gatgatgccc cacatgttaa ccccaacgga ggcgctatcg ccttaggcca tccgttggga 1080
atgagtggtg cccgcctggc actggctgcc agccatgagc tgcatcggcg taacggtcgt 1140
tacgcattgt gcaccatgtg catcggtgtc ggtcagggca tcgccatgat tctggagcgt 1200
gtttga 1206
<210> 181
<211> 1428
<212> DNA
<213> 大肠杆菌
<400> 181
atgatgataa atgtgcaaac tgtggcagtg attgggagcg gcaccatggg ggcaggcatt 60
gctgaagttg ctgccagtca tggacaccag gttttactgt atgacatttc tgctgaagcg 120
ctgacccgcg caatcgacgg gatacacgcg cggctaaatt cacgcgtgac gcggggaaaa 180
ctgactgctg aaacctgtga acgcacattg aaacgcctga tcccggtgac cgatattcac 240
gcgctggcag ctgcggacct ggtcattgaa gcggcgtctg aacgtctgga agtcaaaaaa 300
gcgctctttg cacagctggc ggaagtttgc ccgccacaaa cgctattgac cactaacact 360
tcgtcaatct ctataaccgc gattgctgcg gagataaaaa atcctgaacg tgttgcgggg 420
ctgcattttt ttaacccggc accggtgatg aagttggtgg aggtggtcag tgggctggca 480
acggcggcgg aagttgttga gcagttgtgt gaactaacgt tgagttgggg taagcagcct 540
gtgcgctgtc attcgactcc tggatttatc gttaaccgtg ttgcgcgtcc ttattattcc 600
gaggcctggc gggcactgga agagcaggtt gctgcaccag aagtgattga cgctgcactt 660
cgcgatggcg ctggtttccc gatggggccg ctggaattaa ccgatctgat tggtcaggac 720
gtcaattttg ctgtcacctg ttcggtgttt aacgctttct ggcaggagcg tcgtttttta 780
ccttcgctgg tgcaacagga actggtgatt ggtggacggt tgggcaagaa aagtgggctg 840
ggcgtgtacg actggcgcgc ggaacgtgag gcagttgttg gcctggaagc ggtaagcgac 900
agttttagcc caatgaaagt agaaaagaaa agtgacggtg tcacggaaat tgacgatgtt 960
ttattgattg agacacaagg cgagacggca caggcgctgg caatacgact ggcacgcccg 1020
gtggtagtga tcgataaaat ggcgggcaag gtggtgacca ttgctgctgc agcggtgaac 1080
ccggactcag cgacccgcaa ggccatttat tacctgcaac agcagggcaa aacagtgctg 1140
caaattgcag attacccagg aatgctgatt tggcgaacgg tagcaatgat catcaatgaa 1200
gcccttgatg cgcttcaaaa aggcgtggcc tctgaacagg atatcgatac cgccatgcgt 1260
cttggggtga attatccata tggcccactt gtctggggag cgcaacttgg ctggcagcga 1320
atattaaggc tccttgaaaa tctacagcat cactatggcg aagaacgcta tcgcccatgt 1380
tcattgctgc gccaacgggc gcttctggag agcggttatg agtcataa 1428
<210> 182
<211> 768
<212> DNA
<213> 大肠杆菌
<400> 182
atgagcgaac tgatcgtcag ccgtcagcaa cgagtattgt tgctgaccct taaccgtccc 60
gccgcacgta atgcgctaaa taatgccctg ctgatgcaac tggtaaatga actggaagct 120
gcggctaccg ataccagcat ttcggtctgt gtgattaccg gtaatgcacg cttttttgcc 180
gctggggccg atctcaacga aatggcagaa aaagatctcg cggccacctt aaacgataca 240
cgtccgcagc tatgggcgcg attgcaggcc tttaataaac cacttatcgc agccgtcaat 300
ggttacgcgc ttggggcggg ttgcgaactg gcattgttgt gcgatgtggt ggttgccgga 360
gagaacgcgc gttttgggtt gccggaaatc actctcggca tcatgcctgg cgcaggcgga 420
acgcaacgtt taatccgtag tgtcggtaaa tcgttagcca gcaaaatggt gctgagcgga 480
gaaagtatca ccgctcagca agcacagcag gccgggctgg ttagcgacgt cttccccagc 540
gatttaaccc tcgaatacgc cttacagctg gcatcgaaaa tggcacgtca ctcgccgctg 600
gccttacaag cggcaaagca agcgctgcgc cagtcgcagg aagtggcttt gcaagccgga 660
cttgcccagg agcgacagtt attcaccttg ctggcggcaa cagaagatcg tcatgaaggc 720
atctccgctt tcttacaaaa acgcacgccc gactttaaag gacgctaa 768
<210> 183
<211> 780
<212> DNA
<213> 未知
<220>
<223> crt(Ck)
<400> 183
atggaattca agaacatcat cctggaaaaa gatggtaatg ttgccagcat taccctgaat 60
cgtccgaaag cactgaatgc cctgaatgca gcaaccctga aagaaattga tgcagccatt 120
aatgatatcg ccgaagatga taatgtgtac gccgtgatta ttaccggtag cggtaaagca 180
tttgttgccg gtgcagatat tgcagaaatg aaagatctga ccgcagttga aggtcgcaaa 240
tttagcgttc tgggcaataa aatctttcgc aaactggaaa atctggaaaa accggttatt 300
gccgcaatta atggttttgc attaggtggt ggttgtgaac tgagcctgag ctgtgatatt 360
cgtattgcaa gcagcaaagc aaaatttggt cagccggaag ttggtctggg tattacccct 420
ggttttggtg gcacccagcg tctggcacgt gcaattggtg ttggtatggc aaaagaactg 480
atttataccg gcaaagtgat taatgcagaa gaagcactgc gtattggtct ggttaataaa 540
gttgttgaac cggacaaact gctggaagaa gcaaaagcac tggttgatgc cattattgtt 600
aatgcaccga ttgcagttcg tatgtgtaaa gcagcaatta atcagggtct gcagtgtgat 660
attgataccg gtgttgcata tgaagccgaa gtttttggtg aatgttttgc aaccgaagat 720
cgtgttgaag gtatgaccgc ctttgttgaa aaacgtgata aagcctttaa aaacaaataa 780
<210> 184
<211> 831
<212> DNA
<213> 未知
<220>
<223> ech(Pp)
<400> 184
atgagcaaat atgaaggtcg ttggaccacc gttaaagttg aactggaagc aggtattgca 60
tgggttaccc tgaatcgtcc ggaaaaacgt aatgcaatga gcccgacact gaatcgtgaa 120
atggttgatg ttctggaaac cctggaacag gatgcagatg ccggtgttct ggttctgacc 180
ggtgccggtg aaagctggac cgcaggtatg gatctgaaag aatattttcg tgaagttgat 240
gccggtccgg aaattctgca agaaaaaatt cgtcgtgaag caagccagtg gcagtggaaa 300
ctgctgcgtc tgtatgcaaa accgaccatt gccatggtta atggttggtg ttttggtggt 360
ggttttagtc cgctggttgc atgtgatctg gcaatttgtg caaatgaagc aacctttggt 420
ctgagcgaaa tcaattgggg tattccgcct ggtaatctgg ttagcaaagc aatggcagat 480
accgttggtc atcgtcagag cctgtattat atcatgaccg gtaaaacctt tgatggtcgt 540
aaagcagcag aaatgggttt agttaatgat agcgttccgc tggcagaact gcgtgaaacc 600
acacgtgaac tggcactgaa tctgctggaa aaaaatccgg ttgttctgcg tgcagcaaaa 660
aatggtttta aacgttgtcg tgaactgacc tgggaacaga atgaagatta tctgtacgcc 720
aaactggatc agagccgtct gctggatacc acaggtggtc gtgaacaggg tatgaaacag 780
tttctggatg acaaaagcat taaaccgggt ctgcaggcat ataaacgtta a 831
<210> 185
<211> 1155
<212> DNA
<213> 未知
<220>
<223> dcaA(Ab)
<400> 185
atgattcgtg atgaaggtat gctgcaacag ctgctgagca ccattcgtga ttttgttaaa 60
aatgaactga ttccgcgtga acatgaagtg gcagaaaaag attgtattcc cgaagatatt 120
attcagcaga tgcgtgaact gggtctgttt ggtctgacca ttccggaaga atatggtggt 180
ctgggtatta ccatggaaga agaagttaac gttgcatttg aactgggcca gaccagtccg 240
gcatttcgta gcctgattgg caccaataat ggtattggta gcagcggtct gattattgat 300
ggcaccgaag aacagaaaca gaaatatctg cctcgttatg caagcggtga aattattggt 360
agtttttgtc tgaccgaacc ggaagcaggt agtgatgcag caagcctgaa aaccaccgca 420
gttaaagatg gtgatttcta tattctgaac ggcaccaaac gctttattac caatgcaccg 480
catgcagcaa cctttaccgt tatggcacgt accaatccgg caattaaagg tgccggtggt 540
attagcgcat ttctggttga agcaaataca ccgggtatta cgctgggtaa aattgatcag 600
aaaatgggtc agaaaggtag ccatacctgt gatgtgattt ttgaaaattg tcgtgttccg 660
gcaagcgcac tgattggtgg tgttgaaggt gttggcttta aaaccgcaat gaaagttctg 720
gataaaggtc gtctgcatat tggtgcatat agcgttggtg ttgcagaacg tatgctgaat 780
gatgcactgc attatgccgt tgaacgtaaa cagtttggtc agccgattgc aaattttcag 840
ctgattcagg caatgctggc agatagcaaa gcagaaattt atgcagccaa atgcatggtt 900
ctggatgcag cacgtcgtcg tgatgagggt cagaatatta gcaccgaagc aagctgtgca 960
aaaatgtttg caaccgaaat gtgtggtcgt gttgccgatc gttgtgttca gattcatggt 1020
ggtgcaggtt atattagcga atacagcatt gaacgcttct atcgtgatgt gcgtctgttt 1080
cgcctgtatg aaggtacaac ccaggtgcag cagattatta tcgccaaaaa catgatcaaa 1140
gaggtgacca gctaa 1155
<210> 186
<211> 1158
<212> DNA
<213> 未知
<220>
<223> Tfu1647
<400> 186
atgagtgact tcgacctcta ccggcccacc gaggagcacg aagccctccg cgaagccatc 60
cgctccgtcg ctgaagacaa gatcgccccg cacgcggccg acgtcgacga gcagtcccgc 120
ttcccgcagg aggcctacga ggcgctgcgc gccagcgact tccacgctcc ccacgtcgcg 180
gaggagtacg gcggtgttgg cgcagacgcg ctggcgacct gcatcgtcat tgaggagatc 240
gcccgggtgt gcgcctcctc ttccctcatc cccgcggtca acaagctcgg ctcgatgccg 300
ctgatcctct ccggatctga cgaggtgaag cagcgctacc tgcccgaact ggcatctggc 360
gaggcgatgt tctcctacgg gctctccgag cgcgaggcgg gatccgacac cgcgagcatg 420
cgcacccgtg ccgtccgcga cggcgacgac tggatcctca acggccagaa gtcgtggatc 480
acgaacgccg gaatctccaa gtactacacg gtgatggcgg tcaccgaccc ggacgggccg 540
cgcggacgca acatcagcgc tttcgtcgtc cacatcgacg atcccgggtt ctcttttgga 600
gaaccggaac gcaagctcgg catcaaaggt tcgcccacgc gcgaactgat cttcgacaat 660
gtccgtatcc ccggcgaccg cctggtgggc aaggtcgggg aaggactgcg taccgcgctg 720
cgcaccctcg accacacccg cgtcaccatt ggcgcccagg ctgtgggcat cgcccagggg 780
gcgctcgact acgccctggg ctatgtcaag gagcgcaagc agttcggcaa ggccatcgcc 840
gacttccaag gcatccagtt catgctcgcc gacatggcca tgaagctgga agcggcccgc 900
cagatggttt acgtcgctgc cgccaagtcc gagcgcgacg acgccgacct ctccttctac 960
ggtgccgccg ctaaatgctt cgcctcggat gtggcaatgg agatcaccac tgacgcggtg 1020
caactactcg gcggctacgg ctacacccgg gactacccgg tcgagcggat gatgcgcgac 1080
gccaagatca cccagatcta cgagggcacc aaccagatcc agcgggtggt catggcccgg 1140
cagctcttga agaagtga 1158
<210> 187
<211> 873
<212> DNA
<213> 未知
<220>
<223> tesB(Ab)
<400> 187
atgaatacac taacccaaga actggttgaa cttttatctc tagaaaagct ggaagagaac 60
ctctatcgcg ggatgagccg aaatctggtg gggaagcgcg tattcggtgg tcaggttctt 120
ggacaagcat taagagctgc gtcatatacg acagatcgtc ctgcacattc attacatgct 180
tactttctct atggtggcga tgttaacgcc cctattattt atgaagtaga tcctttacgt 240
gatggtaaaa gttttgtgag tcgtcaggtt cgcgctattc agcatggacg tactattttt 300
tctgcgatgg tttcttttgc atcaccagaa gaagggctta actatcaaaa tgatatgcct 360
gattatcctg cacctgagca gcttaaatct gaagcggagt taaaactggg gctgattgat 420
tttgtacctg aaaatgtacg tgccagtttt atgcgtgaac gccacataga gattcgtcca 480
gttgagccag ttaatccctt tcagcctcaa cctcaagcac cgactaacgc ccactatatc 540
cgtacccatg acaagatcgg aaaagcgttt gaccagattg cgctacacca gtctattgtt 600
gctttttatt cagactttac tttaatgaca acagcactta aaccgcatgg tttaagctat 660
ctttcaccga gtttgcagtg tgccagtata gatcacacca tttattttca tcgtccgtta 720
cgtgcagatg aatggatgct ctacgacatg gatgccacgg tcagtgctgg ctcaagaggt 780
