CN112195110B - 一株重组米曲霉菌株及其曲酸发酵方法与应用 - Google Patents

一株重组米曲霉菌株及其曲酸发酵方法与应用 Download PDF

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CN112195110B
CN112195110B CN202010999032.2A CN202010999032A CN112195110B CN 112195110 B CN112195110 B CN 112195110B CN 202010999032 A CN202010999032 A CN 202010999032A CN 112195110 B CN112195110 B CN 112195110B
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王德培
高紫君
毕付提
史亚楠
薛鲜丽
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Tianjin University of Science and Technology
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Abstract

本发明以基因工程方法构建一株高产曲酸,自源基因重组米曲霉菌株CGMCC No.18557(Aspergillus oryzae)，该米曲霉的次级代谢产物全局调控因子laeA基因、曲酸合成基因簇内kojA,kojR,kojT基因、葡萄糖‑6‑磷酸脱氢酶gdh基因分别整合到自体基因组上pyrG基因、ku70基因、msn2基因得到各基因过表达的重组米曲霉菌株CGMCC No.18557；所述次级代谢产物全局调控因子laeA基因受PsodM启动子调控，所述曲酸合成基因簇内kojA,kojR,kojT基因受PglaA启动子调控，所述葡萄糖‑6‑磷酸脱氢酶gdh基因受PgpdA启动子调控。

Description

一株重组米曲霉菌株及其曲酸发酵方法与应用
技术领域
本发明涉及生物工程技术领域，尤其是涉及一株重组米曲霉菌株及其曲酸发酵方法。
背景技术
曲酸是一种重要的有机酸。曲酸在食品工业、化妆品、卫生、纺织业、禽畜生产、化工、医药、环保等领域都有十分广阔的应用。目前曲酸的工业应用正在被更多地开发和研究，市场需求以每年5-7%递增，未来也将会具有更加广阔的市场前景。
米曲霉作为重要的曲酸发酵菌株拥有良好的发酵特性。以葡萄糖为发酵原料时，米曲霉可利用葡萄糖直接发酵生成曲酸，而不必破坏葡萄糖的碳骨架。米曲霉是一种曲酸发酵的重要菌株，对米曲霉菌株CGMCC3.042进行基因工程改造是提高曲酸发酵水平的重要手段，而米曲霉对多种真菌常用抗性不敏感，因而常用营养缺陷作为标记。
曲酸是由米曲霉分泌的一种次级代谢产物，受多种转录调控因子调控，其中laeA基因，曲酸合成基因簇内kojA,kojR,kojT基因，在米曲霉中可以显著正调控其次级代谢产物的生成，葡萄糖-6-磷酸脱氢酶是戊糖磷酸途径的关键酶，对糖次级代谢必不可少，而msn2基因敲除菌株曲酸产量也会有所提高。
发明内容
针对现有菌株存在的产量较低的问题，本发明提供了一株高水平发酵生产曲酸米曲霉菌株。本发明方法实现了次级代谢产物全局调控因子laeA 基因、曲酸合成基因簇内kojA, kojR, kojT基因、葡萄糖-6-磷酸脱氢酶gdh基因的过量表达及msn2基因的敲除、以增强曲酸次级代谢水平，增加HMP途径关键酶的表达促进曲酸的合成，提高曲酸产量
为实现上述目的，本发明公开了如下的技术内容：
一株重组米曲霉菌株，其分类为米曲霉Aspergillus oryzae，来自米曲霉自源基因重组米曲霉菌株，保藏号CGMCC No.18557（中国微生物菌种保藏管理委员会普通微生物中心CGMCC）。保藏日期2019年10月8日。在中国微生物菌种保藏管理委员会普通微生物中心（China General Microbiological Cultuer Collection Centre）进行保藏，保藏地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮政编码：100101。
本发明进一步公开了提高米曲霉发酵生产曲酸产量的方法，其特征在于该菌株将米曲霉CGMCC3.042（来源：中国微生物菌种保藏管理委员会普通微生物中心CGMCC公布的菌株）的次级代谢产物全局调控因子laeA 基因、曲酸合成基因簇内kojA, kojR, kojT基因、葡萄糖-6-磷酸脱氢酶gdh基因分别整合到米曲霉CGMCC3.042基因组上pyrG基因、ku70基因、msn2基因得到的重组米曲霉CGMCC18557菌株；所述次级代谢产物全局调控因子laeA 基因受PsodM启动子调控，所述曲酸合成基因簇内kojA, kojR, kojT基因受PglaA启动子调控，所述所述葡萄糖-6-磷酸脱氢酶gdh基因受PgpdA启动子调控。最终使全局调控因子laeA基因、曲酸合成基因簇内kojA, kojR, kojT基因、葡萄糖-6-磷酸脱氢酶gdh基因在米曲霉CGMCC3.042中实现过表达，获得基因重组菌株米曲霉CGMCC18557，实现发酵高产曲酸的目的。所述次级代谢产物全局调控因子laeA 基因核苷酸序列如SEQ ID NO.1，其氨基酸序列如SEQ ID NO.2所示。所述PsodM启动子为诱导型启动子，该启动子的核苷酸序列如SEQ IDNO.3所示。
一种次级代谢产物全局调控因子laeA 基因表达框，其特征在于所述表达框包含pyrGL左臂、PsodM启动子、次级代谢产物全局调控因子laeA基因、pyrGR右臂、按照pyrGL-PsodM-laeA-pyrGR的顺序排布。
其特征在于所述pyrGL左臂的核苷酸序列如SEQ ID NO.4所示；所述PsodM启动子的核苷酸序列如SEQ ID NO.3所示；所述次级代谢产物全局调控因子laeA 基因的核苷酸序列如SEQ ID NO.1所示；所述pyrGR右臂的核苷酸序列如SEQ ID NO.5所示。
所述曲酸合成基因簇内kojA, kojR, kojT基因核苷酸序列如SEQ ID NO.6，其氨基酸序列如SEQ ID NO.7所示。所述PglaA启动子为诱导型启动子，该启动子的核苷酸序列如SEQ ID NO.8所示。
一种曲酸合成基因簇内kojA, kojR, kojT基因表达框，其特征在于所述表达框包含ku70左臂、PglaA启动子、曲酸合成基因簇内kojA, kojR, kojT基因、pyrG、曲酸合成基因簇kojT基因3’部分、ku70R、按照ku70L-PglaA-koj (kojA, kojR, kojT)-pyrG-koj3’-ku70R的顺序排布。
所述ku70L左臂的核苷酸序列如SEQ ID NO.9所示；所述PglaA启动子的核苷酸序列如SEQ ID NO.8所示；所述曲酸合成基因簇内kojA,KojR,kojT基因的氨基酸序列如SEQID NO.7所示；所述pyrG的核苷酸序列如SEQ ID NO.10；所述koj3’的核苷酸序列如SEQ IDNO.11；所述ku70R右臂的核苷酸序列如SEQ ID NO.12所示。
所述葡萄糖-6-磷酸脱氢酶gdh基因核苷酸序列如SEQ ID NO.13所示，其氨基酸序列如SEQ ID NO.14所示。所述PgpdA启动子为组成型启动子，该启动子的核苷酸序列如SEQID NO.15所示。
一种葡萄糖-6-磷酸脱氢酶gdh基因表达框，其特征在于所述表达框包含msn2L左臂、PgpdA启动子、葡萄糖-6-磷酸脱氢酶gdh基因、pyrG、msn2R右臂按照msn2L-PgpdA-gdh-pyrG-msn2R的顺序排布。所述msn2L左臂的核苷酸序列如SEQ ID NO.16所示；所述PgpdA启动子的核苷酸序列如SEQ ID NO.15所示；所述葡萄糖-6-磷酸脱氢酶gdh基因的核苷酸序列如SEQ ID NO.14所示；所述pyrG的核苷酸序列如SEQ ID NO.10；所述msn2R右臂的核苷酸序列如SEQ ID NO.17所示。
所述的重组米曲霉的制备方法包括如下步骤：
(1)构建次级代谢产物全局调控因子laeA基因表达框pyrGL-PsodM-laeA-pyrGR；
(2)构建曲酸合成基因簇内kojA, kojR, kojT基因表达框ku70L-PglaA-kojA, kojR, kojT-pyrG-koj3’-ku70R；
(3)构建葡萄糖-6-磷酸脱氢酶gdh基因表达框msn2L-PgpdA-gdh-pyrG-msn2R；
(4)将步骤(1)、(2)、(3)制得的基因表达框依次转化米曲霉，经营养缺陷筛选和PCR鉴定获得所述重组米曲霉菌。
所述的米曲霉高产曲酸发酵培养基制备方法包括如下步骤：
1）碳源添加量10%~15%，氮源添加量1%~1.5%，磷酸二氢钾添加量0.05%~0.15%，硫酸镁添加量0.05%~0.1%；
2）将步骤1）各组分混合，搅拌使各组分在水中充分溶解；
3）调节发酵液pH值为6.0，进行灭菌。
所述的米曲霉高产曲酸发酵培养基的配制方法,其特征在于步骤3）所述的碳源可以是葡萄糖、蔗糖、糊精、麦芽糖、半乳糖、可溶性淀粉、果糖、玉米液化液、米糠、甘油等；氮源可以是：酵母浸出物、胰蛋白胨、蛋白胨、牛肉膏、尿素、豆粕、硝酸铵、硝酸钠等。
本发明所用到的SEQ ID NO.1-SEQ ID NO.17的序列如下：
SEQ ID NO.1
laeA基因核苷酸序列
ATGTTTGGAAACGGCCAGACTGGACAGCGTCTTCCGGCGATGGCCTCACCACCACACGACTCGTACTACTCACAGTCATTGGCATCTAGTCGATCAAGGAATAACTCGGATGCTATGGATATCTACGCCATCACAGACAGAGATCCTCCGGCACGAGAACCCTCTGGTTATAGCCAGTGGTACCGTAATGGTTCTCCAAGTGTGAATTCCATTCATAGCAAGTAATTCTTCCCTGTTTCCTTAAATCTGCCGTTGAATTTCTCTCTGGGCTGCGTCAGTTGTTCACTCTGCCGAAGCTGTTATTGTTGGACGACTAATGTTCTTCTCTGTTGTTACTGTCTCTCTCTAGGAGTTCTGAAAAACAGCCTTTCTATGAAGAAAACGGACGAATGTATCATGCGTACCGCAAAGGGGTATATATGCTACCATGCGATGAGCAGGAGCAAGATCGCCTTGATATCTTCCACAAATTATTCACGGTGGCAAGGGTGTCGGATGGCCTAATGTACGCCCCGCATCCCAGGAACGGCCGATTTCTAGACTTGGGCTGCGGGACCGGGATATGGGCAATTGATGTTGCCAACAAATACCCAGACGCTTTCGTTGTTGGGGTTGACCTTGCTCCCATACAGCCCTCAAACCACCCAAAGAATTGCGAATTTTACGCCCCATTCGACTTCGAGAGTCCTTGGGCCATGGGCGAGGATTCTTGGGACCTGATTCACCTGCAAATGGGGTGTGGAAGTGTGATGGGATGGCCGAACCTGTACCGGAGAATATTCGCTCACCTTCGACCCGGGGCTTGGTTTGAACAGGTGGAGATCGACTTTGAACCGCGGTGCGATGACCGTCCTCTCGAGGGACTAGCTATTCGACAGTGGTATCAGTATCTAAAGCAAGCTACACAAGATGCCATGCGACCTATAAACCACAACTCTCGTGATACAATCCGAGATCTGCAGGAGGCTGGTTTTACCGATATTGATCATCAAATGGTGGGGTTGCCCCTTAACCCATGGCATCAAGACGAGCACGAAAGAAAGGTTGCTCGCTGGTACAATTTGGCTGTCTCGGAGAGTATTGAGTCGCTCAGTATGGCGCCTTTCAGTCGTATTTTTAATTGGGACTTGGACAGAATCAGGCGCATTTCGTCAGAAGTCAAGTCGGAGGCGTTCAACAAAGAAATACACGCCTACAATATCCTTCATATATACCAAGCACGGAAACCTGCGAACTGATTCTTCTACCAACATGCGCACGACGGACATCCAAACATGCGCCAGCAGCGTCACTAGTGCCCAAGCTCCGAGTTATGGGGTGGGCGAATTACCATCCAGGAAGTCACCTTTATCTGCTCTTTTATGAGCTCTCCAAAGATGCAGCGAGTTGATATGAGCTTGTGTGGTGCCACTTGTTAGCTTGCACACAACGGTCCCGAGCAGTCATTTGCGTACTGCAAAAGCAAAATCGAACTCATACGGGTCTGTGATCTCTATCTATCTGGAAAATCCAGTGTGTCGGGAGAGCTCCATTACTGGG
SEQ ID NO.2
laeA氨基酸序列
MFGNGQTGQRLPAMASPPHDSYYSQSLASSRSRNNSDAMDIYAITDRDPPAREPSGYSQWYRNGSPSVNSIHSKSSEKQPFYEENGRMYHAYRKGVYMLPCDEQEQDRLDIFHKLFTVARVSDGLMYAPHPRNGRFLDLGCGTGIWAIDVANKYPDAFVVGVDLAPIQPSNHPKNCEFYAPFDFESPWAMGEDSWDLIHLQMGCGSVMGWPNLYRRIFAHLRPGAWFEQVEIDFEPRCDDRPLEGLAIRQWYQYLKQATQDAMRPINHNSRDTIRDLQEAGFTDIDHQMVGLPLNPWHQDEHERKVARWYNLAVSESIESLSMAPFSRIFNWDLDRIRRISSEVKSEAFNKEIHAYNILHIYQARKPAN*
SEQ ID NO.3
PsodM启动子核苷酸序列
ATCAGAGTTCTACAGAGTACCATGCTAAGCCTGGGGATTTACTGGTTAACATTTGCCTAGTAACTTGTAAGAATCGGAAGTACTTGGCTCCATGAGCCCCACATTATGAAAAACAATGGCATCAACAAATTGTCTGTGCATTATCATAGTATAGTTCTGGGTGGGGGCTATAGTACTTGCTGGTACTTCATGGAGTGACAGGCTGTAGTAGGCGGCCTTAATAAAACCCAGCAGAACGCATTCATTAGACAGATTTATCTACGCATATACCAGCTAACATAGCCTATTTGTAATCCTTGTAATCCACACTCACCTACGAATCACATAGACGAAAGGAAGAATGTGCTCAGTGGTGCGCCAAAGGTCCCCCGAATCCCCTCAAACTCGCTCATAGTCACATCCCGCCAACATGATTGGTAAAAATGAAGGTACCTTGGGGGTCCTCAGCATCCATTCCAGGCTTATATTCTCCCTCGCCATAATCATGACCAGAACTCCCTCACAACCCCTCTTCTCCCTCGTCCTCGTCCTCGCATCTCACCTCTCCAACTACACATATACTCAAAACCCTCAACCAGAATACCAACCAAACCACCCAAA
SEQ ID NO.4
pyrGL左臂核苷酸序列
TGAAATCAAATCCTGCCTACCTAACATGGGATTTAGTCATTTCTTTTGGCATCAAAGCAAGGCGCCACAATCGTTGAGGTAAGAATGATCTGCCAATATGTGAGCGAGCGCGGCTGACAACTATGGTAGTTTGGTAATACAACATCTCACGAATCGTACCTTGCATGTGCACTAAAGTTTGGTCAGATGTACAGTTAACAAGAGATCTCGTGCCCGTCATATACCATGACTTTTCTCTGAGCGAGTCAGGGACTGATATCCCTATTCATGACTTAAATCTTGATCAGGTCAGTATCTCTGAGATTGATTCGAACTGATTCTCACCCGTCAAAGTTCATGCACGCAAGCAATATCCAGTCGCCACGAGGGGATCCCGTATCTGTGCTTGGGAAAGCGAATGCACAGCCTATATCCACTCAGGTTACTTCGACTAAGCCACGATCTCGATCATTAACTAAAGACCACGAGAGGGGAACTAGGGAAATCCGTGATAGAATGAAGTATACAGTTGATTTTGTCAGTAAGGGATTCAAACCTAATACCAGAGGTGACTTTATCCAAGATTCCTTCACGACACTGGAAGAACTGCTTGAAGAGCTGCCTGAGTCAATCAGCTTCAATATCGAGATAAGTAGGTTATATGCTGCCACTGGTGGTTCTCCACTTGCGGGAGACAAAGCTAACAACGTCCCAATGAAGAGTACCCCAGGCTTCATGAAGCTATAGAAGCAGGTGTAGCACCAGTGGCTATTGAAATCAACACCTTCGTCGACAAAGCGCTTGAGAGACTCTTTTCTTACGGCAACAAAAAACGGACCATTATCCTATCCTCATTTACCCCCGAGATCTGCATTTTATTGGCCATCAAACAACAGACGTACCCTGTGATGTTCATCACTAATGCCGGCAAGCCTCCAGTTACGGATCGAGAGATGAGGGCTGCCAGCATACAGTCCGCTGTTCGATTTGCCAAGAGGTGGAATTTATCTGGCCTTGTCTTTGCATCTGAGGCGCTGGTAATGTGCCCCAGGCTTGTCAGATATGTTCAGCGATCAGGATTGATCTGTGGATCCTATGGATCTCAGAACAATATACCAGAAAATGCGAAGGTAAGTGCTTCTATATTAGTTCTTAGTGCTTTCAAATTGTGATGTAGAAGTTGTTCAATAGCTGATGAAATATTCTAGACCCAAGCCGCTGCTGGAATTGACATTATTATGGCCGATAGGGTTGGGCTTATTGCTATGTCCCTGAAAGGATATCAAAAGCAGGCAAAAAGCCAGGCATAATCCCCGCGCGGATGGTACCCTAAGGATAGGCCCTAATCTTATCTACATGTGACTGCATCGATGTGTTTGGTCCAAATGAGGCATGTGGCTCACCCCACAGGCGGAGAAACGTGTGGCTAGTGCA
SEQ ID NO.5
pyrGR右臂核苷酸序列
CAGGGTCAAGGGTCATGTTTCAAAATCACAATAATCTCCAACATAGACGAGAATTTGTACCGGAGTGTCTGAAGGTGCAGCTGGAGATTGGTCTATTTTCTTAGAGTGGGGTATCACTAATATACAGTCGGTCACTATCGTACAAACAATCACAATTATATACAAGATTTCCCACCACCCCCTACTCTAACACGGCACAATTATCCATCGAGTCAGAGCCTATCCACCATTTGGTGCTCTCGTAGAGACCAAAGTATAATCCTGATCCGACAGCGGCCATAAACGTGTTGATAGCACACCCTCGGAATAGTCCTCTCGGGCCATCTGTTCGTACAATCTCCCGTACGGTATTGATCATCCTTTTCTTCTGAGGTGCAGTTGTATCTGCAGCATCGAGCATGATTCGTGTCCGGACCATATCCATGGGTGCTGTCAAGACACTGGCTATACCGCCCGAGACCGCAGCACTTATCGCGGCTGTCGCTGCAGCCTCTCCGATTGTCGAATGGGCCTCTTTCTTTCCATACTCTCTTGGTCTTTCTAGCACCTTCTCTCGATCTCCGAATCTATATTCAAAAATTCGATACCGAAAAGACTCGTACAGAGGCATCTGAATCGCCGACACTGGCAAGCTATGCGCCACAAGAGCCGGGTATCCGCTCCAAAGCTGTCTAGGGTTGATAAACTTCTTGAAAGCTAGCCGTGTCGCTTTCTGGGCTACACCACCTACCCTTCCCCCAGCTACAGGTGCTGATGCGTCTGGATGGTGTGATTGGATCATCTGCGCGTCGTGTTTTAATGCATCAGCCGGAGCAAAGACTCCGCAAGCAGCAAGATCCGCAACGGAGGCTGCGCAAAAATCGGAAAAGAGCCGAGCTGAGCTAGACTCATGCGTTCCAAGTTTTTGATGTATGACTTGGAGTCCTGACTGTGCATACTCGTATGTGATGAAGAATGCGCCCGCTTTGACCCGTTATTCTTTGCTCAATGAATATGAGTTGGGTAAGGATAATGTTGGGTATCACCAACCTGTGGGAAATGAGGCAGCGGTTACGCTCGCGATACCTTGGTAAAGGCCGCGGAATATCCCTGGGTGTCTCCATATGCTGGTTCCTGTGTTGGTTCTCAGGAACTGCGAATATTCGCGAGACTGAATGCGGGTTTTGATCGTATCTAAGGGAAATATGAATAGATCTAGTGAGAGGGATGCGGTTGTGCTTGCCTAGTTCCCTCAGGTTAATGTATGCTGTTTGTCCTAGGGGGTTAGAAAATATACCAGCAATATACTAAGTGGCTCGGCGGCCATATTATTTGTCGATAGAGGCGGG
SEQ ID NO.6
kojA,KojR,kojT基因核苷酸序列
ttagtttgcagtcactagtgaaacataggtctgcaagttgctagtgtggtgctcgcccagccctggcttatcgccaatctccttgctgtgggtgtggaaggcgctgctcacagtgaagcgtacgccattccactcccgaaccacaccgtgccgtcggcgcgcaatgtcattgatcagaggaaccacttcctcggcagccgttacaacgttgctcaccttgccggggaagatgtggatcacgccgtctttttcaaacgtcaaactgcttcggaattcggatgtggtctttaacttggccaggacactgcccttccagccgcggtactggaaccgatcgagaaatccgggccagaaccgggtaatctcccgctcacagtggggcttgatccgagcagcgatagcttcctcgtccagactatccagaagctcttgaccctcctcgtgacggtcgaatgatccgaggatggtgtaactgccgtgtgtcaagatatacttcttctgaaggggctcgtttgtgtcgatcgccggcatcacgcagggaaaccagccatccatgacaatgaaggaaagaggcttttcctgtggctgctggtcttcgtaaacgagagcaatacaggtctgataggtgatgccaatgtcaaccggaagagcatctgcaatattttgtggcagaaggctggtataaccggtggcgttgatcactacgtcaaagacgtactgcccgtccccggtttgaatgcggttggtgtcctcggtgtggatgatacggtcgactgttgcattgaggtgcacgtagatgcctgcacgaccaagtttctctttgaatgtattccggagtcggtcgccaatagcgacgctgggttcatccagattgtatgcgctgatgaggccctggaaaccattcgccttcggatcaaccgcagtacactcaggagattcgtagcatacatccctgaatgcctcatccgagaccttcgatgggttccccagagcgtccgcttcgccgtgtgcatatatcgcggattcatggtggacgactagttcgggatacgtctcacagaacttgaccaaggcttcacggcagctgtctcgagttgctttagaccgagggtagtgtggccccttgtgcaagcggataccaaagtttcctgagacgccttgaaagatatcaggcttctgctcgaacaaagaaactcggtggccctgtttcttgagctctaaagcaagatggcagccgttccatccgccaccgacgatgccgacccttagctgtgtcgcgacacgcattatgaagaggcaggtagttatagtctagagatggttatgttcggttcaagaatcttgagaagaatgattgttgaagtgtagaggcatacccccaaaggatctccatcgctttatatccttcccttcagcaagatagcttgactcactcccatgggcatgtcaaaaatggtccttccctgattgtaacccaaccttgccggataacgacacatgcaccctgaagccaattgcatccaatgtgatacgaacggatgaggtggaatgagcggcaaagtcgggatagatgattgtcgacgactctagaagatcgtaacatactgaatgagcgtttaatgcatgtcagcgcttgataatgtcacatccgcggcgatgcatccgcaagacagtatccaatgagagggtggggggatggcacgaaaggaagcgcactagtcgggatgtgcagcctttgcagtggacagtgcgccctgcagaaaacagaaaaataacattctgcaggtatggtatcatcgcttaaggattccaatcctagcagatacccgtcgggggataacgggatcccgaattagggttatccagttccagcgttggagccgcgcgtcggagttaacatttattccgactgcccgataaccttgtacttttgcccccgtcgttctgactctcccgaaccccccactgatagaccaacgtcgactgcgatagatccccttctttccgcgataaccctgtagtgcagcgtaatgtcgttgaataccgacgattccggtcggataaggacccggcaacgcgccaaaagagcgtgcgaaacgtgcaaactgcgcaagaggaaatgtgacggccatgagccctgcacttactgcttgcgatacgaatatcagtgcactttcaagcctcatccacggagaaagcctgcagcttccaaatcttccgcacggcccagcgaggaagaagactcaccaaagtttctcgacagagttgatgctaaccaagaacacatggaggccaactcaggcaccgctttcccccatctcctagggatgaggttgaacccgcagggtgctcccaaggtgtacgggtttagctggaacctaggtccacgggacgagcctctggagcccttcaccaatcttacagatctgatttcaagagaggaaatggaagacttggctagccattatttaaagaagatacatccggtatatgcggtcctggatcccgacacgttgaggcagaagattgttgccagatggcatgatcctgcaactgcggccagctatgaccccattctttgcggcgtcgcggcattgggctccttgtactcaggccaccaggaacaccccaaagaaggagcactggtgcaatcagcgaaggagatgcttgaaaccacccggatctcaaagactactctcctgcatcatgccaccgcctggatcttacggaccatatatctccgaagcaccaactgcccccatgcttcatggatggccagttgttccaccatgcacatcatagaggccatcggggctcaccaggatccggaactggtgtctttggtctattctgacaccgcagacgttagcgtaaatgatgagagtcaaagacggttattctgggtggccacagtgctgaattcatggatttcgtatgaatatggccgatcgcgcgttattctacgaggtgtctcttgcaaaccacctttaccgcgaacgggtgacttcaccacggacctcatctccatgtaccagatctccgaacgactggatcccgaccagaacaataagctttccgacctagaagatgcactcagccgtgttgagcgcctcacactctcccatgacgccttgatactatcccaaagtaatctggcactcacaatttatcgtcgtctgcgagtcgcctcgtccaatatatcaaatgacatccttacccgcatcatccgactaggcaatgacggccttgaagccgcagtacgcctggcggaggatcgctcgccttggtggcatgtcgccaatataccctttcaatttctctgtatcttgcttgctattgacactagggaatcattgtcgtacgtcggcccagcactgcgctcattcagggccatcaccaggcattacagcacaccgaccctacacaccgcgctggagactatagaatcattggttcgcctatcccagaacaaaaaagaacgggacttgactcttctccgagatagcatgcaacaggaagacccagggctgaccgaacaaggttcaacaacatcccaagccttcaatgacgcttcttggctgggggcgaccggtgatctgaccttaccagataactttgactgggattggaatgtgttcttggacacccaggttccgttcttcgacgagggtgaagcaggtggtcagagatatagataatgcaggaaattgaggactatttacacgatcagaaagtgctcgtatgcgtctacttatagtattagtcgttgaacctagtacctaataattctgcttgaattatatcatagactgacatacctcatgtaagtccttcctcctccttcggcgccgaataaggattttcgaagccattcagcggctaagtcgggcatgacggaaaagtcgggtagggcggaatttccgttgacgcaatggaagcaccactagaaataaatactctgaaagagcacatctatcccagccgaggaataaaaacacttgacagatgctgctcaattaattgcctccctcccaattcattttttttattctgtggcgtatgtcttgaacgcctcgcaacgatgcagtccttccgccaataccgacggatgcgcagagacctgcaagaaagcattaagctgcatggtccgtatgccgctgctggtgatcgtcacgtccagccgaccgatgatattttggaggatgccgacgatgccagattggaaaaaggcatccattcaatgaacggacacggccaatctccatactccacgccaggaatcgttcttcacgacggccagagagtgaccgttcctggagtcaatctccgccgcgcctccgaaatctgtgagcgcgtcaatactaaaacacttttcattgtcgggttcgatggcccggacgaccagctcaatcccaaaaactggtctatagggcggaaatgggcgacattgggcattgtaggcacaacgggtatgcttgttggatgggcttcctccatcgactccactgtgattaaacaagggcaagaggcgttcggagtcagcgaggtcgccgaatctctcgcaactgccctgttcctgttcgcattcggcttcggttctctggtagcagcacctttctctgagactgtagggcggaatccggtctacatagcgacgctgtccattctaatgatctttacgatggcctcaggtctggctccgaattttggagcccagcttgccttccgcttcctggctggactgttcggatgcacaccgatgaccacctttggtggcagtatggcggatatctttgaccctatggaccgcacatacgcctttccagtttgttgcacgctatccttcctggggccgttcctagctccaatggttggagctttcattggccaaagcacgcatatcagctggcgctggactgaatggtgcacactgatcatggctgcattggtaaccggcgctatcttcctgttcgttccagaaacttatggtcccgtactgttgcagtggaaggcaaaacaattgcgtgagattaccggtgacccgcgtttcatggcggaaattgagcttcgccagacatcgctcgtgactcggttgatgcacagctgctcccgtcccttccatttattcttccgggagattatggtagcactttttaccatgtatctggtcgtggtatacattgtcctattcgggttcctgacgggctatgagttcatctttggtagaacatatggctttacccaagggtctgtgggcctgacatttatcggcatgaacatcggattcctgatcgcctttgccatggtgccgcatatttacttctcatacaagaagcgccttcaaaatgcgattgaaaatggacacaatgggttgccaccggaggagcgactttggttcgctatgtacggagcgccttggctaccaatatctcttttctggatgggctggaccagctatccctctatctcgtactggtcgccgctagtcgcctcggtggctttcggattctctgtccaaggaatctttatttccacgtatcagtaccttatcgacacgtatgagctcttcgcagcaagtgccttagtgagtgcaactttcttccgttatatcgccgctggcgcgatggtcatcgtgtcaatcccgatgtacggaaatttgggcgtacattggtcactcacgttgctggggtgtatctcggtcctgatgacccctgtaccttatattttttataagtacggacatgtcattcggcagcggaataaaaagaccccctga
SEQ ID NO.7
kojA,KojR,kojT氨基酸序列
KojA
MRVATQLRVGIVGGGWNGCHLALELKKQGHRVSLFEQKPDIFQGVSGNFGIRLHKGPHYPRSKATRDSCREALVKFCETYPELVVHHESAIYAHGEADALGNPSKVSDEAFRDVCYESPECTAVDPKANGFQGLISAYNLDEPSVAIGDRLRNTFKEKLGRAGIYVHLNATVDRIIHTEDTNRIQTGDGQYVFDVVINATGYTSLLPQNIADALPVDIGITYQTCIALVYEDQQPQEKPLSFIVMDGWFPCVMPAIDTNEPLQKKYILTHGSYTILGSFDRHEEGQELLDSLDEEAIAARIKPHCEREITRFWPGFLDRFQYRGWKGSVLAKLKTTSEFRSSLTFEKDGVIHIFPGKVSNVVTAAEEVVPLINDIARRRHGVVREWNGVRFTVSSAFHTHSKEIGDKPGLGEHHTSNLQTYVSLVTANMSLNTDDSGRIRTRQRAKRACETCKLRKRKCDGHEPCTYCLRYEYQCTFKPHPRRKPAASKSSARPSEEEDSPKFLDRVDANQEHMEANSGTAFPHLLGMRLNPQGAPKVYGFSWNLGPRDEPLEPFTNLTDLISREEMEDLASHYLKKIHPVYAVLDPDTLRQKIVARWHDPATAASYDPILCGVAALGSLYSGHQEHPKEGALVQSAKEMLETTRISKTTLLHHATAWILRTIYLRSTNCPHASWMASCSTMHIIEAIGAHQDPELVSLVYSDTADVSVNDESQRRLFWVATVLNSWISYEYGRSRVILRGVSCKPPLPRTGDFTTDLISMYQISERLDPDQNNKLSDLEDALSRVERLTLSHDALILSQSNLALTIYRRLRVASSNISNDILTRIIRLGNDGLEAAVRLAEDRSPWWHVANIPFQFLCILLAIDTRESLSYVGPALRSFRAITRHYSTPTLHTALETIESLVRLSQNKKERDLTLLRDSMQQEDPGLTEQGSTTSQAFNDASWLGATGDLTLPDNFDWDWNVFLDTQVPFFDEGEAGGQRYRMQSFRQYRRMRRDLQESIKLHGPYAAAGDRHVQPTDDILEDADDARLEKGIHSMNGHGQSPYSTPGIVLHDGQRVTVPGVNLRRASEICERVNTKTLFIVGFDGPDDQLNPKNWSIGRKWATLGIVGTTGMLVGWASSIDSTVIKQGQEAFGVSEVAESLATALFLFAFGFGSLVAAPFSETVGRNPVYIATLSILMIFTMASGLAPNFGAQLAFRFLAGLFGCTPMTTFGGSMADIFDPMDRTYAFPVCCTLSFLGPFLAPMVGAFIGQSTHISWRWTEWCTLIMAALVTGAIFLFVPETYGPVLLQWKAKQLREITGDPRFMAEIELRQTSLVTRLMHSCSRPFHLFFREIMVALFTMYLVVVYIVLFGFLTGYEFIFGRTYGFTQGSVGLTFIGMNIGFLIAFAMVPHIYFSYKKRLQNAIENGHNGLPPEERLWFAMYGAPWLPISLFWMGWTSYPSISYWSPLVASVAFGFSVQGIFISTYQYLIDTYELFAASALVSATFFRYIAAGAMVIVSIPMYGNLGVHWSLTLLGCISVLMTPVPYIFYKYGHVIRQRNKKTP
SEQ ID NO.8
PglaA启动子核苷酸序列