ttgaactttg ggcgaatgtg gcaaaatgga ttattggttt gtagcaccgt acaagaaggt 840
ttaatgcgcc ttcgtgaaat tgaaacacag taa 873
<210> 188
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaJ(Ec) IF
<400> 188
ataaggagat ataccatgcg tgaagccttt atttg 35
<210> 189
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaJ(Ec) IF
<400> 189
tgcggccgca agctttcaaa cacgctccag aatca 35
<210> 190
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pRSFDuet-P1
<400> 190
aagcttgcgg ccgcataatg cttaa 25
<210> 191
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pRSFDuet-P1
<400> 191
catggtatat ctccttatta 20
<210> 192
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaH(Ec) IF
<400> 192
ggagatatac atatgatgat aaatgtgcaa actgt 35
<210> 193
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaH(Ec) IF
<400> 193
agactcgagg gtaccttatg actcataacc gctct 35
<210> 194
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pRSFDuet-P2
<400> 194
ggtaccctcg agtctggtaa agaaa 25
<210> 195
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pRSFDuet-P2
<400> 195
catatgtata tctccttctt 20
<210> 196
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaF(Ec) IF
<400> 196
aggagatata ccatgagcga actgatcgtc agccg 35
<210> 197
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaF(Ec) IF
<400> 197
tgcggccgca agcttttagc gtcctttaaa gtcgg 35
<210> 198
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaF(L3)opt
<400> 198
agaaggagat ataccatgca gaccattgaa aatga 35
<210> 199
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaF(L3)opt
<400> 199
tgcggccgca agcttttaac gaccggcata ggtcg 35
<210> 200
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ crt1(Ck)opt
<400> 200
agaaggagat ataccatgga attcaagaac atcat 35
<210> 201
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ crt1(Ck)opt
<400> 201
tgcggccgca agcttttatt tgtttttaaa ggctt 35
<210> 202
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ ech(Pp)opt
<400> 202
agaaggagat ataccatgag caaatatgaa ggtcg 35
<210> 203
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ ech(Pp)opt
<400> 203
tgcggccgca agcttttaac gtttatatgc ctgca 35
<210> 204
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pETDuet-P1
<400> 204
aagcttgcgg ccgcataatg cttaa 25
<210> 205
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pETDuet-P1
<400> 205
catggtatat ctccttctta aagtt 25
<210> 206
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ dcaA(Ab)
<400> 206
gaaggagata tacatatgat tcgtgatgaa ggtat 35
<210> 207
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ dcaA(Ab)
<400> 207
tggcagcagc ctaggttagc tggtcacctc tttga 35
<210> 208
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ Tfu1647
<400> 208
gaaggagata tacatatgag tgacttcgac ctcta 35
<210> 209
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ Tfu1647
<400> 209
tggcagcagc ctaggtcact tcttcaagag ctgcc 35
<210> 210
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ mmgC(L3)opt
<400> 210
gaaggagata tacatatgat ccgtgatcaa gaaac 35
<210> 211
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ mmgC(L3)opt
<400> 211
tggcagcagc ctaggttaca cctggacatc acgaa 35
<210> 212
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pETDuet-P2
<400> 212
cctaggctgc tgccaccgct gagca 25
<210> 213
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pETDuet-P2
<400> 213
catatgtata tctccttctt atact 25
<210> 214
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ tesB(Ab)
<400> 214
ataaggagat ataccatgaa tacactaacc caaga 35
<210> 215
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ tesB(Ab)
<400> 215
tgcggccgca agcttttact gtgtttcaat ttcac 35
<210> 216
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pCDFDuet
<400> 216
aagcttgcgg ccgcataatg cttaa 25
<210> 217
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pCDFDuet
<400> 217
catggtatat ctccttatta 20
<210> 218
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ ldh
<400> 218
ttgttgaata aatcgtttat cgatattgat ccagg 35
<210> 219
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ ldh
<400> 219
tatcgcatgc agatctgtgt gcattaccca acggc 35
<210> 220
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ atoB
<400> 220
ttgttgaata aatcggcact cggtatcgct tacct 35
<210> 221
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ atoB
<400> 221
tatcgcatgc agatcggctg gtggtcaacg gtgcc 35
<210> 222
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ sucD
<400> 222
ttgttgaata aatcggagcg cgacctggcg ttgat 35
<210> 223
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ sucD
<400> 223
tatcgcatgc agatcacgat atttttcagt taacc 35
<210> 224
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pHAK1
<400> 224
gatctgcatg cgatatctct agaacgcgta agctt 35
<210> 225
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物/ pHAK1
<400> 225
tctcgagccg atttattcaa caaagccgc 29
<210> 226
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物/ del-ldh
<400> 226
tggtttaatc ttgccgctcc 20
<210> 227
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 引物/ del-ldh
<400> 227
gtgctataaa cggcgagttt ca 22
<210> 228
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物/ del-atoB
<400> 228
cgatggtgat tgaacggttg 20
<210> 229
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物/ del-atoB
<400> 229
gcactgacga tgacacaatt 20
<210> 230
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物/ del-sucD
<400> 230
gaaattcgct gctctggaag 20
<210> 231
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物/ del-sucD
<400> 231
cctggcagat aaccttggtg ttt 23
<210> 232
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaJ(L3)opt
<400> 232
ataaggagat ataccatgct ggatgcatat ctgta 35
<210> 233
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaJ(L3)opt
<400> 233
tgcggccgca agcttttaca tacgttcaat aacaa 35
<210> 234
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaH(L3)opt
<400> 234
gaaggagata tacatatgaa caccgatatt cagac 35
<210> 235
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物/ paaH(L3)opt
<400> 235
agactcgagg gtaccttaat tatccggtgt cagca 35
<210> 236
<211> 453
<212> PRT
<213> 克氏梭菌
<400> 236
Met Ser Asn Glu Val Ser Ile Lys Glu Leu Ile Glu Lys Ala Lys Val
1 5 10 15
Ala Gln Lys Lys Leu Glu Ala Tyr Ser Gln Glu Gln Val Asp Val Leu
20 25 30
Val Lys Ala Leu Gly Lys Val Val Tyr Asp Asn Ala Glu Met Phe Ala
35 40 45
Lys Glu Ala Val Glu Glu Thr Glu Met Gly Val Tyr Glu Asp Lys Val
50 55 60
Ala Lys Cys His Leu Lys Ser Gly Ala Ile Trp Asn His Ile Lys Asp
65 70 75 80
Lys Lys Thr Val Gly Ile Ile Lys Glu Glu Pro Glu Arg Ala Leu Val
85 90 95
Tyr Val Ala Lys Pro Lys Gly Val Val Ala Ala Thr Thr Pro Ile Thr
100 105 110
Asn Pro Val Val Thr Pro Met Cys Asn Ala Met Ala Ala Ile Lys Gly
115 120 125
Arg Asn Thr Ile Ile Val Ala Pro His Pro Lys Ala Lys Lys Val Ser
130 135 140
Ala His Thr Val Glu Leu Met Asn Ala Glu Leu Lys Lys Leu Gly Ala
145 150 155 160
Pro Glu Asn Ile Ile Gln Ile Val Glu Ala Pro Ser Arg Glu Ala Ala
165 170 175
Lys Glu Leu Met Glu Ser Ala Asp Val Val Ile Ala Thr Gly Gly Ala
180 185 190
Gly Arg Val Lys Ala Ala Tyr Ser Ser Gly Arg Pro Ala Tyr Gly Val
195 200 205
Gly Pro Gly Asn Ser Gln Val Ile Val Asp Lys Gly Tyr Asp Tyr Asn
210 215 220
Lys Ala Ala Gln Asp Ile Ile Thr Gly Arg Lys Tyr Asp Asn Gly Ile
225 230 235 240
Ile Cys Ser Ser Glu Gln Ser Val Ile Ala Pro Ala Glu Asp Tyr Asp
245 250 255
Lys Val Ile Ala Ala Phe Val Glu Asn Gly Ala Phe Tyr Val Glu Asp
260 265 270
Glu Glu Thr Val Glu Lys Phe Arg Ser Thr Leu Phe Lys Asp Gly Lys
275 280 285
Ile Asn Ser Lys Ile Ile Gly Lys Ser Val Gln Ile Ile Ala Asp Leu
290 295 300
Ala Gly Val Lys Val Pro Glu Gly Thr Lys Val Ile Val Leu Lys Gly
305 310 315 320
Lys Gly Ala Gly Glu Lys Asp Val Leu Cys Lys Glu Lys Met Cys Pro
325 330 335
Val Leu Val Ala Leu Lys Tyr Asp Thr Phe Glu Glu Ala Val Glu Ile
340 345 350
Ala Met Ala Asn Tyr Met Tyr Glu Gly Ala Gly His Thr Ala Gly Ile
355 360 365
His Ser Asp Asn Asp Glu Asn Ile Arg Tyr Ala Gly Thr Val Leu Pro
370 375 380
Ile Ser Arg Leu Val Val Asn Gln Pro Ala Thr Thr Ala Gly Gly Ser
385 390 395 400
Phe Asn Asn Gly Phe Asn Pro Thr Thr Thr Leu Gly Cys Gly Ser Trp
405 410 415
Gly Arg Asn Ser Ile Ser Glu Asn Leu Thr Tyr Glu His Leu Ile Asn
420 425 430
Val Ser Arg Ile Gly Tyr Phe Asn Lys Glu Ala Lys Val Pro Ser Tyr
435 440 445
Glu Glu Ile Trp Gly
450
<210> 237
<211> 1174
<212> PRT
<213> 未知
<220>
<223> CAR
<400> 237
Met Ala Val Asp Ser Pro Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln
1 5 10 15
Leu Phe Ala Glu Asp Glu Gln Val Lys Ala Ala Arg Pro Leu Glu Ala
20 25 30
Val Ser Ala Ala Val Ser Ala Pro Gly Met Arg Leu Ala Gln Ile Ala
35 40 45
Ala Thr Val Met Ala Gly Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg
50 55 60
Ala Phe Glu Leu Asn Thr Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg
65 70 75 80
Leu Leu Pro Arg Phe Glu Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg
85 90 95
Val Gly Glu Val Ala Ala Ala Trp His His Asp Pro Glu Asn Pro Leu
100 105 110
Arg Ala Gly Asp Phe Val Ala Leu Leu Gly Phe Thr Ser Ile Asp Tyr
115 120 125