GCGAGACCGGACCACAATTACTATATCATGGTCTACCCATGATCTAGCCTCCGCCCAGCTCACTATAGATGTTCTTCAGACAGGGGACGGCGAATTCACGGGCGAAATCAATTTGTGGCTGCATCCTCATGTCTTGAAACAAGCAGTGTGATTGGAAGTGGTGGCGTCTTGGCTCCAGCCAGCTGCATGACTATGAGAAAATAGTCCAACTAGTCTTGGCTTGCAGAATGACTTCCGGAAATTTAACCTGGTGGCTGCTCTCAGTATTGGCTGACGCTCCGCACCTCTAATTGTGCTTCAGGTCAGTCAAAGATGTTTTGAACCACCAACGAGCCGAACCCTTCCGCCGCTACAATACTCGGGTTATTATAAAACGCATCGTGGTTCATCGTCCCAGCTATAGGATTATTTTCACATCAGCAAACGAAGTCGAAGCAAG
SEQ ID NO.9
ku70L左臂的核苷酸序列
CTCCAGGCGATTTCTCCATTTCCATAGTTCATTCAGAGAACTGCCTGTAGTCGAACAGGAATATTGTGGGTATTGTGTAAATGCGACAGCACGTCCTCCCTTCCCTCTTCCGAACCCATCATCGAGTAACTGGGTATACATCTCTTCCGTCAAAGGCGCAGCATACCGAAATGCAACGTAGGGTTTATGAGGCGCACTTTCAGGATTGAGTTTGTCTAGCAACTTGCACATTTCCTGGCATTGGTATTCGGACCATTTTCGGATAGGCGAGCCGCCTCCAATGTCCGCGTATTGCTTTTGGATCTTTGGGGTTCTCCGCTTTGCGATAAGTGGTCCAAGATATGTTTGAAGCCGTCCCAAGGGAATCAGATCACCGTCGGCCTTTACCAAAGAGCGAGGGAGACGTTAAAAATGGTCCATGAGAATGTTTGCTAGTCTGAAAACGTACAAATAACCTGCTCAAAAAGTCTTCCACCTCATTCGTCGTAGAGGGGCCGCCCATGTTTAAGAATACCATTGCGGTAGGGCCCTTGGAGCCAGTTGCATCTTGAGTGACTGGAGGGACTGCCGTTGCGAAGCCATTTCGCTGTTCTGTGTTGTATCCAAGCGAGCTGCGAGCTACCGGTCGCAGTAGTTGCCGAGGAAGCGCGAAGGTCCGACGGAGGGCCATTCTAATTAACAGATGGTAGCACAATCCTTTTCCCAGAAAGTAATGACCAAAGCGTGCGAATAGCGGCGATAACAAGACAATGAGGTCGGAGCAAAAAGTGTATAGGGATTTTAATGGGCCATGTGGCAAGGGTCAACAGTAATCAGAAAAGATGCTGACTTGAAGCAAAGAGTCCAGAACCGGTAAAATGACGGATGAGTTGGAGCTGAAGCGAATGGCCAATCAGGATTGACTTGGGGTCATCGAGTCTTGGCGTATGTCACCTGATGTTCGTGTCTTCCCCGCGTCGTCCGATTGAAGTGTCGCGTCACAGGTGACGAAAGAGCGGGAACGCAGGCAGCAGAGCGACAGGCAGACACCTAGGAAAATACCAATGTTGCCGTTCAACGGCACTTTTGCGTCGATTACTTAAATATCGTCCTTGACTACTTTTCCTTTGACGCAATTCATGACTTACGACTCTCAACACATTCACAATGGCTGACGAGGA
SEQ ID NO.10
pyrG的核苷酸序列
CCGATAGGGTTGGGCTTATTGCTATGTCCCTGAAAGGATATCAAAAGCAGGCAAAAAGCCAGGCATAATCCCCGCGCGGATGGTACCCTAAGGATAGGCCCTAATCTTATCTACATGTGACTGCATCGATGTGTTTGGTCCAAATGAGGCATGTGGCTCACCCCACAGGCGGAGAAACGTGTGGCTAGTGCATGACGGTCCCCTCCATAGATTCAATTTAATTTTTTCGCGGCAATTGTCGTGCAGTTTGTATCAACCGTTCATTCCACATATTGAGAGTTAGTAATTGGACATCCTGTTTATTTTGTCTAATTACTGAATATTCGAAGTACTAACCTACTAAATAAGCCAGTTTCAACCACTGAGTGCTCATTTATACAATAGTTGCAGAACCCCGCGCTACCCCTCCATCGCTGACATGTCTTCCAAGTCGCAATTGACCTACAGCGCACGCGCTAGCAAGCACCCCAACGCGCTCGTAAAGAAGCTCTTCGAGGTTGCCGAGGCCAAGAAAACCAATGTCACCGTTTCCGCCGACGTGACAACCACCAAAGAGCTGCTGGATTTGGCTGACCGTATGCGCACCGGGGATGCCACTTACATATGATCTAGTAATGGTTAATGGTGGATTATATAACAGGACTCGGTCCGTACATTGCCGTGATCAAAACTCACATCGATATCCTCTCCGATTTCAGCAAAGAAACCATCACCGGTCTGAAGGCCCTTGCAGAGAAGCACAATTTCCTCATCTTCGAAGATCGCAAGTTCATCGATATCGGAAACACAGTCCAAAAGCAGTACCATGGCGGCACTCTGCGTATCTCTGAGTGGGCCCACATCATCAACTGCAGTATTCTGCCCGGTGAGGGTATCGTCGAGGCTCTAGCCCAGACTGCTTCGGCCGAGGACTTCCCCTACGGCTCCGAGAGGGGCCTTTTGATCCTTGCGGAGATGACCTCCAAGGGATCTTTGGCTACCGGTCAATATACTACTTCTTCTGTTGACTATGCTCGGAAGTATAAGAAGTTTGTGATGGGATTCGTCTCGACACGTCACCTTGGCGAGGTTCAGTCTGAAGTTAGCTCGCCTTCGGAGGAGGAAGATTTTGTCGTCTTCACGACAGGTGTCAACCTCTCCTCGAAGGGAGACAAGCTGGGACAGCAGTACCAAACTCCTGAGTCGGCTGTTGGACGCGGTGCCGACTTTATTATTGCTGGCCGTGGAATTTATGCTGCTCCTGATCCCGTGGAGGCGGCGAAGCAGTACCAGAAGGAGGGATGGGATGCATACCTGAAGCGTGTTGGTGCGCAATAAGTAGTGGTGGATACGTACTCCTTTTATGGCAGTATGTCGCAAGTATGATGCGATTTATAGATTCAGCACTCGAAATGACTACTATGTGTCTACGACAGATACCCTCTCCGTACGAATAAGACACCTGCCTCGATATATGGACAAATTCAAAATCAGGGTCAAGGGTCATGTTTCAAAATCACAATAATCTCCAACATAGACGAGAATTTGTACCGGAGTGTCTGAAGGTGCAGCTGGAGATTGGTCTATTTTCTTAGAGTGGGGTATCACTAATACACAGTCGGTCACTATCGTACAAACAATCACAATTATATACAAGATTTCCCACCACCC
SEQ ID NO.11
koj3’终止子的核苷酸序列
tcgccgctagtcgcctcggtggctttcggattctctgtccaaggaatctttatttccacgtatcagtaccttatcgacacgtatgagctcttcgcagcaagtgccttagtgagtgcaactttcttccgttatatcgccgctggcgcgatggtcatcgtgtcaatcccgatgtacggaaatttgggcgtacattggtcactcacgttgctggggtgtatctcggtcctgatgacccctgtaccttatattttttataagtacggacatgtcattcggcagcggaataaaaagaccccctga
SEQ ID NO.12
ku70R右臂的核苷酸序列
GGAGCAGAAATGAGACAACGCTAGTATTGGTTACGAGAGACAGATTTTAGCAGATAATGTTTCTCTATCCTTTTAATAAAAAATACATAATGCTAGAACACTATCAAGTGTATATTAATGGTCTTTACCTACGTAATCCTACTCAATAATGTCATCTCAGAGAGCAAATGTGTACATGTTCGAGATATTCAAACACTATCGGGAAACCTAGGATGATGCTGTAGGGACTTTTGGTCCTCGTCTATGTAATTTGACCCAAAATCTGAAAATATAGTGGGAGGTTTGTCACCGTAAAAGTCATTGTACCAGCAACGTTGCAGTCGTACATCAGTAAGTAATATAAAATATGAATAGGAATATATTTCGATATATTTACATGTTTTCTAAACCGTACCGGCAGAAATAACACAGATGATTCAGACGGGTATATCCAAACGGACGAAAGCCCTCTAACAACGCCTGTCTAATCCCAGAAATGCATGCTCAAAGCGATAAACAAAAGTATACCGGATAGGTATCTATGACAAAACCCACATAGTAAAAACAGTCGTGGCTGGTGTACAGTGTATATATATCTATGTTGCGATTGAAAAAAAATGTACTAGCATTTGCCATATACTTGGCAGTAGCAGAGTTCCTCGTCTGGGCAACTAGAGGACAAACCATGTTAGTTAATTTTGTTATTGCATAGGTTTGATGCAATGGATGCTTACTATACGGACTCCTTGCCGAATATGACATGCCAGAGGAACTCCATGACTCGTCCGCTACTGGCATCATCTTTCTCTGTGTCGATGACCCATTGTCGGAACTTGATGTAGTCTTCTCGAGGTCGTTTCAGGACCTGTTCGCGCGACACGGCGAATTGCGCGCAGCATGCTGCGCCGACGAGTTTGGGCGCTTGGAGGAATTTTTGTGTTTGTCGGTTCTCCGTCGATGGGTCTTCTGTGATGGCGGGCGAAGTAGTCGAGTTTAGGGGCGGGGTGCTGGTCCCTTCGAACACTTCTTCCCAGACCTGTTCCGTTACATGGCGATTGATGCGATGATCCGCCTTGCATCCGGGGTTCCAATTGCAACGGAGGTTGACGTAGCCATTCTGTTGGACG
SEQ ID NO.13
gdh基因核苷酸序列
atggattcggaagatcaggtgcccaacgtgcagagtctgaagactctggatgaacagacgagggagagtctgctaccgcgcaacccagtcatgtcgagaaccgggtcttcaacgtcgctgtctagtcttgttacgactaacacgacgcactaccaaaattggctgaagacgttttatgcgaacaaactgaacgaggtgatacactttcgcgaagcgggccatgtggtaaccaactataattataagatggacgatggtgacgacatggatgacatagttgagccctgcctgctcgactgccacacggaggagggttttgttatggctgccatgcacctgattctgcatcgcataaaggcgaagcaggctgagtctccgacttcgattgtgactattggtttgtccggtggcaacaccccgagggccgtgttccggtacatgtctaacatggagaatctcgacattgacttcaatcgcataatattcttcctagtggacgagcgttatgttccgcctactgacgagctgtctaatcagcggcttatacggaacacgctgctgaaaaactggccggtgcccgagtcgcacatcattttcccggacacaaccttgcctctggaagaatgtgtgcgcaagtatgaggcggatctggagcgcgttttcggcaagttggagccctcggcaacggctgcggttgattcttcgccccaatccaaagtggttggtatggctatgagcaagagcacgctgaagcccgacttgatcacgctgggcattggcgacgatttccacattgctggcctgttcccggagtttttatccacactcgagccctcgcaagtaacggactgcaaggaacgtgtcatgatcacccatacagacacggcggtggttcatgaccggttgaccctatcactgcctttcctgtgttgcgcaaagagcaagctgtttttcctcaagggggagcgtaagaagcgcatttggacgaccatgctgcaatacagacacgttgacccgatacgtttcccggctactcagatcttcacgaaggctggatgcgttgcagtgttggactcgtcgaccatccgccgaatcagacgtaaagttccgctggagcaatcggactgcctaactttcattttgttcggtagcactggcgatttggctcgtcgcaagctctacccagctctgttccatctcttttatttgggattcttgccaaccaaattccgaattttggcagtgagccggtcgcatcagtcgtttgacgaatttttcgagacggtctccaaggacattttcgcctctatcaacaccacgattttcatgtgcgaagcggcggcgcgattcgatttccccactgtgattgcggaattcaagaagaagctggggagggtgaccgtgaagtacgacaacccccaatcgctcgtgcagctgaacgagagcttgaacgagattgagtcgggttctaccgtgacgcaccgtctcatctacatggccacgccagctgaagcctaccatcccatcatgaagattgcaacttcggtgtgccgccccaaggagggttggttcagggtgatgttggagaagccgttcgggcgtgacatggagagctgccaggatatccagcgtgtgctgcagcagcacgcacagcccgaggagatgttcctcgtggaccactacttggggaagccgctgatatcctgtatcatagccatcaagcgttcgctgcgctacaaccatctcttcagcagacggtacgtgaagagcgtgcacattaaaatgaaggagcgcatcggcacgttcgggcgcaactatttcgagaactacggcattattcgcgacatggtgcagaatcacgggttgcaactgctgtcgttgattgcgatggacaaacccgaccggctgaatgactctgttgctgacgagaagcgcaaggtgctgagggccgtgcgcaccgtgcgtctggaggacaccatcataggtcagtatacggagagcgaggatggcagcgagtgcgcgtacttgcaggagaagggaacgcctccagattcgctctgcagcacgttctgctccatggtcatgtacgtcgataacgacaagtggcagggcgtgccgttcgtgataaccaccggcaaaggtctggatgagcatctgtgtgaggtgcggcttaacatgaaaagcaacttcgcagccgaatttggcgcggacttcgaggcgcgcaacctcgttttccgggttcagcccaaccctactgtcttctggtccgtggactccaggcacttcgacacgcagcaagagagtgagaagttggtgttcaccagcgaccatgacaccgacaacatgggcctgaaggtggaggagcgtatcttacgtgatgtggactacaacctgaccaagcgtcccgtggttgggtgctacgaaatactgctctaccacgccttcagcggcaagcgcaactacttcccttcgctcgaagaagtgagcgagtgttggcgtattctgacccccgtgctgcacgaaattgacgataagcgcgtgcgcccgattctgtacccgcgtgggtcgagcgggcccaaagagcaggagcgtcatttacagctattgaattcaatatga
SEQ ID NO.14
gdh基因氨基酸序列
MDSEDQVPNVQSLKTLDEQTRESLLPRNPVMSRTGSSTSLSSLVTTNTTHYQNWLKTFYANKLNEVIHFREAGHVVTNYNYKMDDGDDMDDIVEPCLLDCHTEEGFVMAAMHLILHRIKAKQAESPTSIVTIGLSGGNTPRAVFRYMSNMENLDIDFNRIIFFLVDERYVPPTDELSNQRLIRNTLLKNWPVPESHIIFPDTTLPLEECVRKYEADLERVFGKLEPSATAAVDSSPQSKVVGMAMSKSTLKPDLITLGIGDDFHIAGLFPEFLSTLEPSQVTDCKERVMITHTDTAVVHDRLTLSLPFLCCAKSKLFFLKGERKKRIWTTMLQYRHVDPIRFPATQIFTKAGCVAVLDSSTIRRIRRKVPLEQSDCLTFILFGSTGDLARRKLYPALFHLFYLGFLPTKFRILAVSRSHQSFDEFFETVSKDIFASINTTIFMCEAAARFDFPTVIAEFKKKLGRVTVKYDNPQSLVQLNESLNEIESGSTVTHRLIYMATPAEAYHPIMKIATSVCRPKEGWFRVMLEKPFGRDMESCQDIQRVLQQHAQPEEMFLVDHYLGKPLISCIIAIKRSLRYNHLFSRRYVKSVHIKMKERIGTFGRNYFENYGIIRDMVQNHGLQLLSLIAMDKPDRLNDSVADEKRKVLRAVRTVRLEDTIIGQYTESEDGSECAYLQEKGTPPDSLCSTFCSMVMYVDNDKWQGVPFVITTGKGLDEHLCEVRLNMKSNFAAEFGADFEARNLVFRVQPNPTVFWSVDSRHFDTQQESEKLVFTSDHDTDNMGLKVEERILRDVDYNLTKRPVVGCYEILLYHAFSGKRNYFPSLEEVSECWRILTPVLHEIDDKRVRPILYPRGSSGPKEQERHLQLLNSI
SEQ ID NO.15
PgpdA启动子核苷酸序列
gcttgatgtcttggtattgagtctacggtgtacaatggtgtactgcgtgtagcggcccccagtatgaccaagatctaaagggcattattttttaggccagattgtttagattgaattagactagccatgtataggtggatattattcagtatcgcctgactatgatatgataaccacattaggtttatataatgacgttttatgtattatacacgcatggggagtgatcactgttatcaaaatcactgagaaagagctaagactattggttcaaaccccagtacagtttcatgcaaagttctagatggcgggatctaggttcccaacaaggcagaaatattgaattgaattttttctaaaaagaataaacaatggtaaaaaaattccaaagagatgattaattgaaattaaaagagaaaataaaaccatgacaagcgcgtgatcctcatttgcctaccggtgaatcttttcaggaatcaggagagcagaagaggcttagagagatcgctaagcccatgatatgagtgtcgagagggaagaggggcagtggaccatagttgatccggtccgatatctcggcccggaaacggaaaggtcacaccgagtgcccctcatttttccattgcttccatccattaagcttgggtgggatgctgtggtctgtagtgttagtctgtatggccagattgtaattacatcatgcccctctatggggatgcctcaggtatgggaccccagggtatcatttccccctcaattgcttgaactacggaacaaaggacaaaaagatagagtaatagccgggatcgtcttcctcgtagcctaggtagtactgccccctcgattccgaaaaactggcaaaagattcacgagatggtaggattgagtacccggcatgctggatttgaggcacgctcattggccagaccggtagctgccgaggagaggcagagtcccaaatatcgtgagtctcctgctttgcccggtgtatgaaaccggaaagggctgctgggagctggggagcggcgcaagccgggaaaacagctgacaaggacccatttcactctggatcttgaggagagctgtagcttttgccccgtctgtccacccggtgactggattagtgacctggtcgttgcgtcagtcaacattgctctttttttatctccccctcccccgccgtccgacttttctccccttttctactctcttcgtatactcaccactgcaatcaccttatccctttgtcttcttacttaaagtgagtcgtctcccgcccatcattccctttggatctcataaagcagagccactttcaagtgcctaccgtttccctttccacacagattgactgacagctaccccgccacaccaacagacacatctaaaca
SEQ ID NO.16
msn2L左臂的核苷酸序列
tcattcatccagaacaagagacatctacattcattcaacctacagtgctcgacatctatcattagcaccaagctgcctcgacatcaatctggaacatcctgacttagactaaacatacttaataatacccaagaggttaagcagtgttcgcttgatcctcatcacattcatcattggttcgtggaaggatgactatatatcatacaggatgcccatactgatccctcgtcttctagtcacaatggacgctacatacaccatggcaccaacggtgcaaggacaaccatcatttgcatactaccccactgctgattcacaaagacaacaatacacaagccacccggctgagccccagccatactatggacagattcaggccttccctcagcaacactgcctcccagagcaacaaccggtctacaatgctcaacccatgatgaacatgcatcagatggctaccaccaatgccttccgcggagcaatgaacatgactcccatcgcctctccccaaccatctcacctgaagcccacaatcgtcgtccagcagggttccccagccctcatgcctttggacacaaggttcgtcggtgactactatagcttcccttctacccctccactctccaccgcgggaagttctatcagcagcccgccatccagcagtggcaccttgcacaccccaatcaacgactgcttcttctcattcgaaaaggtggagggtgtaaaggagggttgtgaaagtgacgtccatgccgagcttttggccagtgccgactggactcggtcagcaacttcgcctcccatgactcctggtatgttttcttctcatcagttgagtagggggttggaaccagtctaacttcttctagtcttcatcaacgccaattctctcaccgccagtcagagctcagacttcctctctgcccacgggtcttgcccctctctttctccttcgccttctcctgtatcttccttgtttactccgtctcagtcggctttccctgttgagcaggccacctccgacttctgtga
SEQ ID NO.17
msn2R右臂的核苷酸序列
gaacaacgcatctgaacggtatgataatcgcgacgcgagtaccatgggtgctgtgctctatgaagctgcccacgctgcggcgaccaagtcgacgactagcgaatcatccgaagatggtgtttctgacgcaccctctatcgatcgtcggtccgccaagaagcgcaagcgtgacgagcacgtttagttgcccccactgccgtttgcaacttcacgacgcgatgtgaccaggagctccccccttactacttaatacactatcataattactccaccattgcactggttggtctggcttttgtctttgcggttttggcgtctgggcatttacattggcggtgtcaggtgggaggctaaggtgtctggcaaaaggatttcgtttctcacaggggaaaagagaaacataatatagcacatggtcttcacaaaggaagatttacattggaggcattagaccggttgtctgggcggcaacggattcggcgcactgggagtccacggtttggactaaacttagagcgcatgacgccttctttgaggcacaggtggtgatttgggatttttctttttttctcgtcgcattttctggcaaagatgtttttgcctcgatcaacagaatggctcgagtcattcttcactcttcttgtacccatttttcttttctttttttttttttttccctctttttctcttctctatacttctcgttcatcttgcattctgtgtccctatttgatgtaatatttgatttgaccttgacccgggttgcaccctcaccacgtctttcctgtgctggccacatgtatacttcaacttgtccccaatcattgcctcatacccctgtgcgcatcatcctgttttcattttgtttgcatccataatattcccccgttgttaattcaagcgcccatttgatttgtggcacagttagtgataatcaaagattcatcaagaaatacctttccaagtatgtagagtagtagagtagtatgttctgtcaacccatggtcagtggtttggcttccgt
本发明进一步公开了重组米曲霉菌株在大幅提高曲酸产量方面的应用，实验结果证明：本发明利用诱导型启动子PsodM启动laeA基因在米曲霉中过表达，次级代谢产物特别曲酸的产量；利用诱导型启动子PglaA启动kojA,KojR,kojT基因在米曲霉中过表达，专一增加曲酸途径的转录及合成；利组成型启动子PgpdA启动gdh基因在米曲霉中过表达同时，持续增强HMP代谢通路增加曲酸合成；同时敲除msn2减少对环境胁迫的耐受以提高曲酸合成量；该出发菌株为米曲霉3.042(CGMCC No. 3.042)来源中国微生物菌种保藏管理委员会普通微生物中心CGMCC，其曲酸产量为27.84g/L，与此相比，该重组米曲霉菌株在多基因过表达条件下经过培养基成分优化后曲酸产量为115.37g/L，产量提高414.4%。
附图说明
图1为米曲霉CGMCC No.18557（重组菌株44）与出发菌株产曲酸对比；
图2为不同碳源对米曲霉CGMCC No.18557（重组菌株44）产曲酸的影响；
图3为不同氮源对米曲霉CGMCC No.18557（重组菌株44）产曲酸的影响；
图4为过表达laeA基因同时敲除pyrG基因pk8质粒构建图；
图5为过表达kojA, kojR, kojT基因同时敲除ku70 pk7质粒构建图；
图6为过表达gdh基因同时敲除 msn2基因pk1质粒构建图。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。本发明所用原料及试剂均有市售。其中米曲霉CGMCC No.3.042的构建中国微生物菌种保藏管理委员会普通微生物中心CGMCC。
实施例1
（1）米曲霉DNA的提取：首先将米曲霉菌丝体通过液氮研磨进行破碎，然后采用有机混合试剂，即苯酚：氯仿：异戊醇=25:24:1的混合液进行基因组的提取。
(2) 次级代谢产物全局调控因子laeA基因过表达质粒构建：
1)利用引物pyrGL-F和pyrGL-R以米曲霉基因组为模板扩增pyrGL左臂序列。
2)利用引物PsodM-F和PsodM-R以米曲霉基因组为模板扩增PsodM核心启动序列。
3)利用引物laeA-F和laeA-R以米曲霉基因组为模板扩增laeA基因及其终止子TlaeA序列。
利用引物pyrGR-F和pyrGR-R以米曲霉基因组为模板扩增pyrGR右臂序列。
用限制性内切酶EcoR I, Hind Ⅲ对已有质粒p44双酶切，利用Vazyme One StepClone Kit进行同源重组，形成pyrGL-PsodM-laeA-pyrGR表达框，，得到pk8表达载体；
用到的引物如下：
pyrGL-F：tatgaccatgattacgaattcTGTCTTTGCATCTGAGGCGC
pyrGL-R：TTAGCATGGTACTCTGTAGAACTCTGATcatacggtcagccaaatc
PsodM-F：gatttggctgaccgtatgATCAGAGTTCTACAGAGTACCATGCTAA
PsodM-R：gtctggccgtttccaaacatTTTGGGTGGTTTGGTTGGTATT
laeA-F：AATACCAACCAAACCACCCAAAatgtttggaaacggccagac
laeA-R：TGTCTCCCTTCGAGGAGAGGTcccagtaatggagctc
pyrGR-F：gagctccattactgggACCTCTCCTCGAAGGGAGACA
pyrGR-R：acgacggccagtgccaagcttCGACAGCCGCGATAAGTGC
(3) 曲酸合成基因簇内kojA,KojR,kojT基因过表达质粒pk7构建：
1)利用引物ku70L-F和ku70L-R以米曲霉基因组为模板扩增ku70L左臂序列。
2)利用引物PglaA-F和PglaA-R以米曲霉基因组为模板扩增PglaA核心启动序列。
3)利用引物koj-F和kojR-R以米曲霉基因组为模板扩增kojA、kojR、kojT序列。
利用引物pyrG-F和pyrG-R以米曲霉基因组为模板扩增pyrG回补标记序列。
利用引物koj3’-F和koj3’-R以米曲霉基因组为模板扩增koj3’序列。
利用引物ku70R-F和ku70R-R以米曲霉基因组为模板扩增ku70R右臂序列。
用限制性内切酶Sma I对已有质粒p44单酶切，利用Vazyme One Step Clone Kit进行同源重组，形成ku70L-PglaA-koj-pyrG-koj3’—ku70R表达框，得到pk7表达载体；
用到的引物如下：
Ku70L-F：aattcgagctcggtacccgggCTCCAGGCGATTTCTCCATTT
Ku70L -R：TCCTCGTCAGCCATTGTGAAT
PglaA-F：ttcacaatggctgacgaggaGCGAGACCGGACCACAATT
PglaA -R：GTGACTGCAAACTAACTTGCTTCGACTTCGTTTGC
koj-F：GCAAACGAAGTCGAAGCAAG ttagtttgcagtcac
koj -R：tatcggTCAGGGGGTCTTTTTATTCCG
pyrG-F：aaaagaccccctgaCCGATAGGGTTGGGCTTATTG
pyrG-R：GGCGACTAGCGGCGAGGGTGGTGGGAAATC
koj3’-F：GATTTCCCACCACCCtcgccgctagtcgcc
koj3’ -R：tctcatttctgctccTCAGGGGGTCTTTTTATTCCG
ku70R-F：cctgaGGAGCAGAAATGAGACAACGCT
ku70R-R：cgactctagaggatccccgggCGTCCAACAGAATGGCTACGT
(4) 葡萄糖-6-磷酸脱氢酶gdh基因过表达质粒pk1构建：
1)利用引物msn2L-F和msn2L-R以米曲霉基因组为模板扩增msn2L左臂序列。
2)利用引物PgpdA-F和PgpdA-R以米曲霉基因组为模板扩增PglaA核心启动序列。
3)利用引物gdh-F和gdh-R以米曲霉基因组为模板扩增gdh基因序列。
4)利用引物pyrG-F和pyrG-R以米曲霉基因组为模板扩增pyrG回补标记序列。
5)利用引物msn2R-F和msn2R-R以米曲霉基因组为模板扩增msn2R右臂序列。
6)用限制性内切酶BamH I, Xba I对已有质粒p44双酶切，利用Vazyme One StepClone Kit进行同源重组，形成msn2L-PgpdA-gdh-pyrG-msn2R表达框，得到pk1表达载体；
用到的引物如下：
msn2L-F：gagctcggtacccggggatccTCATTCATCCAGAACAAGAGACATC
msn2L -R：tggcctctgtTCACAGAAGTCGGAGGTGGC
PgpdA-F：acttctgtgaACAGAGGCCAGAGCATCACCA
PgpdA -R：ctgatcttccgaatccatTGTTTAGATGTGTCTATGTGGCGG
gdh-F：caATGGATTCGGAAGATCAGGTGC
gdh-R：cccaaccctatcggTCATATTGAATTCAATAGCTGTAAATGAC
pyrG-F：atatgaCCGATAGGGTTGGGCTTATTG
pyrG-R：ttgttcGGGTGGTGGGAAATCTTGTATATAA
msn2R-F：atttcccaccacccGAACAACGCATCTGAACGGTATG
msn2R-R：tcagttaacgtcgactctagaACGGAAGCCAAACCACTGACC
(5)将构建的pK8, pK7, pK1质粒分别转化到根癌农杆菌中
根癌农杆菌感受态的制备及转化：
1)农杆菌AG1菌液70 μL(来源实验室保存工具菌株)，接种于3 mL LB培养基中，28℃，180 r/min培养过夜。
2）取过夜培养的菌液2 mL，接种于100 mL LB培养基中，28℃，180 r/min，6 h。
3）将菌液冰浴40 min，4℃，5000 r/min离心10 min，弃上清，用10 mL冰冷的无菌水悬浮。
4）重复步骤3）。
5）4℃，5000 r/min离心10 min，弃上清，用10 mL冷却的10%甘油悬浮。
6）重复步骤5）。
7）4℃，5000 r/min离心10 min，弃上清，用1 mL冷却的10%甘油悬浮，分装，-20℃保存。
8）分别取pK8, pK7, pK1各质粒1.2 μL，加到70 μL农杆菌感受态中，混匀吸取加到间距0.2 cm电转杯中，置于冰上静置3min。
9）将电转杯放入电转仪中，调节电转仪参数如下，进行电转：
Figure 381069DEST_PATH_IMAGE001
10）加入1 mL LB培养基，混匀，吸出加到一无菌EP管中，28℃，180 r/min，培养6h。吸取150 μL，涂布到含有Kan抗性的LB平板上，28℃，培养2 d。挑取农杆菌单菌落接种于3mL含100 μg/mL Kan的LB培养基中，28℃，180 r/min，培养过夜，PCR验证正确即可进行后续实验。
（6）携带不同pK8, pK7, pK1质粒农杆菌介导米曲霉CGMCC No.3.042转化：
挑取农杆菌单菌落接种于3 mL含100 μg/mL Kan的LB培养基中，28℃，180 r/min，培养过夜。取过夜菌液0.5 mL菌液接种于2.5 mL含100 μg/mL Kan和200 μM AS的LB中，28℃，180 r/min，培养6 h。接种PDA斜面，30℃，培养6d。吸取3 mL无菌水，洗脱斜面中的米曲霉孢子，将其经过有三层纱布和塞有脱脂棉的锥形漏斗，获得米曲霉孢子悬液。使用无菌镊子将0.45 μm的Hybond N+滤膜放于含有100 μg/mL Kan和200 μMAS的IM平板上。各取100 μL的OD600nm为0.8的农杆菌菌液和1×107个/mL的米曲霉CGMCC No.3.042孢子悬液，混合均匀，将目的基因整合到米曲霉基因组中，经两次筛选后,提米曲霉基因组验证，得到阳性转化子44号。
测试例：
（1）将上述pK8, pK7, pK1质粒转化后获得的阳性转化子44号即构建成功将米曲霉CGMCC No.18557接种于PDA培养基上30℃培养6天，刮取孢子制成孢子悬液并进行活菌计数。以 104个/mL的接种量接种于装有50 mL发酵液的250mL摇瓶中，进行三个平行，30℃、250 r/min 培养144 h后，进行产酸测定，结果如图1。由图1可以看出，米曲霉CGMCCNo.18557（重组菌株44）产酸性能优于原始出发菌株3.042，曲酸产量有大幅提高。
（2）将实施例米曲霉CGMCC No.18557接种于不同碳源及不同氮源的发酵培养基上30℃培养6天，刮取孢子制成孢子悬液并进行活菌计数。以 104个/mL的接种量接种于装有50 mL发酵液的250mL摇瓶中，每个碳氮源种类进行三个平行，30℃、250 r/min 培养144 h后，进行产酸测定，结果如图2、3。由图2、3可以看出，米曲霉CGMCC No.18557（重组菌株44）在优化后的发酵培养基上，曲酸产量提高了414.4%。
实施例2
曲酸的发酵方法如下：
一种米曲霉曲酸培养基,其中的可利用碳源种类有：葡萄糖、蔗糖、糊精、麦芽糖、半乳糖、可溶性淀粉、果糖、玉米液化液、米糠、甘油。可利用氮源种类有：酵母浸出物、胰蛋白胨、蛋白胨、牛肉膏、尿素、豆粕、硝酸铵、硝酸钠；具体步骤如下
1）葡萄糖为碳源添加量15%；豆粕氮源添加量1.5%，磷酸二氢钾添加量1%，硫酸镁添加量0.075%。
2）将步骤1）各组分混合，搅拌使各组分在水中充分溶解；
3）调节发酵液pH值为6.0，进行灭菌。以 104个/mL的接种量接种于装有50 mL发酵液的250mL摇瓶中，30℃、250 r/min 培养144 h后，进行产曲酸达到110g/L以上。
实施例3
曲酸的发酵方法如下：
一种米曲霉曲酸培养基,其中的可利用碳源种类有：葡萄糖、蔗糖、糊精、麦芽糖、半乳糖、可溶性淀粉、果糖、玉米液化液、米糠、甘油。可利用氮源种类有：酵母浸出物、胰蛋白胨、蛋白胨、牛肉膏、尿素、豆粕、硝酸铵、硝酸钠；具体步骤如下
1）糊精为碳源添加量10%；胰蛋白胨氮源添加量1 %，磷酸二氢钾添加量0.5%，硫酸镁添加量0.075%。
2）将步骤1）各组分混合，搅拌使各组分在水中充分溶解；
3）调节发酵液pH值为6.0，进行灭菌。以 104个/mL的接种量接种于装有50 mL发酵液的250mL摇瓶中，30℃、250 r/min 培养144 h后，进行产曲酸达到110g/L以上。实施例4
曲酸的发酵方法如下：