Ala Thr Leu Asp Leu Ala Asp Ile His Leu Gly Ala Val Thr Val Pro
130 135 140
Leu Gln Ala Ser Ala Ala Val Ser Gln Leu Ile Ala Ile Leu Thr Glu
145 150 155 160
Thr Ser Pro Arg Leu Leu Ala Ser Thr Pro Glu His Leu Asp Ala Ala
165 170 175
Val Glu Cys Leu Leu Ala Gly Thr Thr Pro Glu Arg Leu Val Val Phe
180 185 190
Asp Tyr His Pro Glu Asp Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala
195 200 205
Arg Arg Arg Leu Ala Asp Ala Gly Ser Ser Val Ile Val Glu Thr Leu
210 215 220
Asp Ala Val Arg Ala Arg Gly Arg Asp Leu Pro Ala Ala Pro Leu Phe
225 230 235 240
Val Pro Asp Thr Asp Asp Asp Pro Leu Ala Leu Leu Ile Tyr Thr Ser
245 250 255
Gly Ser Thr Gly Thr Pro Lys Gly Ala Met Tyr Thr Asn Arg Leu Ala
260 265 270
Ala Thr Met Trp Gln Gly Asn Ser Met Leu Gln Gly Asn Ser Gln Arg
275 280 285
Val Gly Ile Asn Leu Asn Tyr Met Pro Met Ser His Ile Ala Gly Arg
290 295 300
Ile Ser Leu Phe Gly Val Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala
305 310 315 320
Ala Lys Ser Asp Met Ser Thr Leu Phe Glu Asp Ile Gly Leu Val Arg
325 330 335
Pro Thr Glu Ile Phe Phe Val Pro Arg Val Cys Asp Met Val Phe Gln
340 345 350
Arg Tyr Gln Ser Glu Leu Asp Arg Arg Ser Val Ala Gly Ala Asp Leu
355 360 365
Asp Thr Leu Asp Arg Glu Val Lys Ala Asp Leu Arg Gln Asn Tyr Leu
370 375 380
Gly Gly Arg Phe Leu Val Ala Val Val Gly Ser Ala Pro Leu Ala Ala
385 390 395 400
Glu Met Lys Thr Phe Met Glu Ser Val Leu Asp Leu Pro Leu His Asp
405 410 415
Gly Tyr Gly Ser Thr Glu Ala Gly Ala Ser Val Leu Leu Asp Asn Gln
420 425 430
Ile Gln Arg Pro Pro Val Leu Asp Tyr Lys Leu Val Asp Val Pro Glu
435 440 445
Leu Gly Tyr Phe Arg Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu
450 455 460
Leu Lys Ala Glu Thr Thr Ile Pro Gly Tyr Tyr Lys Arg Pro Glu Val
465 470 475 480
Thr Ala Glu Ile Phe Asp Glu Asp Gly Phe Tyr Lys Thr Gly Asp Ile
485 490 495
Val Ala Glu Leu Glu His Asp Arg Leu Val Tyr Val Asp Arg Arg Asn
500 505 510
Asn Val Leu Lys Leu Ser Gln Gly Glu Phe Val Thr Val Ala His Leu
515 520 525
Glu Ala Val Phe Ala Ser Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr
530 535 540
Gly Ser Ser Glu Arg Ser Tyr Leu Leu Ala Val Ile Val Pro Thr Asp
545 550 555 560
Asp Ala Leu Arg Gly Arg Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala
565 570 575
Glu Ser Ile Gln Arg Ile Ala Lys Asp Ala Asn Leu Gln Pro Tyr Glu
580 585 590
Ile Pro Arg Asp Phe Leu Ile Glu Thr Glu Pro Phe Thr Ile Ala Asn
595 600 605
Gly Leu Leu Ser Gly Ile Ala Lys Leu Leu Arg Pro Asn Leu Lys Glu
610 615 620
Arg Tyr Gly Ala Gln Leu Glu Gln Met Tyr Thr Asp Leu Ala Thr Gly
625 630 635 640
Gln Ala Asp Glu Leu Leu Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro
645 650 655
Val Leu Glu Thr Val Ser Arg Ala Ala Lys Ala Met Leu Gly Val Ala
660 665 670
Ser Ala Asp Met Arg Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp
675 680 685
Ser Leu Ser Ala Leu Ser Phe Ser Asn Leu Leu His Glu Ile Phe Gly
690 695 700
Val Glu Val Pro Val Gly Val Val Val Ser Pro Ala Asn Glu Leu Arg
705 710 715 720
Asp Leu Ala Asn Tyr Ile Glu Ala Glu Arg Asn Ser Gly Ala Lys Arg
725 730 735
Pro Thr Phe Thr Ser Val His Gly Gly Gly Ser Glu Ile Arg Ala Ala
740 745 750
Asp Leu Thr Leu Asp Lys Phe Ile Asp Ala Arg Thr Leu Ala Ala Ala
755 760 765
Asp Ser Ile Pro His Ala Pro Val Pro Ala Gln Thr Val Leu Leu Thr
770 775 780
Gly Ala Asn Gly Tyr Leu Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu
785 790 795 800
Arg Leu Asp Lys Thr Gly Gly Thr Leu Ile Cys Val Val Arg Gly Ser
805 810 815
Asp Ala Ala Ala Ala Arg Lys Arg Leu Asp Ser Ala Phe Asp Ser Gly
820 825 830
Asp Pro Gly Leu Leu Glu His Tyr Gln Gln Leu Ala Ala Arg Thr Leu
835 840 845
Glu Val Leu Ala Gly Asp Ile Gly Asp Pro Asn Leu Gly Leu Asp Asp
850 855 860
Ala Thr Trp Gln Arg Leu Ala Glu Thr Val Asp Leu Ile Val His Pro
865 870 875 880
Ala Ala Leu Val Asn His Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro
885 890 895
Asn Val Val Gly Thr Ala Glu Ile Val Arg Leu Ala Ile Thr Ala Arg
900 905 910
Arg Lys Pro Val Thr Tyr Leu Ser Thr Val Gly Val Ala Asp Gln Val
915 920 925
Asp Pro Ala Glu Tyr Gln Glu Asp Ser Asp Val Arg Glu Met Ser Ala
930 935 940
Val Arg Val Val Arg Glu Ser Tyr Ala Asn Gly Tyr Gly Asn Ser Lys
945 950 955 960
Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys Gly Leu
965 970 975
Pro Val Ala Val Phe Arg Ser Asp Met Ile Leu Ala His Ser Arg Tyr
980 985 990
Ala Gly Gln Leu Asn Val Gln Asp Val Phe Thr Arg Leu Ile Leu Ser
995 1000 1005
Leu Val Ala Thr Gly Ile Ala Pro Tyr Ser Phe Tyr Arg Thr Asp
1010 1015 1020
Ala Asp Gly Asn Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Ala
1025 1030 1035
Asp Phe Thr Ala Ala Ala Ile Thr Ala Leu Gly Ile Gln Ala Thr
1040 1045 1050
Glu Gly Phe Arg Thr Tyr Asp Val Leu Asn Pro Tyr Asp Asp Gly
1055 1060 1065
Ile Ser Leu Asp Glu Phe Val Asp Trp Leu Val Glu Ser Gly His
1070 1075 1080
Pro Ile Gln Arg Ile Thr Asp Tyr Ser Asp Trp Phe His Arg Phe
1085 1090 1095
Glu Thr Ala Ile Arg Ala Leu Pro Glu Lys Gln Arg Gln Ala Ser
1100 1105 1110
Val Leu Pro Leu Leu Asp Ala Tyr Arg Asn Pro Cys Pro Ala Val
1115 1120 1125
Arg Gly Ala Ile Leu Pro Ala Lys Glu Phe Gln Ala Ala Val Gln
1130 1135 1140
Thr Ala Lys Ile Gly Pro Glu Gln Asp Ile Pro His Leu Ser Ala
1145 1150 1155
Pro Leu Ile Asp Lys Tyr Val Ser Asp Leu Glu Leu Leu Gln Leu
1160 1165 1170
Leu
<210> 238
<211> 1174
<212> PRT
<213> 未知
<220>
<223> CAR
<400> 238
Met Ser Pro Ile Thr Arg Glu Glu Arg Leu Glu Arg Arg Ile Gln Asp
1 5 10 15
Leu Tyr Ala Asn Asp Pro Gln Phe Ala Ala Ala Lys Pro Ala Thr Ala
20 25 30
Ile Thr Ala Ala Ile Glu Arg Pro Gly Leu Pro Leu Pro Gln Ile Ile
35 40 45
Glu Thr Val Met Thr Gly Tyr Ala Asp Arg Pro Ala Leu Ala Gln Arg
50 55 60
Ser Val Glu Phe Val Thr Asp Ala Gly Thr Gly His Thr Thr Leu Arg
65 70 75 80
Leu Leu Pro His Phe Glu Thr Ile Ser Tyr Gly Glu Leu Trp Asp Arg
85 90 95
Ile Ser Ala Leu Ala Asp Val Leu Ser Thr Glu Gln Thr Val Lys Pro
100 105 110
Gly Asp Arg Val Cys Leu Leu Gly Phe Asn Ser Val Asp Tyr Ala Thr
115 120 125
Ile Asp Met Thr Leu Ala Arg Leu Gly Ala Val Ala Val Pro Leu Gln
130 135 140
Thr Ser Ala Ala Ile Thr Gln Leu Gln Pro Ile Val Ala Glu Thr Gln
145 150 155 160
Pro Thr Met Ile Ala Ala Ser Val Asp Ala Leu Ala Asp Ala Thr Glu
165 170 175
Leu Ala Leu Ser Gly Gln Thr Ala Thr Arg Val Leu Val Phe Asp His
180 185 190
His Arg Gln Val Asp Ala His Arg Ala Ala Val Glu Ser Ala Arg Glu
195 200 205
Arg Leu Ala Gly Ser Ala Val Val Glu Thr Leu Ala Glu Ala Ile Ala
210 215 220
Arg Gly Asp Val Pro Arg Gly Ala Ser Ala Gly Ser Ala Pro Gly Thr
225 230 235 240
Asp Val Ser Asp Asp Ser Leu Ala Leu Leu Ile Tyr Thr Ser Gly Ser
245 250 255
Thr Gly Ala Pro Lys Gly Ala Met Tyr Pro Arg Arg Asn Val Ala Thr
260 265 270
Phe Trp Arg Lys Arg Thr Trp Phe Glu Gly Gly Tyr Glu Pro Ser Ile
275 280 285
Thr Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gln Ile Leu
290 295 300
Tyr Gly Thr Leu Cys Asn Gly Gly Thr Ala Tyr Phe Val Ala Lys Ser
305 310 315 320
Asp Leu Ser Thr Leu Phe Glu Asp Leu Ala Leu Val Arg Pro Thr Glu
325 330 335
Leu Thr Phe Val Pro Arg Val Trp Asp Met Val Phe Asp Glu Phe Gln
340 345 350
Ser Glu Val Asp Arg Arg Leu Val Asp Gly Ala Asp Arg Val Ala Leu
355 360 365
Glu Ala Gln Val Lys Ala Glu Ile Arg Asn Asp Val Leu Gly Gly Arg
370 375 380
Tyr Thr Ser Ala Leu Thr Gly Ser Ala Pro Ile Ser Asp Glu Met Lys
385 390 395 400
Ala Trp Val Glu Glu Leu Leu Asp Met His Leu Val Glu Gly Tyr Gly
405 410 415
Ser Thr Glu Ala Gly Met Ile Leu Ile Asp Gly Ala Ile Arg Arg Pro
420 425 430
Ala Val Leu Asp Tyr Lys Leu Val Asp Val Pro Asp Leu Gly Tyr Phe
435 440 445
Leu Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu Val Lys Thr Asp
450 455 460
Ser Leu Phe Pro Gly Tyr Tyr Gln Arg Ala Glu Val Thr Ala Asp Val
465 470 475 480
Phe Asp Ala Asp Gly Phe Tyr Arg Thr Gly Asp Ile Met Ala Glu Val
485 490 495
Gly Pro Glu Gln Phe Val Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys
500 505 510
Leu Ser Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe
515 520 525
Gly Asp Ser Pro Leu Val Arg Gln Ile Tyr Ile Tyr Gly Asn Ser Ala
530 535 540
Arg Ala Tyr Leu Leu Ala Val Ile Val Pro Thr Gln Glu Ala Leu Asp
545 550 555 560
Ala Val Pro Val Glu Glu Leu Lys Ala Arg Leu Gly Asp Ser Leu Gln
565 570 575
Glu Val Ala Lys Ala Ala Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp
580 585 590
Phe Ile Ile Glu Thr Thr Pro Trp Thr Leu Glu Asn Gly Leu Leu Thr
595 600 605
Gly Ile Arg Lys Leu Ala Arg Pro Gln Leu Lys Lys His Tyr Gly Glu
610 615 620
Leu Leu Glu Gln Ile Tyr Thr Asp Leu Ala His Gly Gln Ala Asp Glu
625 630 635 640
Leu Arg Ser Leu Arg Gln Ser Gly Ala Asp Ala Pro Val Leu Val Thr
645 650 655
Val Cys Arg Ala Ala Ala Ala Leu Leu Gly Gly Ser Ala Ser Asp Val
660 665 670
Gln Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala
675 680 685
Leu Ser Phe Thr Asn Leu Leu His Glu Ile Phe Asp Ile Glu Val Pro
690 695 700
Val Gly Val Ile Val Ser Pro Ala Asn Asp Leu Gln Ala Leu Ala Asp
705 710 715 720
Tyr Val Glu Ala Ala Arg Lys Pro Gly Ser Ser Arg Pro Thr Phe Ala
725 730 735
Ser Val His Gly Ala Ser Asn Gly Gln Val Thr Glu Val His Ala Gly
740 745 750
Asp Leu Ser Leu Asp Lys Phe Ile Asp Ala Ala Thr Leu Ala Glu Ala
755 760 765
Pro Arg Leu Pro Ala Ala Asn Thr Gln Val Arg Thr Val Leu Leu Thr
770 775 780
Gly Ala Thr Gly Phe Leu Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu
785 790 795 800
Arg Met Asp Leu Val Asp Gly Lys Leu Ile Cys Leu Val Arg Ala Lys
805 810 815
Ser Asp Thr Glu Ala Arg Ala Arg Leu Asp Lys Thr Phe Asp Ser Gly
820 825 830
Asp Pro Glu Leu Leu Ala His Tyr Arg Ala Leu Ala Gly Asp His Leu
835 840 845
Glu Val Leu Ala Gly Asp Lys Gly Glu Ala Asp Leu Gly Leu Asp Arg
850 855 860
Gln Thr Trp Gln Arg Leu Ala Asp Thr Val Asp Leu Ile Val Asp Pro
865 870 875 880
Ala Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu Phe Gly Pro
885 890 895
Asn Ala Leu Gly Thr Ala Glu Leu Leu Arg Leu Ala Leu Thr Ser Lys
900 905 910
Ile Lys Pro Tyr Ser Tyr Thr Ser Thr Ile Gly Val Ala Asp Gln Ile
915 920 925
Pro Pro Ser Ala Phe Thr Glu Asp Ala Asp Ile Arg Val Ile Ser Ala
930 935 940
Thr Arg Ala Val Asp Asp Ser Tyr Ala Asn Gly Tyr Ser Asn Ser Lys
945 950 955 960
Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys Gly Leu
965 970 975
Pro Val Ala Val Phe Arg Cys Asp Met Ile Leu Ala Asp Thr Thr Trp
980 985 990
Ala Gly Gln Leu Asn Val Pro Asp Met Phe Thr Arg Met Ile Leu Ser
995 1000 1005
Leu Ala Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Glu Leu Ala
1010 1015 1020
Ala Asp Gly Ala Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Val
1025 1030 1035
Glu Phe Ile Ala Glu Ala Ile Ser Thr Leu Gly Ala Gln Ser Gln
1040 1045 1050
Asp Gly Phe His Thr Tyr His Val Met Asn Pro Tyr Asp Asp Gly
1055 1060 1065
Ile Gly Leu Asp Glu Phe Val Asp Trp Leu Asn Glu Ser Gly Cys
1070 1075 1080
Pro Ile Gln Arg Ile Ala Asp Tyr Gly Asp Trp Leu Gln Arg Phe
1085 1090 1095
Glu Thr Ala Leu Arg Ala Leu Pro Asp Arg Gln Arg His Ser Ser
1100 1105 1110
Leu Leu Pro Leu Leu His Asn Tyr Arg Gln Pro Glu Arg Pro Val
1115 1120 1125
Arg Gly Ser Ile Ala Pro Thr Asp Arg Phe Arg Ala Ala Val Gln
1130 1135 1140
Glu Ala Lys Ile Gly Pro Asp Lys Asp Ile Pro His Val Gly Ala
1145 1150 1155
Pro Ile Ile Val Lys Tyr Val Ser Asp Leu Arg Leu Leu Gly Leu
1160 1165 1170
Leu
<210> 239
<211> 1178
<212> PRT
<213> 脓肿分枝杆菌
<400> 239
Met Thr Glu Thr Ile Ser Thr Ala Ala Val Pro Thr Thr Asp Leu Glu
1 5 10 15
Glu Gln Val Lys Arg Arg Ile Glu Gln Val Val Ser Asn Asp Pro Gln
20 25 30
Leu Ala Ala Leu Leu Pro Glu Asp Ser Val Thr Glu Ala Val Asn Glu
35 40 45
Pro Asp Leu Pro Leu Val Glu Val Ile Arg Arg Leu Leu Glu Gly Tyr
50 55 60
Gly Asp Arg Pro Ala Leu Gly Gln Arg Ala Phe Glu Phe Val Thr Gly
65 70 75 80
Asp Asp Gly Ala Thr Val Ile Ala Leu Lys Pro Glu Tyr Thr Thr Val
85 90 95
Ser Tyr Arg Glu Leu Trp Glu Arg Ala Glu Ala Ile Ala Ala Ala Trp
100 105 110
His Glu Gln Gly Ile Arg Asp Gly Asp Phe Val Ala Gln Leu Gly Phe
115 120 125
Thr Ser Thr Asp Phe Ala Ser Leu Asp Val Ala Gly Leu Arg Leu Gly
130 135 140
Thr Val Ser Val Pro Leu Gln Thr Gly Ala Ser Leu Gln Gln Arg Asn
145 150 155 160
Ala Ile Leu Glu Glu Thr Arg Pro Ala Val Phe Ala Ala Ser Ile Glu
165 170 175
Tyr Leu Asp Ala Ala Val Asp Ser Val Leu Ala Thr Pro Ser Val Arg
180 185 190
Leu Leu Ser Val Phe Asp Tyr His Ala Glu Val Asp Ser Gln Arg Glu
195 200 205
Ala Leu Glu Ala Val Arg Ala Arg Leu Glu Ser Ala Gly Arg Thr Ile
210 215 220
Val Val Glu Ala Leu Ala Glu Ala Leu Ala Arg Gly Arg Asp Leu Pro
225 230 235 240
Ala Ala Pro Leu Pro Ser Ala Asp Pro Asp Ala Leu Arg Leu Leu Ile
245 250 255
Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys Gly Ala Met Tyr Pro Gln
260 265 270
Trp Leu Val Ala Asn Leu Trp Gln Lys Lys Trp Leu Thr Asp Asp Val
275 280 285
Ile Pro Ser Ile Gly Val Asn Phe Met Pro Met Ser His Leu Ala Gly
290 295 300
Arg Leu Thr Leu Met Gly Thr Leu Ser Gly Gly Gly Thr Ala Tyr Tyr
305 310 315 320
Ile Ala Ser Ser Asp Leu Ser Thr Phe Phe Glu Asp Ile Ala Leu Ile
325 330 335
Arg Pro Ser Glu Val Leu Phe Val Pro Arg Val Val Glu Met Val Phe
340 345 350
Gln Arg Phe Gln Ala Glu Leu Asp Arg Ser Leu Ala Pro Gly Glu Ser
355 360 365
Asn Ser Glu Ile Ala Glu Arg Ile Lys Val Arg Ile Arg Glu Gln Asp
370 375 380
Phe Gly Gly Arg Val Leu Ser Ala Gly Ser Gly Ser Ala Pro Leu Ser
385 390 395 400
Pro Glu Met Thr Glu Phe Met Glu Ser Leu Leu Gln Val Pro Leu Arg
405 410 415
Asp Gly Tyr Gly Ser Thr Glu Ala Gly Gly Val Trp Arg Asp Gly Val
420 425 430
Leu Gln Arg Pro Pro Val Thr Asp Tyr Lys Leu Val Asp Val Pro Glu
435 440 445
Leu Gly Tyr Phe Thr Thr Asp Ser Pro His Pro Arg Gly Glu Leu Arg
450 455 460
Leu Lys Ser Glu Thr Met Phe Pro Gly Tyr Tyr Lys Arg Pro Glu Thr
465 470 475 480
Thr Ala Asp Val Phe Asp Asp Glu Gly Tyr Tyr Lys Thr Gly Asp Val
485 490 495
Val Ala Glu Leu Gly Pro Asp His Leu Lys Tyr Leu Asp Arg Val Lys
500 505 510
Asn Val Leu Lys Leu Ala Gln Gly Glu Phe Val Ala Val Ser Lys Leu
515 520 525
Glu Ala Ala Tyr Thr Gly Ser Pro Leu Val Arg Gln Ile Phe Val Tyr
530 535 540
Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val Val Val Pro Thr Pro
545 550 555 560
Glu Val Leu Glu Arg Tyr Ala Asp Ser Pro Asp Ala Leu Lys Pro Leu
565 570 575
Ile Gln Asp Ser Leu Gln Gln Val Ala Lys Asp Ala Glu Leu Gln Ser
580 585 590
Tyr Glu Ile Pro Arg Asp Phe Ile Val Glu Thr Val Pro Phe Thr Val
595 600 605
Glu Ser Gly Leu Leu Ser Asp Ala Arg Lys Leu Leu Arg Pro Lys Leu
610 615 620
Lys Asp His Tyr Gly Glu Arg Leu Glu Ala Leu Tyr Ala Glu Leu Ala
625 630 635 640
Glu Ser Gln Asn Glu Arg Leu Arg Gln Leu Ala Arg Glu Ala Ala Thr
645 650 655
Arg Pro Val Leu Glu Thr Val Thr Asp Ala Ala Ala Ala Leu Leu Gly
660 665 670
Ala Ser Ser Ser Asp Leu Ala Pro Asp Val Arg Phe Ile Asp Leu Gly
675 680 685
Gly Asp Ser Leu Ser Ala Leu Ser Tyr Ser Glu Leu Leu Arg Asp Ile
690 695 700
Phe Glu Val Asp Val Pro Val Gly Val Ile Asn Ser Val Ala Asn Asp
705 710 715 720
Leu Ala Ala Ile Ala Arg His Ile Glu Ala Gln Arg Thr Gly Ala Ala
725 730 735
Thr Gln Pro Thr Phe Ala Ser Val His Gly Lys Asp Ala Thr Val Ile
740 745 750
Thr Ala Gly Glu Leu Thr Leu Asp Lys Phe Leu Asp Glu Ser Leu Leu
755 760 765
Lys Ala Ala Lys Asp Val Gln Pro Ala Thr Ala Asp Val Lys Thr Val
770 775 780
Leu Val Thr Gly Gly Asn Gly Trp Leu Gly Arg Trp Leu Val Leu Asp
785 790 795 800
Trp Leu Glu Arg Leu Ala Pro Asn Gly Gly Lys Val Tyr Ala Leu Ile
805 810 815
Arg Gly Ala Asp Ala Glu Ala Ala Arg Ala Arg Leu Asp Ala Val Tyr
820 825 830
Glu Ser Gly Asp Pro Lys Leu Ser Ala His Tyr Arg Gln Leu Ala Gln
835 840 845
Gln Ser Leu Glu Val Ile Ala Gly Asp Phe Gly Asp Gln Asp Leu Gly
850 855 860
Leu Ser Gln Glu Val Trp Gln Lys Leu Ala Lys Asp Val Asp Leu Ile
865 870 875 880
Val His Ser Gly Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu
885 890 895
Phe Gly Pro Asn Val Ala Gly Thr Ala Glu Ile Ile Lys Leu Ala Ile
900 905 910
Ser Glu Arg Leu Lys Pro Val Thr Tyr Leu Ser Thr Val Gly Ile Ala
915 920 925
Asp Gln Ile Pro Val Thr Glu Phe Glu Glu Asp Ser Asp Val Arg Val
930 935 940
Met Ser Ala Glu Arg Gln Ile Asn Asp Gly Tyr Ala Asn Gly Tyr Gly
945 950 955 960
Asn Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu
965 970 975
Ala Gly Leu Pro Val Arg Val Phe Arg Ser Asp Met Ile Leu Ala His
980 985 990
Ser Asp Tyr His Gly Gln Leu Asn Val Thr Asp Val Phe Thr Arg Ser
995 1000 1005
Ile Gln Ser Leu Leu Leu Thr Gly Val Ala Pro Ala Ser Phe Tyr
1010 1015 1020
Glu Leu Asp Ala Asp Gly Asn Arg Gln Arg Ala His Tyr Asp Gly
1025 1030 1035
Val Pro Gly Asp Phe Thr Ala Ala Ser Ile Thr Ala Ile Gly Gly
1040 1045 1050
Val Asn Val Val Asp Gly Tyr Arg Ser Phe Asp Val Phe Asn Pro
1055 1060 1065
His His Asp Gly Val Ser Met Asp Thr Phe Val Asp Trp Leu Ile
1070 1075 1080
Asp Ala Gly Tyr Lys Ile Ala Arg Ile Asp Asp Tyr Asp Gln Trp
1085 1090 1095
Leu Ala Arg Phe Glu Leu Ala Leu Lys Gly Leu Pro Glu Gln Gln
1100 1105 1110
Arg Gln Gln Ser Val Leu Pro Leu Leu Lys Met Tyr Glu Lys Pro
1115 1120 1125
Gln Pro Ala Ile Asp Gly Ser Ala Leu Pro Thr Ala Glu Phe Ser
1130 1135 1140
Arg Ala Val His Glu Ala Lys Val Gly Asp Ser Gly Glu Ile Pro
1145 1150 1155
His Val Thr Lys Glu Leu Ile Leu Lys Tyr Ala Ser Asp Ile Gln
1160 1165 1170
Leu Leu Gly Leu Val
1175
<210> 240
<211> 1193
<212> PRT
<213> 未知
<220>
<223> CAR
<400> 240
Met His Gln Leu Thr Val Thr Gly Met Asn Ile Cys Glu Val Gln Arg
1 5 10 15
Leu Phe Pro Arg Met Thr Ser Asp Val His Asp Ala Thr Asp Gly Val
20 25 30
Thr Glu Thr Ala Leu Asp Asp Glu Gln Ser Thr Arg Arg Ile Ala Glu
35 40 45
Leu Tyr Ala Thr Asp Pro Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala
50 55 60
Val Val Asp Ala Ala His Lys Pro Gly Leu Arg Leu Ala Glu Ile Leu
65 70 75 80
Gln Thr Leu Phe Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly Tyr Arg
85 90 95
Ala Arg Glu Leu Ala Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Leu
100 105 110
Leu Pro Arg Phe Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg Val
115 120 125
Gln Ala Val Ala Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr
130 135 140