一种米曲霉曲酸培养基,其中的可利用碳源种类有：葡萄糖、蔗糖、糊精、麦芽糖、半乳糖、可溶性淀粉、果糖、玉米液化液、米糠、甘油。可利用氮源种类有：酵母浸出物、胰蛋白胨、蛋白胨、牛肉膏、尿素、豆粕、硝酸铵、硝酸钠；具体步骤如下
1）蔗糖为碳源添加量12%；氮源添加量酵母浸出物1.5%，磷酸二氢钾添加量1%，硫酸镁添加量0.05%。
2）将步骤1）各组分混合，搅拌使各组分在水中充分溶解；
3）调节发酵液pH值为6.0，进行灭菌。调节发酵液pH值为6.0，进行灭菌。以 104个/mL的接种量接种于装有50 mL发酵液的250mL摇瓶中，30℃、250 r/min 培养144 h后，进行产曲酸达到110g/L以上。
序列表
<110> 天津科技大学
<120> 一株重组米曲霉菌株及其曲酸发酵方法与应用
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1539
<212> DNA
<213> 人工序列()
<400> 1
atgtttggaa acggccagac tggacagcgt cttccggcga tggcctcacc accacacgac 60
tcgtactact cacagtcatt ggcatctagt cgatcaagga ataactcgga tgctatggat 120
atctacgcca tcacagacag agatcctccg gcacgagaac cctctggtta tagccagtgg 180
taccgtaatg gttctccaag tgtgaattcc attcatagca agtaattctt ccctgtttcc 240
ttaaatctgc cgttgaattt ctctctgggc tgcgtcagtt gttcactctg ccgaagctgt 300
tattgttgga cgactaatgt tcttctctgt tgttactgtc tctctctagg agttctgaaa 360
aacagccttt ctatgaagaa aacggacgaa tgtatcatgc gtaccgcaaa ggggtatata 420
tgctaccatg cgatgagcag gagcaagatc gccttgatat cttccacaaa ttattcacgg 480
tggcaagggt gtcggatggc ctaatgtacg ccccgcatcc caggaacggc cgatttctag 540
acttgggctg cgggaccggg atatgggcaa ttgatgttgc caacaaatac ccagacgctt 600
tcgttgttgg ggttgacctt gctcccatac agccctcaaa ccacccaaag aattgcgaat 660
tttacgcccc attcgacttc gagagtcctt gggccatggg cgaggattct tgggacctga 720
ttcacctgca aatggggtgt ggaagtgtga tgggatggcc gaacctgtac cggagaatat 780
tcgctcacct tcgacccggg gcttggtttg aacaggtgga gatcgacttt gaaccgcggt 840
gcgatgaccg tcctctcgag ggactagcta ttcgacagtg gtatcagtat ctaaagcaag 900
ctacacaaga tgccatgcga cctataaacc acaactctcg tgatacaatc cgagatctgc 960
aggaggctgg ttttaccgat attgatcatc aaatggtggg gttgcccctt aacccatggc 1020
atcaagacga gcacgaaaga aaggttgctc gctggtacaa tttggctgtc tcggagagta 1080
ttgagtcgct cagtatggcg cctttcagtc gtatttttaa ttgggacttg gacagaatca 1140
ggcgcatttc gtcagaagtc aagtcggagg cgttcaacaa agaaatacac gcctacaata 1200
tccttcatat ataccaagca cggaaacctg cgaactgatt cttctaccaa catgcgcacg 1260
acggacatcc aaacatgcgc cagcagcgtc actagtgccc aagctccgag ttatggggtg 1320
ggcgaattac catccaggaa gtcaccttta tctgctcttt tatgagctct ccaaagatgc 1380
agcgagttga tatgagcttg tgtggtgcca cttgttagct tgcacacaac ggtcccgagc 1440
agtcatttgc gtactgcaaa agcaaaatcg aactcatacg ggtctgtgat ctctatctat 1500
ctggaaaatc cagtgtgtcg ggagagctcc attactggg 1539
<210> 2
<211> 369
<212> PRT
<213> laeA氨基酸序列()
<400> 2
Met Phe Gly Asn Gly Gln Thr Gly Gln Arg Leu Pro Ala Met Ala Ser
1 5 10 15
Pro Pro His Asp Ser Tyr Tyr Ser Gln Ser Leu Ala Ser Ser Arg Ser
20 25 30
Arg Asn Asn Ser Asp Ala Met Asp Ile Tyr Ala Ile Thr Asp Arg Asp
35 40 45
Pro Pro Ala Arg Glu Pro Ser Gly Tyr Ser Gln Trp Tyr Arg Asn Gly
50 55 60
Ser Pro Ser Val Asn Ser Ile His Ser Lys Ser Ser Glu Lys Gln Pro
65 70 75 80
Phe Tyr Glu Glu Asn Gly Arg Met Tyr His Ala Tyr Arg Lys Gly Val
85 90 95
Tyr Met Leu Pro Cys Asp Glu Gln Glu Gln Asp Arg Leu Asp Ile Phe
100 105 110
His Lys Leu Phe Thr Val Ala Arg Val Ser Asp Gly Leu Met Tyr Ala
115 120 125
Pro His Pro Arg Asn Gly Arg Phe Leu Asp Leu Gly Cys Gly Thr Gly
130 135 140
Ile Trp Ala Ile Asp Val Ala Asn Lys Tyr Pro Asp Ala Phe Val Val
145 150 155 160
Gly Val Asp Leu Ala Pro Ile Gln Pro Ser Asn His Pro Lys Asn Cys
165 170 175
Glu Phe Tyr Ala Pro Phe Asp Phe Glu Ser Pro Trp Ala Met Gly Glu
180 185 190
Asp Ser Trp Asp Leu Ile His Leu Gln Met Gly Cys Gly Ser Val Met
195 200 205
Gly Trp Pro Asn Leu Tyr Arg Arg Ile Phe Ala His Leu Arg Pro Gly
210 215 220
Ala Trp Phe Glu Gln Val Glu Ile Asp Phe Glu Pro Arg Cys Asp Asp
225 230 235 240
Arg Pro Leu Glu Gly Leu Ala Ile Arg Gln Trp Tyr Gln Tyr Leu Lys
245 250 255
Gln Ala Thr Gln Asp Ala Met Arg Pro Ile Asn His Asn Ser Arg Asp
260 265 270
Thr Ile Arg Asp Leu Gln Glu Ala Gly Phe Thr Asp Ile Asp His Gln
275 280 285
Met Val Gly Leu Pro Leu Asn Pro Trp His Gln Asp Glu His Glu Arg
290 295 300
Lys Val Ala Arg Trp Tyr Asn Leu Ala Val Ser Glu Ser Ile Glu Ser
305 310 315 320
Leu Ser Met Ala Pro Phe Ser Arg Ile Phe Asn Trp Asp Leu Asp Arg
325 330 335
Ile Arg Arg Ile Ser Ser Glu Val Lys Ser Glu Ala Phe Asn Lys Glu
340 345 350
Ile His Ala Tyr Asn Ile Leu His Ile Tyr Gln Ala Arg Lys Pro Ala
355 360 365
Asn
<210> 3
<211> 600
<212> DNA
<213> 人工序列()
<400> 3
atcagagttc tacagagtac catgctaagc ctggggattt actggttaac atttgcctag 60
taacttgtaa gaatcggaag tacttggctc catgagcccc acattatgaa aaacaatggc 120
atcaacaaat tgtctgtgca ttatcatagt atagttctgg gtgggggcta tagtacttgc 180
tggtacttca tggagtgaca ggctgtagta ggcggcctta ataaaaccca gcagaacgca 240
ttcattagac agatttatct acgcatatac cagctaacat agcctatttg taatccttgt 300
aatccacact cacctacgaa tcacatagac gaaaggaaga atgtgctcag tggtgcgcca 360
aaggtccccc gaatcccctc aaactcgctc atagtcacat cccgccaaca tgattggtaa 420
aaatgaaggt accttggggg tcctcagcat ccattccagg cttatattct ccctcgccat 480
aatcatgacc agaactccct cacaacccct cttctccctc gtcctcgtcc tcgcatctca 540
cctctccaac tacacatata ctcaaaaccc tcaaccagaa taccaaccaa accacccaaa 600
<210> 4
<211> 1413
<212> DNA
<213> 人工序列()
<400> 4
tgaaatcaaa tcctgcctac ctaacatggg atttagtcat ttcttttggc atcaaagcaa 60
ggcgccacaa tcgttgaggt aagaatgatc tgccaatatg tgagcgagcg cggctgacaa 120
ctatggtagt ttggtaatac aacatctcac gaatcgtacc ttgcatgtgc actaaagttt 180
ggtcagatgt acagttaaca agagatctcg tgcccgtcat ataccatgac ttttctctga 240
gcgagtcagg gactgatatc cctattcatg acttaaatct tgatcaggtc agtatctctg 300
agattgattc gaactgattc tcacccgtca aagttcatgc acgcaagcaa tatccagtcg 360
ccacgagggg atcccgtatc tgtgcttggg aaagcgaatg cacagcctat atccactcag 420
gttacttcga ctaagccacg atctcgatca ttaactaaag accacgagag gggaactagg 480
gaaatccgtg atagaatgaa gtatacagtt gattttgtca gtaagggatt caaacctaat 540
accagaggtg actttatcca agattccttc acgacactgg aagaactgct tgaagagctg 600
cctgagtcaa tcagcttcaa tatcgagata agtaggttat atgctgccac tggtggttct 660
ccacttgcgg gagacaaagc taacaacgtc ccaatgaaga gtaccccagg cttcatgaag 720
ctatagaagc aggtgtagca ccagtggcta ttgaaatcaa caccttcgtc gacaaagcgc 780
ttgagagact cttttcttac ggcaacaaaa aacggaccat tatcctatcc tcatttaccc 840
ccgagatctg cattttattg gccatcaaac aacagacgta ccctgtgatg ttcatcacta 900
atgccggcaa gcctccagtt acggatcgag agatgagggc tgccagcata cagtccgctg 960
ttcgatttgc caagaggtgg aatttatctg gccttgtctt tgcatctgag gcgctggtaa 1020
tgtgccccag gcttgtcaga tatgttcagc gatcaggatt gatctgtgga tcctatggat 1080
ctcagaacaa tataccagaa aatgcgaagg taagtgcttc tatattagtt cttagtgctt 1140
tcaaattgtg atgtagaagt tgttcaatag ctgatgaaat attctagacc caagccgctg 1200
ctggaattga cattattatg gccgataggg ttgggcttat tgctatgtcc ctgaaaggat 1260
atcaaaagca ggcaaaaagc caggcataat ccccgcgcgg atggtaccct aaggataggc 1320
cctaatctta tctacatgtg actgcatcga tgtgtttggt ccaaatgagg catgtggctc 1380
accccacagg cggagaaacg tgtggctagt gca 1413
<210> 5
<211> 1328
<212> DNA
<213> 人工序列()
<400> 5
cagggtcaag ggtcatgttt caaaatcaca ataatctcca acatagacga gaatttgtac 60
cggagtgtct gaaggtgcag ctggagattg gtctattttc ttagagtggg gtatcactaa 120
tatacagtcg gtcactatcg tacaaacaat cacaattata tacaagattt cccaccaccc 180
cctactctaa cacggcacaa ttatccatcg agtcagagcc tatccaccat ttggtgctct 240
cgtagagacc aaagtataat cctgatccga cagcggccat aaacgtgttg atagcacacc 300
ctcggaatag tcctctcggg ccatctgttc gtacaatctc ccgtacggta ttgatcatcc 360
ttttcttctg aggtgcagtt gtatctgcag catcgagcat gattcgtgtc cggaccatat 420
ccatgggtgc tgtcaagaca ctggctatac cgcccgagac cgcagcactt atcgcggctg 480
tcgctgcagc ctctccgatt gtcgaatggg cctctttctt tccatactct cttggtcttt 540
ctagcacctt ctctcgatct ccgaatctat attcaaaaat tcgataccga aaagactcgt 600
acagaggcat ctgaatcgcc gacactggca agctatgcgc cacaagagcc gggtatccgc 660
tccaaagctg tctagggttg ataaacttct tgaaagctag ccgtgtcgct ttctgggcta 720
caccacctac ccttccccca gctacaggtg ctgatgcgtc tggatggtgt gattggatca 780
tctgcgcgtc gtgttttaat gcatcagccg gagcaaagac tccgcaagca gcaagatccg 840
caacggaggc tgcgcaaaaa tcggaaaaga gccgagctga gctagactca tgcgttccaa 900
gtttttgatg tatgacttgg agtcctgact gtgcatactc gtatgtgatg aagaatgcgc 960
ccgctttgac ccgttattct ttgctcaatg aatatgagtt gggtaaggat aatgttgggt 1020
atcaccaacc tgtgggaaat gaggcagcgg ttacgctcgc gataccttgg taaaggccgc 1080
ggaatatccc tgggtgtctc catatgctgg ttcctgtgtt ggttctcagg aactgcgaat 1140
attcgcgaga ctgaatgcgg gttttgatcg tatctaaggg aaatatgaat agatctagtg 1200
agagggatgc ggttgtgctt gcctagttcc ctcaggttaa tgtatgctgt ttgtcctagg 1260
gggttagaaa atataccagc aatatactaa gtggctcggc ggccatatta tttgtcgata 1320
gaggcggg 1328
<210> 6
<211> 5776
<212> DNA
<213> 人工序列()
<400> 6
ttagtttgca gtcactagtg aaacataggt ctgcaagttg ctagtgtggt gctcgcccag 60
ccctggctta tcgccaatct ccttgctgtg ggtgtggaag gcgctgctca cagtgaagcg 120
tacgccattc cactcccgaa ccacaccgtg ccgtcggcgc gcaatgtcat tgatcagagg 180
aaccacttcc tcggcagccg ttacaacgtt gctcaccttg ccggggaaga tgtggatcac 240
gccgtctttt tcaaacgtca aactgcttcg gaattcggat gtggtcttta acttggccag 300
gacactgccc ttccagccgc ggtactggaa ccgatcgaga aatccgggcc agaaccgggt 360