Pro Gly Asp Ala Val Ala Thr Ile Gly Phe Ala Ser Pro Asp Tyr Leu
145 150 155 160
Thr Leu Asp Leu Val Cys Ala Tyr Leu Gly Leu Val Ser Val Pro Leu
165 170 175
Gln His Asn Ala Pro Val Ser Arg Leu Ala Pro Ile Leu Ala Glu Val
180 185 190
Glu Pro Arg Ile Leu Thr Val Ser Ala Glu Tyr Leu Asp Leu Ala Val
195 200 205
Glu Ser Val Arg Asp Val Asn Ser Val Ser Gln Leu Val Val Phe Asp
210 215 220
His His Pro Glu Val Asp Asp His Arg Asp Ala Leu Ala Arg Ala Arg
225 230 235 240
Glu Gln Leu Ala Gly Lys Gly Ile Ala Val Thr Thr Leu Asp Ala Ile
245 250 255
Ala Asp Glu Gly Ala Gly Leu Pro Ala Glu Pro Ile Tyr Thr Ala Asp
260 265 270
His Asp Gln Arg Leu Ala Met Ile Leu Tyr Thr Ser Gly Ser Thr Gly
275 280 285
Ala Pro Lys Gly Ala Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp
290 295 300
Thr Met Ser Phe Ile Thr Gly Asp Pro Thr Pro Val Ile Asn Val Asn
305 310 315 320
Phe Met Pro Leu Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr Ala
325 330 335
Val Gln Asn Gly Gly Thr Ser Tyr Phe Val Pro Glu Ser Asp Met Ser
340 345 350
Thr Leu Phe Glu Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Gly Leu
355 360 365
Val Pro Arg Val Ala Asp Met Leu Tyr Gln His His Leu Ala Thr Val
370 375 380
Asp Arg Leu Val Thr Gln Gly Ala Asp Glu Leu Thr Ala Glu Lys Gln
385 390 395 400
Ala Gly Ala Glu Leu Arg Glu Gln Val Leu Gly Gly Arg Val Ile Thr
405 410 415
Gly Phe Val Ser Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu
420 425 430
Asp Ile Thr Leu Gly Ala His Ile Val Asp Gly Tyr Gly Leu Thr Glu
435 440 445
Thr Gly Ala Val Thr Arg Asp Gly Val Ile Val Arg Pro Pro Val Ile
450 455 460
Asp Tyr Lys Leu Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser Thr Asp
465 470 475 480
Lys Pro Tyr Pro Arg Gly Glu Leu Leu Val Arg Ser Gln Thr Leu Thr
485 490 495
Pro Gly Tyr Tyr Lys Arg Pro Glu Val Thr Ala Ser Val Phe Asp Arg
500 505 510
Asp Gly Tyr Tyr His Thr Gly Asp Val Met Ala Glu Thr Ala Pro Asp
515 520 525
His Leu Val Tyr Val Asp Arg Arg Asn Asn Val Leu Lys Leu Ala Gln
530 535 540
Gly Glu Phe Val Ala Val Ala Asn Leu Glu Ala Val Phe Ser Gly Ala
545 550 555 560
Ala Leu Val Arg Gln Ile Phe Val Tyr Gly Asn Ser Glu Arg Ser Phe
565 570 575
Leu Leu Ala Val Val Val Pro Thr Pro Glu Ala Leu Glu Gln Tyr Asp
580 585 590
Pro Ala Ala Leu Lys Ala Ala Leu Ala Asp Ser Leu Gln Arg Thr Ala
595 600 605
Arg Asp Ala Glu Leu Gln Ser Tyr Glu Val Pro Ala Asp Phe Ile Val
610 615 620
Glu Thr Glu Pro Phe Ser Ala Ala Asn Gly Leu Leu Ser Gly Val Gly
625 630 635 640
Lys Leu Leu Arg Pro Asn Leu Lys Asp Arg Tyr Gly Gln Arg Leu Glu
645 650 655
Gln Met Tyr Ala Asp Ile Ala Ala Thr Gln Ala Asn Gln Leu Arg Glu
660 665 670
Leu Arg Arg Ala Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln
675 680 685
Ala Ala Ala Thr Ile Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala
690 695 700
His Phe Thr Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Thr Leu Ser
705 710 715 720
Asn Leu Leu Ser Asp Phe Phe Gly Phe Glu Val Pro Val Gly Thr Ile
725 730 735
Val Asn Pro Ala Thr Asn Leu Ala Gln Leu Ala Gln His Ile Glu Ala
740 745 750
Gln Arg Thr Ala Gly Asp Arg Arg Pro Ser Phe Thr Thr Val His Gly
755 760 765
Ala Asp Ala Thr Glu Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe
770 775 780
Ile Asp Ala Glu Thr Leu Arg Ala Ala Pro Gly Leu Pro Lys Val Thr
785 790 795 800
Thr Glu Pro Arg Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly
805 810 815
Arg Phe Leu Thr Leu Gln Trp Leu Glu Arg Leu Ala Pro Val Gly Gly
820 825 830
Thr Leu Ile Thr Ile Val Arg Gly Arg Asp Asp Ala Ala Ala Arg Ala
835 840 845
Arg Leu Thr Gln Ala Tyr Asp Thr Asp Pro Glu Leu Ser Arg Arg Phe
850 855 860
Ala Glu Leu Ala Asp Arg His Leu Arg Val Val Ala Gly Asp Ile Gly
865 870 875 880
Asp Pro Asn Leu Gly Leu Thr Pro Glu Ile Trp His Arg Leu Ala Ala
885 890 895
Glu Val Asp Leu Val Val His Pro Ala Ala Leu Val Asn His Val Leu
900 905 910
Pro Tyr Arg Gln Leu Phe Gly Pro Asn Val Val Gly Thr Ala Glu Val
915 920 925
Ile Lys Leu Ala Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr Leu Ser
930 935 940
Thr Val Ser Val Ala Met Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp
945 950 955 960
Ile Arg Thr Val Ser Pro Val Arg Pro Leu Asp Gly Gly Tyr Ala Asn
965 970 975
Gly Tyr Gly Asn Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala
980 985 990
His Asp Leu Cys Gly Leu Pro Val Ala Thr Phe Arg Ser Asp Met Ile
995 1000 1005
Leu Ala His Pro Arg Tyr Arg Gly Gln Val Asn Val Pro Asp Met
1010 1015 1020
Phe Thr Arg Leu Leu Leu Ser Leu Leu Ile Thr Gly Val Ala Pro
1025 1030 1035
Arg Ser Phe Tyr Ile Gly Asp Gly Glu Arg Pro Arg Ala His Tyr
1040 1045 1050
Pro Gly Leu Thr Val Asp Phe Val Ala Glu Ala Val Thr Thr Leu
1055 1060 1065
Gly Ala Gln Gln Arg Glu Gly Tyr Val Ser Tyr Asp Val Met Asn
1070 1075 1080
Pro His Asp Asp Gly Ile Ser Leu Asp Val Phe Val Asp Trp Leu
1085 1090 1095
Ile Arg Ala Gly His Pro Ile Asp Arg Val Asp Asp Tyr Asp Asp
1100 1105 1110
Trp Val Arg Arg Phe Glu Thr Ala Leu Thr Ala Leu Pro Glu Lys
1115 1120 1125
Arg Arg Ala Gln Thr Val Leu Pro Leu Leu His Ala Phe Arg Ala
1130 1135 1140
Pro Gln Ala Pro Leu Arg Gly Ala Pro Glu Pro Thr Glu Val Phe
1145 1150 1155
His Ala Ala Val Arg Thr Ala Lys Val Gly Pro Gly Asp Ile Pro
1160 1165 1170
His Leu Asp Glu Ala Leu Ile Asp Lys Tyr Ile Arg Asp Leu Arg
1175 1180 1185
Glu Phe Gly Leu Ile
1190
<210> 241
<211> 1178
<212> PRT
<213> 人工序列
<220>
<223> MaCar(m)
<400> 241
Met Thr Glu Thr Ile Ser Thr Ala Ala Val Pro Thr Thr Asp Leu Glu
1 5 10 15
Glu Gln Val Lys Arg Arg Ile Glu Gln Val Val Ser Asn Asp Pro Gln
20 25 30
Leu Ala Ala Leu Leu Pro Glu Asp Ser Val Thr Glu Ala Val Asn Glu
35 40 45
Pro Asp Leu Pro Leu Val Glu Val Ile Arg Arg Leu Leu Glu Gly Tyr
50 55 60
Gly Asp Arg Pro Ala Leu Gly Gln Arg Ala Phe Glu Phe Val Thr Gly
65 70 75 80
Asp Asp Gly Ala Thr Val Ile Ala Leu Lys Pro Glu Tyr Thr Thr Val
85 90 95
Ser Tyr Arg Glu Leu Trp Glu Arg Ala Glu Ala Ile Ala Ala Ala Trp
100 105 110
His Glu Gln Gly Ile Arg Asp Gly Asp Phe Val Ala Gln Leu Gly Phe
115 120 125
Thr Ser Thr Asp Phe Ala Ser Leu Asp Val Ala Gly Leu Arg Leu Gly
130 135 140
Thr Val Ser Val Pro Leu Gln Thr Gly Ala Ser Leu Gln Gln Arg Asn
145 150 155 160
Ala Ile Leu Glu Glu Thr Arg Pro Ala Val Phe Ala Ala Ser Ile Glu
165 170 175
Tyr Leu Asp Ala Ala Val Asp Ser Val Leu Ala Thr Pro Ser Val Arg
180 185 190
Leu Leu Ser Val Phe Asp Tyr His Ala Glu Val Asp Ser Gln Arg Glu
195 200 205
Ala Leu Glu Ala Val Arg Ala Arg Leu Glu Ser Ala Gly Arg Thr Ile
210 215 220
Val Val Glu Ala Leu Ala Glu Ala Leu Ala Arg Gly Arg Asp Leu Pro
225 230 235 240
Ala Ala Pro Leu Pro Ser Ala Asp Pro Asp Ala Leu Arg Leu Leu Ile
245 250 255
Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys Gly Ala Met Tyr Pro Gln
260 265 270
Trp Leu Val Ala Asn Leu Trp Gln Lys Lys Arg Leu Thr Asp Asp Val
275 280 285
Ile Pro Ser Ile Gly Val Asn Phe Met Pro Met Ser His Leu Met Gly
290 295 300
Arg Leu Thr Leu Met Gly Thr Leu Ser Gly Gly Gly Thr Ala Tyr Tyr
305 310 315 320
Ile Ala Ser Ser Asp Leu Ser Thr Phe Phe Glu Asp Ile Ala Leu Ile
325 330 335
Arg Pro Ser Glu Val Leu Phe Val Pro Arg Val Val Glu Met Val Phe
340 345 350
Gln Arg Phe Gln Ala Glu Leu Asp Arg Ser Leu Ala Pro Gly Glu Ser
355 360 365
Asn Ser Glu Ile Ala Glu Arg Ile Lys Val Arg Ile Arg Glu Gln Asp
370 375 380
Phe Gly Gly Arg Val Leu Ser Ala Gly Ser Gly Ser Ala Pro Leu Ser
385 390 395 400
Pro Glu Met Thr Glu Phe Met Glu Ser Leu Leu Gln Val Pro Leu Arg
405 410 415
Asp Gly Tyr Gly Ser Thr Glu Ala Gly Gly Val Trp Arg Asp Gly Val
420 425 430
Leu Gln Arg Pro Pro Val Thr Asp Tyr Lys Leu Val Asp Val Pro Glu
435 440 445
Leu Gly Tyr Phe Thr Thr Asp Ser Pro His Pro Arg Gly Glu Leu Arg
450 455 460
Leu Lys Ser Glu Thr Met Phe Pro Gly Tyr Tyr Lys Arg Pro Glu Thr
465 470 475 480
Thr Ala Asp Val Phe Asp Asp Glu Gly Tyr Tyr Lys Thr Gly Asp Val
485 490 495
Val Ala Glu Leu Gly Pro Asp His Leu Lys Tyr Leu Asp Arg Val Lys
500 505 510
Asn Val Leu Lys Leu Ala Gln Gly Glu Phe Val Ala Val Ser Lys Leu
515 520 525
Glu Ala Ala Tyr Thr Gly Ser Pro Leu Val Arg Gln Ile Phe Val Tyr
530 535 540
Gly Asn Ser Glu Arg Ser Phe Leu Leu Ala Val Val Val Pro Thr Pro
545 550 555 560
Glu Val Leu Glu Arg Tyr Ala Asp Ser Pro Asp Ala Leu Lys Pro Leu
565 570 575
Ile Gln Asp Ser Leu Gln Gln Val Ala Lys Asp Ala Glu Leu Gln Ser
580 585 590
Tyr Glu Ile Pro Arg Asp Phe Ile Val Glu Thr Val Pro Phe Thr Val
595 600 605
Glu Ser Gly Leu Leu Ser Asp Ala Arg Lys Leu Leu Arg Pro Lys Leu
610 615 620
Lys Asp His Tyr Gly Glu Arg Leu Glu Ala Leu Tyr Ala Glu Leu Ala
625 630 635 640
Glu Ser Gln Asn Glu Arg Leu Arg Gln Leu Ala Arg Glu Ala Ala Thr
645 650 655
Arg Pro Val Leu Glu Thr Val Thr Asp Ala Ala Ala Ala Leu Leu Gly
660 665 670
Ala Ser Ser Ser Asp Leu Ala Pro Asp Val Arg Phe Ile Asp Leu Gly
675 680 685
Gly Asp Ser Leu Ser Ala Leu Ser Tyr Ser Glu Leu Leu Arg Asp Ile
690 695 700
Phe Glu Val Asp Val Pro Val Gly Val Ile Asn Ser Val Ala Asn Asp
705 710 715 720
Leu Ala Ala Ile Ala Arg His Ile Glu Ala Gln Arg Thr Gly Ala Ala