aatctcccgc tcacagtggg gcttgatccg agcagcgata gcttcctcgt ccagactatc 420
cagaagctct tgaccctcct cgtgacggtc gaatgatccg aggatggtgt aactgccgtg 480
tgtcaagata tacttcttct gaaggggctc gtttgtgtcg atcgccggca tcacgcaggg 540
aaaccagcca tccatgacaa tgaaggaaag aggcttttcc tgtggctgct ggtcttcgta 600
aacgagagca atacaggtct gataggtgat gccaatgtca accggaagag catctgcaat 660
attttgtggc agaaggctgg tataaccggt ggcgttgatc actacgtcaa agacgtactg 720
cccgtccccg gtttgaatgc ggttggtgtc ctcggtgtgg atgatacggt cgactgttgc 780
attgaggtgc acgtagatgc ctgcacgacc aagtttctct ttgaatgtat tccggagtcg 840
gtcgccaata gcgacgctgg gttcatccag attgtatgcg ctgatgaggc cctggaaacc 900
attcgccttc ggatcaaccg cagtacactc aggagattcg tagcatacat ccctgaatgc 960
ctcatccgag accttcgatg ggttccccag agcgtccgct tcgccgtgtg catatatcgc 1020
ggattcatgg tggacgacta gttcgggata cgtctcacag aacttgacca aggcttcacg 1080
gcagctgtct cgagttgctt tagaccgagg gtagtgtggc cccttgtgca agcggatacc 1140
aaagtttcct gagacgcctt gaaagatatc aggcttctgc tcgaacaaag aaactcggtg 1200
gccctgtttc ttgagctcta aagcaagatg gcagccgttc catccgccac cgacgatgcc 1260
gacccttagc tgtgtcgcga cacgcattat gaagaggcag gtagttatag tctagagatg 1320
gttatgttcg gttcaagaat cttgagaaga atgattgttg aagtgtagag gcataccccc 1380
aaaggatctc catcgcttta tatccttccc ttcagcaaga tagcttgact cactcccatg 1440
ggcatgtcaa aaatggtcct tccctgattg taacccaacc ttgccggata acgacacatg 1500
caccctgaag ccaattgcat ccaatgtgat acgaacggat gaggtggaat gagcggcaaa 1560
gtcgggatag atgattgtcg acgactctag aagatcgtaa catactgaat gagcgtttaa 1620
tgcatgtcag cgcttgataa tgtcacatcc gcggcgatgc atccgcaaga cagtatccaa 1680
tgagagggtg gggggatggc acgaaaggaa gcgcactagt cgggatgtgc agcctttgca 1740
gtggacagtg cgccctgcag aaaacagaaa aataacattc tgcaggtatg gtatcatcgc 1800
ttaaggattc caatcctagc agatacccgt cgggggataa cgggatcccg aattagggtt 1860
atccagttcc agcgttggag ccgcgcgtcg gagttaacat ttattccgac tgcccgataa 1920
ccttgtactt ttgcccccgt cgttctgact ctcccgaacc ccccactgat agaccaacgt 1980
cgactgcgat agatcccctt ctttccgcga taaccctgta gtgcagcgta atgtcgttga 2040
ataccgacga ttccggtcgg ataaggaccc ggcaacgcgc caaaagagcg tgcgaaacgt 2100
gcaaactgcg caagaggaaa tgtgacggcc atgagccctg cacttactgc ttgcgatacg 2160
aatatcagtg cactttcaag cctcatccac ggagaaagcc tgcagcttcc aaatcttccg 2220
cacggcccag cgaggaagaa gactcaccaa agtttctcga cagagttgat gctaaccaag 2280
aacacatgga ggccaactca ggcaccgctt tcccccatct cctagggatg aggttgaacc 2340
cgcagggtgc tcccaaggtg tacgggttta gctggaacct aggtccacgg gacgagcctc 2400
tggagccctt caccaatctt acagatctga tttcaagaga ggaaatggaa gacttggcta 2460
gccattattt aaagaagata catccggtat atgcggtcct ggatcccgac acgttgaggc 2520
agaagattgt tgccagatgg catgatcctg caactgcggc cagctatgac cccattcttt 2580
gcggcgtcgc ggcattgggc tccttgtact caggccacca ggaacacccc aaagaaggag 2640
cactggtgca atcagcgaag gagatgcttg aaaccacccg gatctcaaag actactctcc 2700
tgcatcatgc caccgcctgg atcttacgga ccatatatct ccgaagcacc aactgccccc 2760
atgcttcatg gatggccagt tgttccacca tgcacatcat agaggccatc ggggctcacc 2820
aggatccgga actggtgtct ttggtctatt ctgacaccgc agacgttagc gtaaatgatg 2880
agagtcaaag acggttattc tgggtggcca cagtgctgaa ttcatggatt tcgtatgaat 2940
atggccgatc gcgcgttatt ctacgaggtg tctcttgcaa accaccttta ccgcgaacgg 3000
gtgacttcac cacggacctc atctccatgt accagatctc cgaacgactg gatcccgacc 3060
agaacaataa gctttccgac ctagaagatg cactcagccg tgttgagcgc ctcacactct 3120
cccatgacgc cttgatacta tcccaaagta atctggcact cacaatttat cgtcgtctgc 3180
gagtcgcctc gtccaatata tcaaatgaca tccttacccg catcatccga ctaggcaatg 3240
acggccttga agccgcagta cgcctggcgg aggatcgctc gccttggtgg catgtcgcca 3300
atataccctt tcaatttctc tgtatcttgc ttgctattga cactagggaa tcattgtcgt 3360
acgtcggccc agcactgcgc tcattcaggg ccatcaccag gcattacagc acaccgaccc 3420
tacacaccgc gctggagact atagaatcat tggttcgcct atcccagaac aaaaaagaac 3480
gggacttgac tcttctccga gatagcatgc aacaggaaga cccagggctg accgaacaag 3540
gttcaacaac atcccaagcc ttcaatgacg cttcttggct gggggcgacc ggtgatctga 3600
ccttaccaga taactttgac tgggattgga atgtgttctt ggacacccag gttccgttct 3660
tcgacgaggg tgaagcaggt ggtcagagat atagataatg caggaaattg aggactattt 3720
acacgatcag aaagtgctcg tatgcgtcta cttatagtat tagtcgttga acctagtacc 3780
taataattct gcttgaatta tatcatagac tgacatacct catgtaagtc cttcctcctc 3840
cttcggcgcc gaataaggat tttcgaagcc attcagcggc taagtcgggc atgacggaaa 3900
agtcgggtag ggcggaattt ccgttgacgc aatggaagca ccactagaaa taaatactct 3960
gaaagagcac atctatccca gccgaggaat aaaaacactt gacagatgct gctcaattaa 4020
ttgcctccct cccaattcat tttttttatt ctgtggcgta tgtcttgaac gcctcgcaac 4080
gatgcagtcc ttccgccaat accgacggat gcgcagagac ctgcaagaaa gcattaagct 4140
gcatggtccg tatgccgctg ctggtgatcg tcacgtccag ccgaccgatg atattttgga 4200
ggatgccgac gatgccagat tggaaaaagg catccattca atgaacggac acggccaatc 4260
tccatactcc acgccaggaa tcgttcttca cgacggccag agagtgaccg ttcctggagt 4320
caatctccgc cgcgcctccg aaatctgtga gcgcgtcaat actaaaacac ttttcattgt 4380
cgggttcgat ggcccggacg accagctcaa tcccaaaaac tggtctatag ggcggaaatg 4440
ggcgacattg ggcattgtag gcacaacggg tatgcttgtt ggatgggctt cctccatcga 4500
ctccactgtg attaaacaag ggcaagaggc gttcggagtc agcgaggtcg ccgaatctct 4560
cgcaactgcc ctgttcctgt tcgcattcgg cttcggttct ctggtagcag cacctttctc 4620
tgagactgta gggcggaatc cggtctacat agcgacgctg tccattctaa tgatctttac 4680
gatggcctca ggtctggctc cgaattttgg agcccagctt gccttccgct tcctggctgg 4740
actgttcgga tgcacaccga tgaccacctt tggtggcagt atggcggata tctttgaccc 4800
tatggaccgc acatacgcct ttccagtttg ttgcacgcta tccttcctgg ggccgttcct 4860
agctccaatg gttggagctt tcattggcca aagcacgcat atcagctggc gctggactga 4920
atggtgcaca ctgatcatgg ctgcattggt aaccggcgct atcttcctgt tcgttccaga 4980
aacttatggt cccgtactgt tgcagtggaa ggcaaaacaa ttgcgtgaga ttaccggtga 5040
cccgcgtttc atggcggaaa ttgagcttcg ccagacatcg ctcgtgactc ggttgatgca 5100
cagctgctcc cgtcccttcc atttattctt ccgggagatt atggtagcac tttttaccat 5160
gtatctggtc gtggtataca ttgtcctatt cgggttcctg acgggctatg agttcatctt 5220
tggtagaaca tatggcttta cccaagggtc tgtgggcctg acatttatcg gcatgaacat 5280
cggattcctg atcgcctttg ccatggtgcc gcatatttac ttctcataca agaagcgcct 5340
tcaaaatgcg attgaaaatg gacacaatgg gttgccaccg gaggagcgac tttggttcgc 5400
tatgtacgga gcgccttggc taccaatatc tcttttctgg atgggctgga ccagctatcc 5460
ctctatctcg tactggtcgc cgctagtcgc ctcggtggct ttcggattct ctgtccaagg 5520
aatctttatt tccacgtatc agtaccttat cgacacgtat gagctcttcg cagcaagtgc 5580
cttagtgagt gcaactttct tccgttatat cgccgctggc gcgatggtca tcgtgtcaat 5640
cccgatgtac ggaaatttgg gcgtacattg gtcactcacg ttgctggggt gtatctcggt 5700
cctgatgacc cctgtacctt atatttttta taagtacgga catgtcattc ggcagcggaa 5760
taaaaagacc ccctga 5776
<210> 7
<211> 1547
<212> PRT
<213> kojA,KojR,kojT氨基酸序列()
<400> 7
Met Arg Val Ala Thr Gln Leu Arg Val Gly Ile Val Gly Gly Gly Trp
1 5 10 15
Asn Gly Cys His Leu Ala Leu Glu Leu Lys Lys Gln Gly His Arg Val
20 25 30
Ser Leu Phe Glu Gln Lys Pro Asp Ile Phe Gln Gly Val Ser Gly Asn
35 40 45
Phe Gly Ile Arg Leu His Lys Gly Pro His Tyr Pro Arg Ser Lys Ala
50 55 60
Thr Arg Asp Ser Cys Arg Glu Ala Leu Val Lys Phe Cys Glu Thr Tyr
65 70 75 80
Pro Glu Leu Val Val His His Glu Ser Ala Ile Tyr Ala His Gly Glu
85 90 95
Ala Asp Ala Leu Gly Asn Pro Ser Lys Val Ser Asp Glu Ala Phe Arg
100 105 110
Asp Val Cys Tyr Glu Ser Pro Glu Cys Thr Ala Val Asp Pro Lys Ala
115 120 125
Asn Gly Phe Gln Gly Leu Ile Ser Ala Tyr Asn Leu Asp Glu Pro Ser
130 135 140
Val Ala Ile Gly Asp Arg Leu Arg Asn Thr Phe Lys Glu Lys Leu Gly
145 150 155 160
Arg Ala Gly Ile Tyr Val His Leu Asn Ala Thr Val Asp Arg Ile Ile
165 170 175
His Thr Glu Asp Thr Asn Arg Ile Gln Thr Gly Asp Gly Gln Tyr Val
180 185 190
Phe Asp Val Val Ile Asn Ala Thr Gly Tyr Thr Ser Leu Leu Pro Gln
195 200 205
Asn Ile Ala Asp Ala Leu Pro Val Asp Ile Gly Ile Thr Tyr Gln Thr
210 215 220
Cys Ile Ala Leu Val Tyr Glu Asp Gln Gln Pro Gln Glu Lys Pro Leu
225 230 235 240
Ser Phe Ile Val Met Asp Gly Trp Phe Pro Cys Val Met Pro Ala Ile
245 250 255
Asp Thr Asn Glu Pro Leu Gln Lys Lys Tyr Ile Leu Thr His Gly Ser
260 265 270
Tyr Thr Ile Leu Gly Ser Phe Asp Arg His Glu Glu Gly Gln Glu Leu
275 280 285
Leu Asp Ser Leu Asp Glu Glu Ala Ile Ala Ala Arg Ile Lys Pro His
290 295 300
Cys Glu Arg Glu Ile Thr Arg Phe Trp Pro Gly Phe Leu Asp Arg Phe
305 310 315 320
Gln Tyr Arg Gly Trp Lys Gly Ser Val Leu Ala Lys Leu Lys Thr Thr
325 330 335
Ser Glu Phe Arg Ser Ser Leu Thr Phe Glu Lys Asp Gly Val Ile His
340 345 350
Ile Phe Pro Gly Lys Val Ser Asn Val Val Thr Ala Ala Glu Glu Val
355 360 365
Val Pro Leu Ile Asn Asp Ile Ala Arg Arg Arg His Gly Val Val Arg
370 375 380
Glu Trp Asn Gly Val Arg Phe Thr Val Ser Ser Ala Phe His Thr His
385 390 395 400
Ser Lys Glu Ile Gly Asp Lys Pro Gly Leu Gly Glu His His Thr Ser
405 410 415
Asn Leu Gln Thr Tyr Val Ser Leu Val Thr Ala Asn Met Ser Leu Asn