725 730 735
Thr Gln Pro Thr Phe Ala Ser Val His Gly Lys Asp Ala Thr Val Ile
740 745 750
Thr Ala Gly Glu Leu Thr Leu Asp Lys Phe Leu Asp Glu Ser Leu Leu
755 760 765
Lys Ala Ala Lys Asp Val Gln Pro Ala Thr Ala Asp Val Lys Thr Val
770 775 780
Leu Val Thr Gly Gly Asn Gly Trp Leu Gly Arg Trp Leu Val Leu Asp
785 790 795 800
Trp Leu Glu Arg Leu Ala Pro Asn Gly Gly Lys Val Tyr Ala Leu Ile
805 810 815
Arg Gly Ala Asp Ala Glu Ala Ala Arg Ala Arg Leu Asp Ala Val Tyr
820 825 830
Glu Ser Gly Asp Pro Lys Leu Ser Ala His Tyr Arg Gln Leu Ala Gln
835 840 845
Gln Ser Leu Glu Val Ile Ala Gly Asp Phe Gly Asp Gln Asp Leu Gly
850 855 860
Leu Ser Gln Glu Val Trp Gln Lys Leu Ala Lys Asp Val Asp Leu Ile
865 870 875 880
Val His Ser Gly Ala Leu Val Asn His Val Leu Pro Tyr Ser Gln Leu
885 890 895
Phe Gly Pro Asn Val Ala Gly Thr Ala Glu Ile Ile Lys Leu Ala Ile
900 905 910
Ser Glu Arg Leu Lys Pro Val Thr Tyr Leu Ser Thr Val Gly Ile Ala
915 920 925
Asp Gln Ile Pro Val Thr Glu Phe Glu Glu Asp Ser Asp Val Arg Val
930 935 940
Met Ser Ala Glu Arg Gln Ile Asn Asp Gly Tyr Ala Asn Gly Tyr Gly
945 950 955 960
Asn Ser Lys Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His Asp Leu
965 970 975
Ala Gly Leu Pro Val Arg Val Phe Arg Ser Asp Met Ile Leu Ala His
980 985 990
Ser Asp Tyr His Gly Gln Leu Asn Val Thr Asp Val Phe Thr Arg Ser
995 1000 1005
Ile Gln Ser Leu Leu Leu Thr Gly Val Ala Pro Ala Ser Phe Tyr
1010 1015 1020
Glu Leu Asp Ala Asp Gly Asn Arg Gln Arg Ala His Tyr Asp Gly
1025 1030 1035
Val Pro Gly Asp Phe Thr Ala Ala Ser Ile Thr Ala Ile Gly Gly
1040 1045 1050
Val Asn Val Val Asp Gly Tyr Arg Ser Phe Asp Val Phe Asn Pro
1055 1060 1065
His His Asp Gly Val Ser Met Asp Thr Phe Val Asp Trp Leu Ile
1070 1075 1080
Asp Ala Gly Tyr Lys Ile Ala Arg Ile Asp Asp Tyr Asp Gln Trp
1085 1090 1095
Leu Ala Arg Phe Glu Leu Ala Leu Lys Gly Leu Pro Glu Gln Gln
1100 1105 1110
Arg Gln Gln Ser Val Leu Pro Leu Leu Lys Met Tyr Glu Lys Pro
1115 1120 1125
Gln Pro Ala Ile Asp Gly Ser Ala Leu Pro Thr Ala Glu Phe Ser
1130 1135 1140
Arg Ala Val His Glu Ala Lys Val Gly Asp Ser Gly Glu Ile Pro
1145 1150 1155
His Val Thr Lys Glu Leu Ile Leu Lys Tyr Ala Ser Asp Ile Gln
1160 1165 1170
Leu Leu Gly Leu Val
1175
<210> 242
<211> 224
<212> PRT
<213> 未知
<220>
<223> PT
<400> 242
Met Lys Ile Tyr Gly Ile Tyr Met Asp Arg Pro Leu Ser Gln Glu Glu
1 5 10 15
Asn Glu Arg Phe Met Thr Phe Ile Ser Pro Glu Lys Arg Glu Lys Cys
20 25 30
Arg Arg Phe Tyr His Lys Glu Asp Ala His Arg Thr Leu Leu Gly Asp
35 40 45
Val Leu Val Arg Ser Val Ile Ser Arg Gln Tyr Gln Leu Asp Lys Ser
50 55 60
Asp Ile Arg Phe Ser Thr Gln Glu Tyr Gly Lys Pro Cys Ile Pro Asp
65 70 75 80
Leu Pro Asp Ala His Phe Asn Ile Ser His Ser Gly Arg Trp Val Ile
85 90 95
Gly Ala Phe Asp Ser Gln Pro Ile Gly Ile Asp Ile Glu Lys Thr Lys
100 105 110
Pro Ile Ser Leu Glu Ile Ala Lys Arg Phe Phe Ser Lys Thr Glu Tyr
115 120 125
Ser Asp Leu Leu Ala Lys Asp Lys Asp Glu Gln Thr Asp Tyr Phe Tyr
130 135 140
His Leu Trp Ser Met Lys Glu Ser Phe Ile Lys Gln Glu Gly Lys Gly
145 150 155 160
Leu Ser Leu Pro Leu Asp Ser Phe Ser Val Arg Leu His Gln Asp Gly
165 170 175
Gln Val Ser Ile Glu Leu Pro Asp Ser His Ser Pro Cys Tyr Ile Lys
180 185 190
Thr Tyr Glu Val Asp Pro Gly Tyr Lys Met Ala Val Cys Ala Ala His
195 200 205
Pro Asp Phe Pro Glu Asp Ile Thr Met Val Ser Tyr Glu Glu Leu Leu
210 215 220
<210> 243
<211> 222
<212> PRT
<213> 艾阿华诺卡氏菌
<400> 243
Met Ile Glu Thr Ile Leu Pro Ala Gly Val Glu Ser Ala Glu Leu Leu
1 5 10 15
Glu Tyr Pro Glu Asp Leu Lys Ala His Pro Ala Glu Glu His Leu Ile
20 25 30
Ala Lys Ser Val Glu Lys Arg Arg Arg Asp Phe Ile Gly Ala Arg His
35 40 45
Cys Ala Arg Leu Ala Leu Ala Glu Leu Gly Glu Pro Pro Val Ala Ile
50 55 60
Gly Lys Gly Glu Arg Gly Ala Pro Ile Trp Pro Arg Gly Val Val Gly
65 70 75 80
Ser Leu Thr His Cys Asp Gly Tyr Arg Ala Ala Ala Val Ala His Lys
85 90 95
Met Arg Phe Arg Ser Ile Gly Ile Asp Ala Glu Pro His Ala Thr Leu
100 105 110
Pro Glu Gly Val Leu Asp Ser Val Ser Leu Pro Pro Glu Arg Glu Trp
115 120 125
Leu Lys Thr Thr Asp Ser Ala Leu His Leu Asp Arg Leu Leu Phe Cys
130 135 140
Ala Lys Glu Ala Thr Tyr Lys Ala Trp Trp Pro Leu Thr Ala Arg Trp
145 150 155 160
Leu Gly Phe Glu Glu Ala His Ile Thr Phe Glu Ile Glu Asp Gly Ser
165 170 175
Ala Asp Ser Gly Asn Gly Thr Phe His Ser Glu Leu Leu Val Pro Gly
180 185 190
Gln Thr Asn Asp Gly Gly Thr Pro Leu Leu Ser Phe Asp Gly Arg Trp
195 200 205
Leu Ile Ala Asp Gly Phe Ile Leu Thr Ala Ile Ala Tyr Ala
210 215 220
<210> 244
<211> 272
<212> PRT
<213> 未知
<220>
<223> PT
<400> 244
Met Val Lys Thr Thr Glu Val Val Ser Glu Val Ser Lys Val Ala Gly
1 5 10 15
Val Arg Pro Trp Ala Gly Ile Phe Val Val Glu Ile Gln Glu Asp Ile
20 25 30
Leu Ala Asp Glu Phe Thr Phe Glu Ala Leu Met Arg Thr Leu Pro Leu
35 40 45
Ala Ser Gln Ala Arg Ile Leu Asn Lys Lys Ser Phe His Asp Arg Cys
50 55 60
Ser Asn Leu Cys Ser Gln Leu Leu Gln Leu Phe Gly Cys Ser Ile Val
65 70 75 80
Thr Gly Leu Asn Phe Gln Glu Leu Lys Phe Asp Lys Gly Ser Phe Gly
85 90 95
Lys Pro Phe Leu Asp Asn Asn Arg Phe Leu Pro Phe Ser Met Thr Ile
100 105 110
Gly Glu Gln Tyr Val Ala Met Phe Leu Val Lys Cys Val Ser Thr Asp
115 120 125
Glu Tyr Gln Asp Val Gly Ile Asp Ile Ala Ser Pro Cys Asn Tyr Gly
130 135 140
Gly Arg Glu Glu Leu Glu Leu Phe Lys Glu Val Phe Ser Glu Arg Glu
145 150 155 160
Phe Asn Gly Leu Leu Lys Ala Ser Asp Pro Cys Thr Ile Phe Thr Tyr
165 170 175
Leu Trp Ser Leu Lys Glu Ser Tyr Thr Lys Phe Thr Gly Thr Gly Leu
180 185 190
Asn Thr Asp Leu Ser Leu Ile Asp Phe Gly Ala Ile Ser Phe Phe Pro
195 200 205
Ala Glu Gly Ala Ser Met Cys Ile Thr Leu Asp Glu Val Pro Leu Ile
210 215 220
Phe His Ser Gln Trp Phe Asn Asn Glu Ile Val Thr Ile Cys Met Pro
225 230 235 240
Lys Ser Ile Ser Asp Lys Ile Asn Thr Asn Arg Pro Lys Leu Tyr Asn
245 250 255
Ile Ser Leu Ser Thr Leu Ile Asp Tyr Phe Ile Glu Asn Asp Gly Leu
260 265 270
<210> 245
<211> 209
<212> PRT
<213> 未知
<220>
<223> PT
<400> 245
Met Val Asp Met Lys Thr Thr His Thr Ser Leu Pro Phe Ala Gly His
1 5 10 15
Thr Leu His Phe Val Glu Phe Asp Pro Ala Asn Phe Cys Glu Gln Asp
20 25 30
Leu Leu Trp Leu Pro His Tyr Ala Gln Leu Gln His Ala Gly Arg Lys
35 40 45
Arg Lys Thr Glu His Leu Ala Gly Arg Ile Ala Ala Val Tyr Ala Leu
50 55 60
Arg Glu Tyr Gly Tyr Lys Cys Val Pro Ala Ile Gly Glu Leu Arg Gln
65 70 75 80
Pro Val Trp Pro Ala Glu Val Tyr Gly Ser Ile Ser His Cys Gly Thr
85 90 95
Thr Ala Leu Ala Val Val Ser Arg Gln Pro Ile Gly Ile Asp Ile Glu
100 105 110
Glu Ile Phe Ser Val Gln Thr Ala Arg Glu Leu Thr Asp Asn Ile Ile
115 120 125
Thr Pro Ala Glu His Glu Arg Leu Ala Asp Cys Gly Leu Ala Phe Ser
130 135 140
Leu Ala Leu Thr Leu Ala Phe Ser Ala Lys Glu Ser Ala Phe Lys Ala
145 150 155 160
Ser Glu Ile Gln Thr Asp Ala Gly Phe Leu Asp Tyr Gln Ile Ile Ser
165 170 175
Trp Asn Lys Gln Gln Val Ile Ile His Arg Glu Asn Glu Met Phe Ala
180 185 190
Val His Trp Gln Ile Lys Glu Lys Ile Val Ile Thr Leu Cys Gln His
195 200 205
Asp
<210> 246
<211> 468
<212> PRT
<213> 未知
<220>
<223> AT
<400> 246
Met Ile Arg Glu Pro Pro Glu His Ile Leu Asn Arg Leu Pro Ser Ser
1 5 10 15
Ala Ser Ala Leu Ala Cys Ser Ala His Ala Leu Asn Leu Ile Glu Lys
20 25 30
Arg Thr Leu Asp His Glu Glu Met Lys Ala Leu Asn Arg Glu Val Ile
35 40 45
Glu Tyr Phe Lys Glu His Val Asn Pro Gly Phe Leu Glu Tyr Arg Lys
50 55 60
Ser Val Thr Ala Gly Gly Asp Tyr Gly Ala Val Glu Trp Gln Ala Gly
65 70 75 80
Ser Leu Asn Thr Leu Val Asp Thr Gln Gly Gln Glu Phe Ile Asp Cys
85 90 95
Leu Gly Gly Phe Gly Ile Phe Asn Val Gly His Arg Asn Pro Val Val
100 105 110
Val Ser Ala Val Gln Asn Gln Leu Ala Lys Gln Pro Leu His Ser Gln
115 120 125
Glu Leu Leu Asp Pro Leu Arg Ala Met Leu Ala Lys Thr Leu Ala Ala
130 135 140
Leu Thr Pro Gly Lys Leu Lys Tyr Ser Phe Phe Cys Asn Ser Gly Thr
145 150 155 160
Glu Ser Val Glu Ala Ala Leu Lys Leu Ala Lys Ala Tyr Gln Ser Pro
165 170 175
Arg Gly Lys Phe Thr Phe Ile Ala Thr Ser Gly Ala Phe His Gly Lys
180 185 190
Ser Leu Gly Ala Leu Ser Ala Thr Ala Lys Ser Thr Phe Arg Lys Pro
195 200 205
Phe Met Pro Leu Leu Pro Gly Phe Arg His Val Pro Phe Gly Asn Ile
210 215 220
Glu Ala Met Arg Thr Ala Leu Asn Glu Cys Lys Lys Thr Gly Asp Asp
225 230 235 240
Val Ala Ala Val Ile Leu Glu Pro Ile Gln Gly Glu Gly Gly Val Ile
245 250 255
Leu Pro Pro Pro Gly Tyr Leu Thr Ala Val Arg Lys Leu Cys Asp Glu
260 265 270
Phe Gly Ala Leu Met Ile Leu Asp Glu Val Gln Thr Gly Met Gly Arg
275 280 285
Thr Gly Lys Met Phe Ala Cys Glu His Glu Asn Val Gln Pro Asp Ile
290 295 300
Leu Cys Leu Ala Lys Ala Leu Gly Gly Gly Val Met Pro Ile Gly Ala
305 310 315 320
Thr Ile Ala Thr Glu Glu Val Phe Ser Val Leu Phe Asp Asn Pro Phe
325 330 335
Leu His Thr Thr Thr Phe Gly Gly Asn Pro Leu Ala Cys Ala Ala Ala
340 345 350
Leu Ala Thr Ile Asn Val Leu Leu Glu Gln Asn Leu Pro Ala Gln Ala
355 360 365
Glu Gln Lys Gly Asp Met Leu Leu Asp Gly Phe Arg Gln Leu Ala Arg
370 375 380
Glu Tyr Pro Asp Leu Val Gln Glu Ala Arg Gly Lys Gly Met Leu Met
385 390 395 400