420 425 430
Thr Asp Asp Ser Gly Arg Ile Arg Thr Arg Gln Arg Ala Lys Arg Ala
435 440 445
Cys Glu Thr Cys Lys Leu Arg Lys Arg Lys Cys Asp Gly His Glu Pro
450 455 460
Cys Thr Tyr Cys Leu Arg Tyr Glu Tyr Gln Cys Thr Phe Lys Pro His
465 470 475 480
Pro Arg Arg Lys Pro Ala Ala Ser Lys Ser Ser Ala Arg Pro Ser Glu
485 490 495
Glu Glu Asp Ser Pro Lys Phe Leu Asp Arg Val Asp Ala Asn Gln Glu
500 505 510
His Met Glu Ala Asn Ser Gly Thr Ala Phe Pro His Leu Leu Gly Met
515 520 525
Arg Leu Asn Pro Gln Gly Ala Pro Lys Val Tyr Gly Phe Ser Trp Asn
530 535 540
Leu Gly Pro Arg Asp Glu Pro Leu Glu Pro Phe Thr Asn Leu Thr Asp
545 550 555 560
Leu Ile Ser Arg Glu Glu Met Glu Asp Leu Ala Ser His Tyr Leu Lys
565 570 575
Lys Ile His Pro Val Tyr Ala Val Leu Asp Pro Asp Thr Leu Arg Gln
580 585 590
Lys Ile Val Ala Arg Trp His Asp Pro Ala Thr Ala Ala Ser Tyr Asp
595 600 605
Pro Ile Leu Cys Gly Val Ala Ala Leu Gly Ser Leu Tyr Ser Gly His
610 615 620
Gln Glu His Pro Lys Glu Gly Ala Leu Val Gln Ser Ala Lys Glu Met
625 630 635 640
Leu Glu Thr Thr Arg Ile Ser Lys Thr Thr Leu Leu His His Ala Thr
645 650 655
Ala Trp Ile Leu Arg Thr Ile Tyr Leu Arg Ser Thr Asn Cys Pro His
660 665 670
Ala Ser Trp Met Ala Ser Cys Ser Thr Met His Ile Ile Glu Ala Ile
675 680 685
Gly Ala His Gln Asp Pro Glu Leu Val Ser Leu Val Tyr Ser Asp Thr
690 695 700
Ala Asp Val Ser Val Asn Asp Glu Ser Gln Arg Arg Leu Phe Trp Val
705 710 715 720
Ala Thr Val Leu Asn Ser Trp Ile Ser Tyr Glu Tyr Gly Arg Ser Arg
725 730 735
Val Ile Leu Arg Gly Val Ser Cys Lys Pro Pro Leu Pro Arg Thr Gly
740 745 750
Asp Phe Thr Thr Asp Leu Ile Ser Met Tyr Gln Ile Ser Glu Arg Leu
755 760 765
Asp Pro Asp Gln Asn Asn Lys Leu Ser Asp Leu Glu Asp Ala Leu Ser
770 775 780
Arg Val Glu Arg Leu Thr Leu Ser His Asp Ala Leu Ile Leu Ser Gln
785 790 795 800
Ser Asn Leu Ala Leu Thr Ile Tyr Arg Arg Leu Arg Val Ala Ser Ser
805 810 815
Asn Ile Ser Asn Asp Ile Leu Thr Arg Ile Ile Arg Leu Gly Asn Asp
820 825 830
Gly Leu Glu Ala Ala Val Arg Leu Ala Glu Asp Arg Ser Pro Trp Trp
835 840 845
His Val Ala Asn Ile Pro Phe Gln Phe Leu Cys Ile Leu Leu Ala Ile
850 855 860
Asp Thr Arg Glu Ser Leu Ser Tyr Val Gly Pro Ala Leu Arg Ser Phe
865 870 875 880
Arg Ala Ile Thr Arg His Tyr Ser Thr Pro Thr Leu His Thr Ala Leu
885 890 895
Glu Thr Ile Glu Ser Leu Val Arg Leu Ser Gln Asn Lys Lys Glu Arg
900 905 910
Asp Leu Thr Leu Leu Arg Asp Ser Met Gln Gln Glu Asp Pro Gly Leu
915 920 925
Thr Glu Gln Gly Ser Thr Thr Ser Gln Ala Phe Asn Asp Ala Ser Trp
930 935 940
Leu Gly Ala Thr Gly Asp Leu Thr Leu Pro Asp Asn Phe Asp Trp Asp
945 950 955 960
Trp Asn Val Phe Leu Asp Thr Gln Val Pro Phe Phe Asp Glu Gly Glu
965 970 975
Ala Gly Gly Gln Arg Tyr Arg Met Gln Ser Phe Arg Gln Tyr Arg Arg
980 985 990
Met Arg Arg Asp Leu Gln Glu Ser Ile Lys Leu His Gly Pro Tyr Ala
995 1000 1005
Ala Ala Gly Asp Arg His Val Gln Pro Thr Asp Asp Ile Leu Glu Asp
1010 1015 1020
Ala Asp Asp Ala Arg Leu Glu Lys Gly Ile His Ser Met Asn Gly His
1025 1030 1035 1040
Gly Gln Ser Pro Tyr Ser Thr Pro Gly Ile Val Leu His Asp Gly Gln
1045 1050 1055
Arg Val Thr Val Pro Gly Val Asn Leu Arg Arg Ala Ser Glu Ile Cys
1060 1065 1070
Glu Arg Val Asn Thr Lys Thr Leu Phe Ile Val Gly Phe Asp Gly Pro
1075 1080 1085
Asp Asp Gln Leu Asn Pro Lys Asn Trp Ser Ile Gly Arg Lys Trp Ala
1090 1095 1100
Thr Leu Gly Ile Val Gly Thr Thr Gly Met Leu Val Gly Trp Ala Ser
1105 1110 1115 1120
Ser Ile Asp Ser Thr Val Ile Lys Gln Gly Gln Glu Ala Phe Gly Val
1125 1130 1135
Ser Glu Val Ala Glu Ser Leu Ala Thr Ala Leu Phe Leu Phe Ala Phe
1140 1145 1150
Gly Phe Gly Ser Leu Val Ala Ala Pro Phe Ser Glu Thr Val Gly Arg
1155 1160 1165
Asn Pro Val Tyr Ile Ala Thr Leu Ser Ile Leu Met Ile Phe Thr Met
1170 1175 1180
Ala Ser Gly Leu Ala Pro Asn Phe Gly Ala Gln Leu Ala Phe Arg Phe
1185 1190 1195 1200
Leu Ala Gly Leu Phe Gly Cys Thr Pro Met Thr Thr Phe Gly Gly Ser
1205 1210 1215
Met Ala Asp Ile Phe Asp Pro Met Asp Arg Thr Tyr Ala Phe Pro Val
1220 1225 1230
Cys Cys Thr Leu Ser Phe Leu Gly Pro Phe Leu Ala Pro Met Val Gly
1235 1240 1245
Ala Phe Ile Gly Gln Ser Thr His Ile Ser Trp Arg Trp Thr Glu Trp
1250 1255 1260
Cys Thr Leu Ile Met Ala Ala Leu Val Thr Gly Ala Ile Phe Leu Phe
1265 1270 1275 1280
Val Pro Glu Thr Tyr Gly Pro Val Leu Leu Gln Trp Lys Ala Lys Gln
1285 1290 1295
Leu Arg Glu Ile Thr Gly Asp Pro Arg Phe Met Ala Glu Ile Glu Leu
1300 1305 1310
Arg Gln Thr Ser Leu Val Thr Arg Leu Met His Ser Cys Ser Arg Pro
1315 1320 1325
Phe His Leu Phe Phe Arg Glu Ile Met Val Ala Leu Phe Thr Met Tyr
1330 1335 1340
Leu Val Val Val Tyr Ile Val Leu Phe Gly Phe Leu Thr Gly Tyr Glu
1345 1350 1355 1360
Phe Ile Phe Gly Arg Thr Tyr Gly Phe Thr Gln Gly Ser Val Gly Leu
1365 1370 1375
Thr Phe Ile Gly Met Asn Ile Gly Phe Leu Ile Ala Phe Ala Met Val
1380 1385 1390
Pro His Ile Tyr Phe Ser Tyr Lys Lys Arg Leu Gln Asn Ala Ile Glu
1395 1400 1405
Asn Gly His Asn Gly Leu Pro Pro Glu Glu Arg Leu Trp Phe Ala Met
1410 1415 1420
Tyr Gly Ala Pro Trp Leu Pro Ile Ser Leu Phe Trp Met Gly Trp Thr
1425 1430 1435 1440
Ser Tyr Pro Ser Ile Ser Tyr Trp Ser Pro Leu Val Ala Ser Val Ala
1445 1450 1455
Phe Gly Phe Ser Val Gln Gly Ile Phe Ile Ser Thr Tyr Gln Tyr Leu
1460 1465 1470
Ile Asp Thr Tyr Glu Leu Phe Ala Ala Ser Ala Leu Val Ser Ala Thr
1475 1480 1485
Phe Phe Arg Tyr Ile Ala Ala Gly Ala Met Val Ile Val Ser Ile Pro
1490 1495 1500
Met Tyr Gly Asn Leu Gly Val His Trp Ser Leu Thr Leu Leu Gly Cys
1505 1510 1515 1520
Ile Ser Val Leu Met Thr Pro Val Pro Tyr Ile Phe Tyr Lys Tyr Gly
1525 1530 1535
His Val Ile Arg Gln Arg Asn Lys Lys Thr Pro
1540 1545
<210> 8
<211> 439
<212> DNA
<213> 人工序列()
<400> 8
gcgagaccgg accacaatta ctatatcatg gtctacccat gatctagcct ccgcccagct 60
cactatagat gttcttcaga caggggacgg cgaattcacg ggcgaaatca atttgtggct 120
gcatcctcat gtcttgaaac aagcagtgtg attggaagtg gtggcgtctt ggctccagcc 180
agctgcatga ctatgagaaa atagtccaac tagtcttggc ttgcagaatg acttccggaa 240
atttaacctg gtggctgctc tcagtattgg ctgacgctcc gcacctctaa ttgtgcttca 300
ggtcagtcaa agatgttttg aaccaccaac gagccgaacc cttccgccgc tacaatactc 360
gggttattat aaaacgcatc gtggttcatc gtcccagcta taggattatt ttcacatcag 420
caaacgaagt cgaagcaag 439
<210> 9
<211> 1160
<212> DNA
<213> 人工序列()
<400> 9
ctccaggcga tttctccatt tccatagttc attcagagaa ctgcctgtag tcgaacagga 60
atattgtggg tattgtgtaa atgcgacagc acgtcctccc ttccctcttc cgaacccatc 120
atcgagtaac tgggtataca tctcttccgt caaaggcgca gcataccgaa atgcaacgta 180
gggtttatga ggcgcacttt caggattgag tttgtctagc aacttgcaca tttcctggca 240
ttggtattcg gaccattttc ggataggcga gccgcctcca atgtccgcgt attgcttttg 300
gatctttggg gttctccgct ttgcgataag tggtccaaga tatgtttgaa gccgtcccaa 360
gggaatcaga tcaccgtcgg cctttaccaa agagcgaggg agacgttaaa aatggtccat 420
gagaatgttt gctagtctga aaacgtacaa ataacctgct caaaaagtct tccacctcat 480
tcgtcgtaga ggggccgccc atgtttaaga ataccattgc ggtagggccc ttggagccag 540
ttgcatcttg agtgactgga gggactgccg ttgcgaagcc atttcgctgt tctgtgttgt 600
atccaagcga gctgcgagct accggtcgca gtagttgccg aggaagcgcg aaggtccgac 660
ggagggccat tctaattaac agatggtagc acaatccttt tcccagaaag taatgaccaa 720
agcgtgcgaa tagcggcgat aacaagacaa tgaggtcgga gcaaaaagtg tatagggatt 780
ttaatgggcc atgtggcaag ggtcaacagt aatcagaaaa gatgctgact tgaagcaaag 840
agtccagaac cggtaaaatg acggatgagt tggagctgaa gcgaatggcc aatcaggatt 900
gacttggggt catcgagtct tggcgtatgt cacctgatgt tcgtgtcttc cccgcgtcgt 960
ccgattgaag tgtcgcgtca caggtgacga aagagcggga acgcaggcag cagagcgaca 1020
ggcagacacc taggaaaata ccaatgttgc cgttcaacgg cacttttgcg tcgattactt 1080
aaatatcgtc cttgactact tttcctttga cgcaattcat gacttacgac tctcaacaca 1140
ttcacaatgg ctgacgagga 1160
<210> 10
<211> 1650
<212> DNA
<213> 人工序列()
<400> 10
ccgatagggt tgggcttatt gctatgtccc tgaaaggata tcaaaagcag gcaaaaagcc 60
aggcataatc cccgcgcgga tggtacccta aggataggcc ctaatcttat ctacatgtga 120
ctgcatcgat gtgtttggtc caaatgaggc atgtggctca ccccacaggc ggagaaacgt 180
gtggctagtg catgacggtc ccctccatag attcaattta attttttcgc ggcaattgtc 240
gtgcagtttg tatcaaccgt tcattccaca tattgagagt tagtaattgg acatcctgtt 300
tattttgtct aattactgaa tattcgaagt actaacctac taaataagcc agtttcaacc 360
actgagtgct catttataca atagttgcag aaccccgcgc tacccctcca tcgctgacat 420
gtcttccaag tcgcaattga cctacagcgc acgcgctagc aagcacccca acgcgctcgt 480
aaagaagctc ttcgaggttg ccgaggccaa gaaaaccaat gtcaccgttt ccgccgacgt 540
gacaaccacc aaagagctgc tggatttggc tgaccgtatg cgcaccgggg atgccactta 600
catatgatct agtaatggtt aatggtggat tatataacag gactcggtcc gtacattgcc 660
gtgatcaaaa ctcacatcga tatcctctcc gatttcagca aagaaaccat caccggtctg 720
aaggcccttg cagagaagca caatttcctc atcttcgaag atcgcaagtt catcgatatc 780
ggaaacacag tccaaaagca gtaccatggc ggcactctgc gtatctctga gtgggcccac 840
atcatcaact gcagtattct gcccggtgag ggtatcgtcg aggctctagc ccagactgct 900