Ala Ile Glu Phe Val Asp Asn Glu Ile Gly Tyr Asn Phe Ala Ser Glu
405 410 415
Met Phe Arg Gln Arg Val Leu Val Ala Gly Thr Leu Asn Asn Ala Lys
420 425 430
Thr Ile Arg Ile Glu Pro Pro Leu Thr Leu Thr Ile Glu Gln Cys Glu
435 440 445
Leu Val Ile Lys Ala Ala Arg Lys Ala Leu Ala Ala Met Arg Val Ser
450 455 460
Val Glu Glu Ala
465
<210> 247
<211> 456
<212> PRT
<213> 未知
<220>
<223> AT
<400> 247
Met Asn Ser Gln Ile Thr Asn Ala Lys Thr Arg Glu Trp Gln Ala Leu
1 5 10 15
Ser Arg Asp His His Leu Pro Pro Phe Thr Asp Tyr Lys Gln Leu Asn
20 25 30
Glu Lys Gly Ala Arg Ile Ile Thr Lys Ala Glu Gly Val Tyr Ile Trp
35 40 45
Asp Ser Glu Gly Asn Lys Ile Leu Asp Ala Met Ala Gly Leu Trp Cys
50 55 60
Val Asn Val Gly Tyr Gly Arg Glu Glu Leu Val Gln Ala Ala Thr Arg
65 70 75 80
Gln Met Arg Glu Leu Pro Phe Tyr Asn Leu Phe Phe Gln Thr Ala His
85 90 95
Pro Pro Val Val Glu Leu Ala Lys Ala Ile Ala Asp Val Ala Pro Glu
100 105 110
Gly Met Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Ala Asn Asp
115 120 125
Thr Val Leu Arg Met Val Arg His Tyr Trp Ala Thr Lys Gly Gln Pro
130 135 140
Gln Lys Lys Val Val Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr
145 150 155 160
Val Ala Gly Val Ser Leu Gly Gly Met Lys Ala Leu His Glu Gln Gly
165 170 175
Asp Phe Pro Ile Pro Gly Ile Val His Ile Ala Gln Pro Tyr Trp Tyr
180 185 190
Gly Glu Gly Gly Asp Met Ser Pro Asp Glu Phe Gly Val Trp Ala Ala
195 200 205
Glu Gln Leu Glu Lys Lys Ile Leu Glu Val Gly Glu Glu Asn Val Ala
210 215 220
Ala Phe Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro
225 230 235 240
Pro Asp Thr Tyr Trp Pro Lys Ile Arg Glu Ile Leu Ala Lys Tyr Asp
245 250 255
Ile Leu Phe Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly
260 265 270
Glu Trp Phe Gly Ser Gln Tyr Tyr Gly Asn Ala Pro Asp Leu Met Pro
275 280 285
Ile Ala Lys Gly Leu Thr Ser Gly Tyr Ile Pro Met Gly Gly Val Val
290 295 300
Val Arg Asp Glu Ile Val Glu Val Leu Asn Gln Gly Gly Glu Phe Tyr
305 310 315 320
His Gly Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Ile Arg Ile Leu Arg Glu Glu Lys Ile Ile Glu Lys Val Lys
340 345 350
Ala Glu Thr Ala Pro Tyr Leu Gln Lys Arg Trp Gln Glu Leu Ala Asp
355 360 365
His Pro Leu Val Gly Glu Ala Arg Gly Val Gly Met Val Ala Ala Leu
370 375 380
Glu Leu Val Lys Asn Lys Lys Thr Arg Glu Arg Phe Thr Asp Lys Gly
385 390 395 400
Val Gly Met Leu Cys Arg Glu His Cys Phe Arg Asn Gly Leu Ile Met
405 410 415
Arg Ala Val Gly Asp Thr Met Ile Ile Ser Pro Pro Leu Val Ile Asp
420 425 430
Pro Ser Gln Ile Asp Glu Leu Ile Thr Leu Ala Arg Lys Cys Leu Asp
435 440 445
Gln Thr Ala Ala Ala Val Leu Ala
450 455
<210> 248
<211> 426
<212> PRT
<213> 未知
<220>
<223> AT
<400> 248
Met Asn Ser Asn Lys Glu Leu Met Gln Arg Arg Ser Gln Ala Ile Pro
1 5 10 15
Arg Gly Val Gly Gln Ile His Pro Ile Phe Ala Asp Arg Ala Glu Asn
20 25 30
Cys Arg Val Trp Asp Val Glu Gly Arg Glu Tyr Leu Asp Phe Ala Gly
35 40 45
Gly Ile Ala Val Leu Asn Thr Gly His Leu His Pro Lys Val Val Ala
50 55 60
Ala Val Glu Ala Gln Leu Lys Lys Leu Ser His Thr Cys Phe Gln Val
65 70 75 80
Leu Ala Tyr Glu Pro Tyr Leu Glu Leu Cys Glu Ile Met Asn Gln Lys
85 90 95
Val Pro Gly Asp Phe Ala Lys Lys Thr Leu Leu Val Thr Thr Gly Ser
100 105 110
Glu Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala Ala Thr Lys Arg
115 120 125
Ser Gly Thr Ile Ala Phe Ser Gly Ala Tyr His Gly Arg Thr His Tyr
130 135 140
Thr Leu Ala Leu Thr Gly Lys Val Asn Pro Tyr Ser Ala Gly Met Gly
145 150 155 160
Leu Met Pro Gly His Val Tyr Arg Ala Leu Tyr Pro Cys Pro Leu His
165 170 175
Gly Ile Ser Glu Asp Asp Ala Ile Ala Ser Ile His Arg Ile Phe Lys
180 185 190
Asn Asp Ala Ala Pro Glu Asp Ile Ala Ala Ile Val Ile Glu Pro Val
195 200 205
Gln Gly Glu Gly Gly Phe Tyr Ala Ser Ser Pro Ala Phe Met Gln Arg
210 215 220
Leu Arg Ala Leu Cys Asp Glu His Gly Ile Met Leu Ile Ala Asp Glu
225 230 235 240
Val Gln Ser Gly Ala Gly Arg Thr Gly Thr Leu Phe Ala Met Glu Gln
245 250 255
Met Gly Val Ala Pro Asp Leu Thr Thr Phe Ala Lys Ser Ile Ala Gly
260 265 270
Gly Phe Pro Leu Ala Gly Val Thr Gly Arg Ala Glu Val Met Asp Ala
275 280 285
Val Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Asn Pro Ile Ala
290 295 300
Cys Val Ala Ala Leu Glu Val Leu Lys Val Phe Glu Gln Glu Asn Leu
305 310 315 320
Leu Gln Lys Ala Asn Asp Leu Gly Gln Lys Leu Lys Asp Gly Leu Leu
325 330 335
Ala Ile Ala Glu Lys His Pro Glu Ile Gly Asp Val Arg Gly Leu Gly
340 345 350
Ala Met Ile Ala Ile Glu Leu Phe Glu Asp Gly Asp His Asn Lys Pro
355 360 365
Asp Ala Lys Leu Thr Ala Glu Ile Val Ala Arg Ala Arg Asp Lys Gly
370 375 380
Leu Ile Leu Leu Ser Cys Gly Pro Tyr Tyr Asn Val Leu Arg Ile Leu
385 390 395 400
Val Pro Leu Thr Ile Glu Asp Ala Gln Ile Arg Gln Gly Leu Glu Ile
405 410 415
Ile Ser Gln Cys Phe Asp Glu Ala Lys Gln
420 425
<210> 249
<211> 421
<212> PRT
<213> 未知
<220>
<223> AT
<400> 249
Met Ser Asn Asn Glu Phe His Gln Arg Arg Leu Ser Ala Thr Pro Arg
1 5 10 15
Gly Val Gly Val Met Cys Asn Phe Phe Ala Gln Ser Ala Glu Asn Ala
20 25 30
Thr Leu Lys Asp Val Glu Gly Asn Glu Tyr Ile Asp Phe Ala Ala Gly
35 40 45
Ile Ala Val Leu Asn Thr Gly His Arg His Pro Asp Leu Val Ala Ala
50 55 60
Val Glu Gln Gln Leu Gln Gln Phe Thr His Thr Ala Tyr Gln Ile Val
65 70 75 80
Pro Tyr Glu Ser Tyr Val Thr Leu Ala Glu Lys Ile Asn Ala Leu Ala
85 90 95
Pro Val Ser Gly Gln Ala Lys Thr Ala Phe Phe Thr Thr Gly Ala Glu
100 105 110
Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala His Thr Gly Arg Pro
115 120 125
Gly Val Ile Ala Phe Ser Gly Gly Phe His Gly Arg Thr Tyr Met Thr
130 135 140
Met Ala Leu Thr Gly Lys Val Ala Pro Tyr Lys Ile Gly Phe Gly Pro
145 150 155 160
Phe Pro Gly Ser Val Tyr His Val Pro Tyr Pro Ser Asp Leu His Gly
165 170 175
Ile Ser Thr Gln Asp Ser Leu Asp Ala Ile Glu Arg Leu Phe Lys Ser
180 185 190
Asp Ile Glu Ala Lys Gln Val Ala Ala Ile Ile Phe Glu Pro Val Gln
195 200 205
Gly Glu Gly Gly Phe Asn Val Ala Pro Lys Glu Leu Val Ala Ala Ile
210 215 220
Arg Arg Leu Cys Asp Glu His Gly Ile Val Met Ile Ala Asp Glu Val
225 230 235 240
Gln Ser Gly Phe Ala Arg Thr Gly Lys Leu Phe Ala Met Asp His Tyr
245 250 255
Ala Asp Lys Pro Asp Leu Met Thr Met Ala Lys Ser Leu Ala Gly Gly
260 265 270
Met Pro Leu Ser Gly Val Val Gly Asn Ala Asn Ile Met Asp Ala Pro
275 280 285
Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Asn Pro Leu Ala Val
290 295 300
Ala Ala Ala His Ala Val Leu Asn Ile Ile Asp Lys Glu Ser Leu Cys
305 310 315 320
Glu Arg Ala Asn Gln Leu Gly Gln Arg Leu Lys Asn Thr Leu Ile Asp
325 330 335
Ala Lys Glu Ser Val Pro Ala Ile Ala Ala Val Arg Gly Leu Gly Ser
340 345 350
Met Ile Ala Val Glu Phe Asn Asp Pro Gln Thr Gly Glu Pro Ser Ala
355 360 365
Ala Ile Ala Gln Lys Ile Gln Gln Arg Ala Leu Ala Gln Gly Leu Leu
370 375 380
Leu Leu Thr Cys Gly Ala Tyr Gly Asn Val Ile Arg Phe Leu Tyr Pro
385 390 395 400
Leu Thr Ile Pro Asp Ala Gln Phe Asp Ala Ala Met Lys Ile Leu Gln
405 410 415
Asp Ala Leu Ser Asp
420
<210> 250
<211> 464
<212> PRT
<213> 未知
<220>
<223> AT
<400> 250
Met Ala Thr Ile Thr Asn His Met Pro Thr Ala Glu Leu Gln Ala Leu
1 5 10 15
Asp Ala Ala His His Leu His Pro Phe Ser Ala Asn Asn Ala Leu Gly
20 25 30
Glu Glu Gly Thr Arg Val Ile Thr Arg Ala Arg Gly Val Trp Leu Asn
35 40 45
Asp Ser Glu Gly Glu Glu Ile Leu Asp Ala Met Ala Gly Leu Trp Cys
50 55 60
Val Asn Ile Gly Tyr Gly Arg Asp Glu Leu Ala Glu Val Ala Ala Arg
65 70 75 80
Gln Met Arg Glu Leu Pro Tyr Tyr Asn Thr Phe Phe Lys Thr Thr His
85 90 95
Val Pro Ala Ile Ala Leu Ala Gln Lys Leu Ala Glu Leu Ala Pro Gly
100 105 110
Asp Leu Asn His Val Phe Phe Ala Gly Gly Gly Ser Glu Ala Asn Asp
115 120 125
Thr Asn Ile Arg Met Val Arg Thr Tyr Trp Gln Asn Lys Gly Gln Pro
130 135 140
Glu Lys Thr Val Ile Ile Ser Arg Lys Asn Ala Tyr His Gly Ser Thr
145 150 155 160
Val Ala Ser Ser Ala Leu Gly Gly Met Ala Gly Met His Ala Gln Ser
165 170 175
Gly Leu Ile Pro Asp Val His His Ile Asn Gln Pro Asn Trp Trp Ala
180 185 190
Glu Gly Gly Asp Met Asp Pro Glu Glu Phe Gly Leu Ala Arg Ala Arg
195 200 205
Glu Leu Glu Glu Ala Ile Leu Glu Leu Gly Glu Asn Arg Val Ala Ala
210 215 220
Phe Ile Ala Glu Pro Val Gln Gly Ala Gly Gly Val Ile Val Ala Pro
225 230 235 240
Asp Ser Tyr Trp Pro Glu Ile Gln Arg Ile Cys Asp Lys Tyr Asp Ile
245 250 255
Leu Leu Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn
260 265 270
Trp Phe Gly Thr Gln Thr Met Gly Ile Arg Pro His Ile Met Thr Ile
275 280 285
Ala Lys Gly Leu Ser Ser Gly Tyr Ala Pro Ile Gly Gly Ser Ile Val
290 295 300
Cys Asp Glu Val Ala His Val Ile Gly Lys Asp Glu Phe Asn His Gly
305 310 315 320
Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn
325 330 335
Leu Arg Ile Leu Glu Glu Glu Asn Ile Leu Asp His Val Arg Asn Val
340 345 350
Ala Ala Pro Tyr Leu Lys Glu Lys Trp Glu Ala Leu Thr Asp His Pro
355 360 365
Leu Val Gly Glu Ala Lys Ile Val Gly Met Met Ala Ser Ile Ala Leu
370 375 380
Thr Pro Asn Lys Ala Ser Arg Ala Lys Phe Ala Ser Glu Pro Gly Thr
385 390 395 400
Ile Gly Tyr Ile Cys Arg Glu Arg Cys Phe Ala Asn Asn Leu Ile Met
405 410 415
Arg His Val Gly Asp Arg Met Ile Ile Ser Pro Pro Leu Val Ile Thr
420 425 430
Pro Ala Glu Ile Asp Glu Met Phe Val Arg Ile Arg Lys Ser Leu Asp
435 440 445
Glu Ala Gln Ala Glu Ile Glu Lys Gln Gly Leu Met Lys Ser Ala Ala
450 455 460
<210> 251
<211> 339
<212> PRT
<213> 大肠杆菌
<400> 251
Met Ser Met Ile Lys Ser Tyr Ala Ala Lys Glu Ala Gly Gly Glu Leu
1 5 10 15
Glu Val Tyr Glu Tyr Asp Pro Gly Glu Leu Arg Pro Gln Asp Val Glu
20 25 30
Val Gln Val Asp Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Ile
35 40 45
Asp Asn Glu Trp Gly Phe Ser Gln Tyr Pro Leu Val Ala Gly His Glu
50 55 60
Val Ile Gly Arg Val Val Ala Leu Gly Ser Ala Ala Gln Asp Lys Gly
65 70 75 80
Leu Gln Val Gly Gln Arg Val Gly Ile Gly Trp Thr Ala Arg Ser Cys
85 90 95
Gly His Cys Asp Ala Cys Ile Ser Gly Asn Gln Ile Asn Cys Glu Gln
100 105 110
Gly Ala Val Pro Thr Ile Met Asn Arg Gly Gly Phe Ala Glu Lys Leu
115 120 125
Arg Ala Asp Trp Gln Trp Val Ile Pro Leu Pro Glu Asn Ile Asp Ile
130 135 140
Glu Ser Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val Phe Lys Pro
145 150 155 160
Leu Leu Met His His Ile Thr Ala Thr Ser Arg Val Gly Val Ile Gly
165 170 175
Ile Gly Gly Leu Gly His Ile Ala Ile Lys Leu Leu His Ala Met Gly
180 185 190
Cys Glu Val Thr Ala Phe Ser Ser Asn Pro Ala Lys Glu Gln Glu Val
195 200 205
Leu Ala Met Gly Ala Asp Lys Val Val Asn Ser Arg Asp Pro Gln Ala
210 215 220
Leu Lys Ala Leu Ala Gly Gln Phe Asp Leu Ile Ile Asn Thr Val Asn
225 230 235 240
Val Ser Leu Asp Trp Gln Pro Tyr Phe Glu Ala Leu Thr Tyr Gly Gly
245 250 255
Asn Phe His Thr Val Gly Ala Val Leu Thr Pro Leu Ser Val Pro Ala
260 265 270
Phe Thr Leu Ile Ala Gly Asp Arg Ser Val Ser Gly Ser Ala Thr Gly
275 280 285
Thr Pro Tyr Glu Leu Arg Lys Leu Met Arg Phe Ala Ala Arg Ser Lys
290 295 300
Val Ala Pro Thr Thr Glu Leu Phe Pro Met Ser Lys Ile Asn Asp Ala
305 310 315 320
Ile Gln His Val Arg Asp Gly Lys Ala Arg Tyr Arg Val Val Leu Lys
325 330 335
Ala Asp Phe
Claims (29)
1.一种重组微生物,其包含外源基因,该外源基因编码具有将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性的蛋白质,其中,
所述外源基因为:
(i)编码由与序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列具有80%以上的序列一致性的氨基酸序列构成的蛋白质的DNA;
(ii)编码由在序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的蛋白质的DNA;
(iii)由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列具有80%以上的序列一致性的多核苷酸序列构成的DNA;
(iv)编码由在利用序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列编码的蛋白质的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的蛋白质的DNA;
(v)与由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列互补的多核苷酸序列构成的DNA在严谨条件下杂交的DNA;或者
(vi)由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列的简并异构体构成的DNA。
2.如权利要求1所述的重组微生物,其中,所述外源基因为:
(xi)编码由序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列构成的蛋白质的DNA;或者
(xiii)由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列构成的DNA。
3.如权利要求1或2所述的重组微生物,其中,所述外源基因来源于选自耳念珠菌(Candida auris)、马克斯克鲁维酵母(Kluyveromyces marxianus)、库德里阿兹威毕赤酵母(Pichia kudriavzevii)、喜热梭孢壳(Thermothelomyces thermophilus)、太瑞斯梭孢壳霉(Thermothielavioides terrestris)、嗜热毛壳菌(Chaetomium thermophilum)、丝状真菌柄孢霉(Podospora anserina)、淡紫拟青霉菌(Purpureocillium lilacinum)和圆核腔菌(Pyrenophora teres)中的至少一者。
4.如权利要求1~3中任一项所述的重组微生物,其中,
所述重组微生物具有C6化合物的生产路径,
所述C6化合物为选自由己二酸、六亚甲基二胺、1,6-己二醇、6-氨基己酸、6-氨基-1-己醇和6-羟基己酸组成的组中的至少一者。
5.如权利要求1~4中任一项所述的重组微生物,其中,所述重组微生物属于埃希氏菌(Escherichia)属、芽孢杆菌(Bacillus)属、棒状杆菌(Corynebacterium)属、节杆菌(Arthrobacter)属、短杆菌(Brevibacterium)属、梭菌(Clostridium)属、发酵单胞菌(Zymomonas)属、假单胞菌(Pseudomonas)属、伯克霍尔德菌(Burkholderia)属、链霉菌(Streptomyces)属、红球菌(Rhodococcus)属、集胞藻(Synechocystis)属、嗜碱盐芽孢杆菌(Alkalihalobacillus)属、酵母菌(Saccharomyces)属、裂殖酵母(Schizosaccharomyces)属、亚罗酵母(Yarrowia)属、念珠菌(Candida)属、毕赤酵母(Pichia)属、或曲霉(Aspergillus)属。
6.如权利要求1~5中任一项所述的重组微生物,其中,所述重组微生物是大肠杆菌(Escherichia coli)。
7.如权利要求1~6中任一项所述的重组微生物,其进一步包含选自由
·编码3-氧代己二酰CoA硫解酶的基因、
·编码3-羟基己二酰CoA脱氢酶的基因、
·编码3-羟基己二酰CoA脱水酶的基因、
·编码羧酸还原酶的基因、
·编码醇脱氢酶的基因、以及
·编码转氨酶(氨基转移酶)的基因
组成的组中的至少一者。
8.一种C6化合物的制造方法,其包括对权利要求1~7中任一项所述的重组微生物进行培养的培养工序,其中,所述C6化合物为选自由己二酸、六亚甲基二胺、1,6-己二醇、6-氨基己酸、6-氨基-1-己醇和6-羟基己酸组成的组中的至少一者。
9.一种重组蛋白,其中,
(a)具有将2,3-脱氢己二酰-CoA还原而转换成己二酰CoA的酶活性;
(b)为下述任一者:
(i)由与序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列具有80%以上的序列一致性的氨基酸序列构成;
(ii)由在序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成;
(iii)利用由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列具有80%以上的序列一致性的多核苷酸序列构成的DNA进行编码;
(iv)由在利用序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列编码的蛋白质的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成;
(v)利用与由与序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列互补的多核苷酸序列构成的DNA在严谨条件下杂交的DNA进行编码;以及
(vi)利用由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列的简并异构体构成的DNA进行编码。
10.一种重组蛋白,其中,
(xi)由序列编号8、9、10、11、12、13、14、15或16所示的氨基酸序列构成,或者
(xiii)利用由序列编号57、58、59、60、61、62、63、64或65所示的多核苷酸序列构成的DNA进行编码。
11.一种C6化合物的制造方法,其包括使用权利要求9或10所述的重组蛋白的步骤。
12.一种重组微生物,其包含编码3-羟基己二酰CoA脱水酶的外源基因和编码2,3-脱氢己二酰CoA还原酶的外源基因中的至少一者。
13.如权利要求12所述的重组微生物,其中,所述3-羟基己二酰CoA脱水酶和2,3-脱氢己二酰CoA还原酶中的至少一者来源于伯克霍尔德菌属(Burkholderiasp.)LEBP-3株(保藏编号:NITE BP-03334)。
14.如权利要求12或13所述的重组微生物,其中,
所述3-羟基己二酰CoA脱水酶被与下述PCR扩增物的序列一致性为80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码,
该DNA具有626bp~940bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(1a)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(1b)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的。
15.如权利要求12~14中任一项所述的重组微生物,其中,
所述2,3-脱氢己二酰CoA还原酶被与下述PCR扩增物的序列一致性为80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码,
该DNA具有924bp~1386bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(2a)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(2b)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的。
16.如权利要求12或13所述的重组微生物,其中,
所述3-羟基己二酰CoA脱水酶被作为下述PCR扩增物的DNA所编码,
该DNA具有783bp或784bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(1a)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(1b)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的。
17.如权利要求12~14中任一项所述的重组微生物,其中,
所述2,3-脱氢己二酰CoA还原酶被作为下述PCR扩增物的DNA所编码,
该DNA具有1155bp或1156bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(2a)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(2b)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的。
18.如权利要求12~17中任一项所述的重组微生物,其中,所述重组微生物属于选自由埃希氏菌属、棒状杆菌属、芽孢杆菌属、不动杆菌属、伯克霍尔德菌属、假单胞菌属、梭菌属、酵母菌属、裂殖酵母属、亚罗酵母属、念珠菌属、毕赤酵母属和曲霉属组成的组中的属。
19.如权利要求12~18中任一项所述的重组微生物,其具有己二酸生产路径。
20.如权利要求12~19中任一项所述的重组微生物,其具有六亚甲基二胺生产路径。
21.如权利要求12~19中任一项所述的重组微生物,其具有1,6-己二醇生产路径。
22.如权利要求12~19中任一项所述的重组微生物,其具有6-氨基-1-己醇生产路径。
23.一种目标化合物的制造方法,其包括对权利要求12~18中任一项所述的重组微生物进行培养的培养工序,其中,
所述目标化合物选自由己二酸、己二酸衍生物、六亚甲基二胺、1,6-己二醇和6-氨基-1-己醇组成的组,
所述己二酸衍生物选自由氧代己二酸、乙酰丙酸、3-羟基己二酸和2,3-脱氢己二酸组成的组。
24.一种己二酸的制造方法,其包括对权利要求19所述的重组微生物进行培养的培养工序。
25.一种六亚甲基二胺的制造方法,其包括对权利要求20所述的重组微生物进行培养的培养工序。
26.一种1,6-己二醇的制造方法,其包括对权利要求21所述的重组微生物进行培养的培养工序。
27.一种6-氨基-1-己醇的制造方法,其包括对权利要求22所述的重组微生物进行培养的培养工序。
28.一种重组多肽,其是下述(A-1)、(A-2)或(A-3)的重组多肽:
(A-1)被与下述PCR扩增物的序列一致性为80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码的重组多肽,
具有3-羟基己二酰CoA脱水酶活性,
所述DNA具有626bp~940bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(a1)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(a2)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的;
(A-2)由在利用作为下述PCR扩增物的DNA所编码的多肽的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的重组多肽,
具有3-羟基己二酰CoA脱水酶活性,
所述DNA具有626bp~940bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(a1)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(a2)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的;
(A-3)被由下述PCR扩增物的简并异构体构成的DNA所编码的重组多肽,
具有3-羟基己二酰CoA脱水酶活性,
所述DNA具有626bp~940bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(a1)由序列编号168所示的碱基序列构成的核苷酸以及由序列编号169所示的碱基序列构成的核苷酸;或者
(a2)包含序列编号168所示的碱基序列的核苷酸以及包含序列编号169所示的碱基序列的核苷酸
作为引物得到的。
29.一种重组多肽,其是下述(B-1)、(B-2)或(B-3)的重组多肽:
(B-1)被与下述PCR扩增物的序列一致性为80%以上、85%以上、88%以上、90%以上、93%以上、95%以上、97%以上、98%以上或99%以上的DNA所编码的重组多肽,
具有2,3-脱氢己二酰CoA还原酶活性,
所述DNA具有924bp~1386bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(b1)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(b2)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的;
(B-2)由在利用作为下述PCR扩增物的DNA所编码的多肽的氨基酸序列中缺失、置换、插入和/或添加1~10个氨基酸而成的氨基酸序列构成的重组多肽,
具有2,3-脱氢己二酰CoA还原酶活性,
所述DNA具有924bp~1386bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(b1)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(b2)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的;
(B-3)被由下述PCR扩增物的简并异构体构成的DNA所编码的重组多肽,
具有2,3-脱氢己二酰CoA还原酶活性,
所述DNA具有924bp~1386bp,
该PCR扩增物是以伯克霍尔德菌属(Burkholderia sp.)LEBP-3株的染色体DNA作为模板,使用
(b1)由序列编号170所示的碱基序列构成的核苷酸以及由序列编号171所示的碱基序列构成的核苷酸;或者
(b2)包含序列编号170所示的碱基序列的核苷酸以及包含序列编号171所示的碱基序列的核苷酸
作为引物得到的。
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CN111386339A (zh) * | 2017-11-30 | 2020-07-07 | 东丽株式会社 | 用于生产3-羟基己二酸、α-氢化己二烯二酸及/或己二酸的基因修饰微生物以及该化学产品的制造方法 |
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CN111386339A (zh) * | 2017-11-30 | 2020-07-07 | 东丽株式会社 | 用于生产3-羟基己二酸、α-氢化己二烯二酸及/或己二酸的基因修饰微生物以及该化学产品的制造方法 |
CN111386339B (zh) * | 2017-11-30 | 2024-05-10 | 东丽株式会社 | 用于生产3-羟基己二酸、α-氢化己二烯二酸及/或己二酸的基因修饰微生物以及该化学产品的制造方法 |
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