tcggccgagg acttccccta cggctccgag aggggccttt tgatccttgc ggagatgacc 960
tccaagggat ctttggctac cggtcaatat actacttctt ctgttgacta tgctcggaag 1020
tataagaagt ttgtgatggg attcgtctcg acacgtcacc ttggcgaggt tcagtctgaa 1080
gttagctcgc cttcggagga ggaagatttt gtcgtcttca cgacaggtgt caacctctcc 1140
tcgaagggag acaagctggg acagcagtac caaactcctg agtcggctgt tggacgcggt 1200
gccgacttta ttattgctgg ccgtggaatt tatgctgctc ctgatcccgt ggaggcggcg 1260
aagcagtacc agaaggaggg atgggatgca tacctgaagc gtgttggtgc gcaataagta 1320
gtggtggata cgtactcctt ttatggcagt atgtcgcaag tatgatgcga tttatagatt 1380
cagcactcga aatgactact atgtgtctac gacagatacc ctctccgtac gaataagaca 1440
cctgcctcga tatatggaca aattcaaaat cagggtcaag ggtcatgttt caaaatcaca 1500
ataatctcca acatagacga gaatttgtac cggagtgtct gaaggtgcag ctggagattg 1560
gtctattttc ttagagtggg gtatcactaa tacacagtcg gtcactatcg tacaaacaat 1620
cacaattata tacaagattt cccaccaccc 1650
<210> 11
<211> 300
<212> DNA
<213> 人工序列()
<400> 11
tcgccgctag tcgcctcggt ggctttcgga ttctctgtcc aaggaatctt tatttccacg 60
tatcagtacc ttatcgacac gtatgagctc ttcgcagcaa gtgccttagt gagtgcaact 120
ttcttccgtt atatcgccgc tggcgcgatg gtcatcgtgt caatcccgat gtacggaaat 180
ttgggcgtac attggtcact cacgttgctg gggtgtatct cggtcctgat gacccctgta 240
ccttatattt tttataagta cggacatgtc attcggcagc ggaataaaaa gaccccctga 300
<210> 12
<211> 1106
<212> DNA
<213> 人工序列()
<400> 12
ggagcagaaa tgagacaacg ctagtattgg ttacgagaga cagattttag cagataatgt 60
ttctctatcc ttttaataaa aaatacataa tgctagaaca ctatcaagtg tatattaatg 120
gtctttacct acgtaatcct actcaataat gtcatctcag agagcaaatg tgtacatgtt 180
cgagatattc aaacactatc gggaaaccta ggatgatgct gtagggactt ttggtcctcg 240
tctatgtaat ttgacccaaa atctgaaaat atagtgggag gtttgtcacc gtaaaagtca 300
ttgtaccagc aacgttgcag tcgtacatca gtaagtaata taaaatatga ataggaatat 360
atttcgatat atttacatgt tttctaaacc gtaccggcag aaataacaca gatgattcag 420
acgggtatat ccaaacggac gaaagccctc taacaacgcc tgtctaatcc cagaaatgca 480
tgctcaaagc gataaacaaa agtataccgg ataggtatct atgacaaaac ccacatagta 540
aaaacagtcg tggctggtgt acagtgtata tatatctatg ttgcgattga aaaaaaatgt 600
actagcattt gccatatact tggcagtagc agagttcctc gtctgggcaa ctagaggaca 660
aaccatgtta gttaattttg ttattgcata ggtttgatgc aatggatgct tactatacgg 720
actccttgcc gaatatgaca tgccagagga actccatgac tcgtccgcta ctggcatcat 780
ctttctctgt gtcgatgacc cattgtcgga acttgatgta gtcttctcga ggtcgtttca 840
ggacctgttc gcgcgacacg gcgaattgcg cgcagcatgc tgcgccgacg agtttgggcg 900
cttggaggaa tttttgtgtt tgtcggttct ccgtcgatgg gtcttctgtg atggcgggcg 960
aagtagtcga gtttaggggc ggggtgctgg tcccttcgaa cacttcttcc cagacctgtt 1020
ccgttacatg gcgattgatg cgatgatccg ccttgcatcc ggggttccaa ttgcaacgga 1080
ggttgacgta gccattctgt tggacg 1106
<210> 13
<211> 2619
<212> DNA
<213> 人工序列()
<400> 13
atggattcgg aagatcaggt gcccaacgtg cagagtctga agactctgga tgaacagacg 60
agggagagtc tgctaccgcg caacccagtc atgtcgagaa ccgggtcttc aacgtcgctg 120
tctagtcttg ttacgactaa cacgacgcac taccaaaatt ggctgaagac gttttatgcg 180
aacaaactga acgaggtgat acactttcgc gaagcgggcc atgtggtaac caactataat 240
tataagatgg acgatggtga cgacatggat gacatagttg agccctgcct gctcgactgc 300
cacacggagg agggttttgt tatggctgcc atgcacctga ttctgcatcg cataaaggcg 360
aagcaggctg agtctccgac ttcgattgtg actattggtt tgtccggtgg caacaccccg 420
agggccgtgt tccggtacat gtctaacatg gagaatctcg acattgactt caatcgcata 480
atattcttcc tagtggacga gcgttatgtt ccgcctactg acgagctgtc taatcagcgg 540
cttatacgga acacgctgct gaaaaactgg ccggtgcccg agtcgcacat cattttcccg 600
gacacaacct tgcctctgga agaatgtgtg cgcaagtatg aggcggatct ggagcgcgtt 660
ttcggcaagt tggagccctc ggcaacggct gcggttgatt cttcgcccca atccaaagtg 720
gttggtatgg ctatgagcaa gagcacgctg aagcccgact tgatcacgct gggcattggc 780
gacgatttcc acattgctgg cctgttcccg gagtttttat ccacactcga gccctcgcaa 840
gtaacggact gcaaggaacg tgtcatgatc acccatacag acacggcggt ggttcatgac 900
cggttgaccc tatcactgcc tttcctgtgt tgcgcaaaga gcaagctgtt tttcctcaag 960
ggggagcgta agaagcgcat ttggacgacc atgctgcaat acagacacgt tgacccgata 1020
cgtttcccgg ctactcagat cttcacgaag gctggatgcg ttgcagtgtt ggactcgtcg 1080
accatccgcc gaatcagacg taaagttccg ctggagcaat cggactgcct aactttcatt 1140
ttgttcggta gcactggcga tttggctcgt cgcaagctct acccagctct gttccatctc 1200
ttttatttgg gattcttgcc aaccaaattc cgaattttgg cagtgagccg gtcgcatcag 1260
tcgtttgacg aatttttcga gacggtctcc aaggacattt tcgcctctat caacaccacg 1320
attttcatgt gcgaagcggc ggcgcgattc gatttcccca ctgtgattgc ggaattcaag 1380
aagaagctgg ggagggtgac cgtgaagtac gacaaccccc aatcgctcgt gcagctgaac 1440
gagagcttga acgagattga gtcgggttct accgtgacgc accgtctcat ctacatggcc 1500
acgccagctg aagcctacca tcccatcatg aagattgcaa cttcggtgtg ccgccccaag 1560
gagggttggt tcagggtgat gttggagaag ccgttcgggc gtgacatgga gagctgccag 1620
gatatccagc gtgtgctgca gcagcacgca cagcccgagg agatgttcct cgtggaccac 1680
tacttgggga agccgctgat atcctgtatc atagccatca agcgttcgct gcgctacaac 1740
catctcttca gcagacggta cgtgaagagc gtgcacatta aaatgaagga gcgcatcggc 1800
acgttcgggc gcaactattt cgagaactac ggcattattc gcgacatggt gcagaatcac 1860
gggttgcaac tgctgtcgtt gattgcgatg gacaaacccg accggctgaa tgactctgtt 1920
gctgacgaga agcgcaaggt gctgagggcc gtgcgcaccg tgcgtctgga ggacaccatc 1980
ataggtcagt atacggagag cgaggatggc agcgagtgcg cgtacttgca ggagaaggga 2040
acgcctccag attcgctctg cagcacgttc tgctccatgg tcatgtacgt cgataacgac 2100
aagtggcagg gcgtgccgtt cgtgataacc accggcaaag gtctggatga gcatctgtgt 2160
gaggtgcggc ttaacatgaa aagcaacttc gcagccgaat ttggcgcgga cttcgaggcg 2220
cgcaacctcg ttttccgggt tcagcccaac cctactgtct tctggtccgt ggactccagg 2280
cacttcgaca cgcagcaaga gagtgagaag ttggtgttca ccagcgacca tgacaccgac 2340
aacatgggcc tgaaggtgga ggagcgtatc ttacgtgatg tggactacaa cctgaccaag 2400
cgtcccgtgg ttgggtgcta cgaaatactg ctctaccacg ccttcagcgg caagcgcaac 2460
tacttccctt cgctcgaaga agtgagcgag tgttggcgta ttctgacccc cgtgctgcac 2520
gaaattgacg ataagcgcgt gcgcccgatt ctgtacccgc gtgggtcgag cgggcccaaa 2580
gagcaggagc gtcatttaca gctattgaat tcaatatga 2619
<210> 14
<211> 872
<212> PRT
<213> gdh基因氨基酸序列()
<400> 14
Met Asp Ser Glu Asp Gln Val Pro Asn Val Gln Ser Leu Lys Thr Leu
1 5 10 15
Asp Glu Gln Thr Arg Glu Ser Leu Leu Pro Arg Asn Pro Val Met Ser
20 25 30
Arg Thr Gly Ser Ser Thr Ser Leu Ser Ser Leu Val Thr Thr Asn Thr
35 40 45
Thr His Tyr Gln Asn Trp Leu Lys Thr Phe Tyr Ala Asn Lys Leu Asn
50 55 60
Glu Val Ile His Phe Arg Glu Ala Gly His Val Val Thr Asn Tyr Asn
65 70 75 80
Tyr Lys Met Asp Asp Gly Asp Asp Met Asp Asp Ile Val Glu Pro Cys
85 90 95
Leu Leu Asp Cys His Thr Glu Glu Gly Phe Val Met Ala Ala Met His
100 105 110
Leu Ile Leu His Arg Ile Lys Ala Lys Gln Ala Glu Ser Pro Thr Ser
115 120 125
Ile Val Thr Ile Gly Leu Ser Gly Gly Asn Thr Pro Arg Ala Val Phe
130 135 140
Arg Tyr Met Ser Asn Met Glu Asn Leu Asp Ile Asp Phe Asn Arg Ile
145 150 155 160
Ile Phe Phe Leu Val Asp Glu Arg Tyr Val Pro Pro Thr Asp Glu Leu
165 170 175
Ser Asn Gln Arg Leu Ile Arg Asn Thr Leu Leu Lys Asn Trp Pro Val
180 185 190
Pro Glu Ser His Ile Ile Phe Pro Asp Thr Thr Leu Pro Leu Glu Glu
195 200 205
Cys Val Arg Lys Tyr Glu Ala Asp Leu Glu Arg Val Phe Gly Lys Leu
210 215 220
Glu Pro Ser Ala Thr Ala Ala Val Asp Ser Ser Pro Gln Ser Lys Val
225 230 235 240
Val Gly Met Ala Met Ser Lys Ser Thr Leu Lys Pro Asp Leu Ile Thr
245 250 255
Leu Gly Ile Gly Asp Asp Phe His Ile Ala Gly Leu Phe Pro Glu Phe
260 265 270
Leu Ser Thr Leu Glu Pro Ser Gln Val Thr Asp Cys Lys Glu Arg Val
275 280 285
Met Ile Thr His Thr Asp Thr Ala Val Val His Asp Arg Leu Thr Leu
290 295 300
Ser Leu Pro Phe Leu Cys Cys Ala Lys Ser Lys Leu Phe Phe Leu Lys
305 310 315 320
Gly Glu Arg Lys Lys Arg Ile Trp Thr Thr Met Leu Gln Tyr Arg His
325 330 335
Val Asp Pro Ile Arg Phe Pro Ala Thr Gln Ile Phe Thr Lys Ala Gly
340 345 350
Cys Val Ala Val Leu Asp Ser Ser Thr Ile Arg Arg Ile Arg Arg Lys
355 360 365
Val Pro Leu Glu Gln Ser Asp Cys Leu Thr Phe Ile Leu Phe Gly Ser
370 375 380
Thr Gly Asp Leu Ala Arg Arg Lys Leu Tyr Pro Ala Leu Phe His Leu
385 390 395 400
Phe Tyr Leu Gly Phe Leu Pro Thr Lys Phe Arg Ile Leu Ala Val Ser
405 410 415
Arg Ser His Gln Ser Phe Asp Glu Phe Phe Glu Thr Val Ser Lys Asp
420 425 430
Ile Phe Ala Ser Ile Asn Thr Thr Ile Phe Met Cys Glu Ala Ala Ala
435 440 445
Arg Phe Asp Phe Pro Thr Val Ile Ala Glu Phe Lys Lys Lys Leu Gly
450 455 460
Arg Val Thr Val Lys Tyr Asp Asn Pro Gln Ser Leu Val Gln Leu Asn
465 470 475 480
Glu Ser Leu Asn Glu Ile Glu Ser Gly Ser Thr Val Thr His Arg Leu
485 490 495
Ile Tyr Met Ala Thr Pro Ala Glu Ala Tyr His Pro Ile Met Lys Ile
500 505 510
Ala Thr Ser Val Cys Arg Pro Lys Glu Gly Trp Phe Arg Val Met Leu
515 520 525
Glu Lys Pro Phe Gly Arg Asp Met Glu Ser Cys Gln Asp Ile Gln Arg
530 535 540
Val Leu Gln Gln His Ala Gln Pro Glu Glu Met Phe Leu Val Asp His
545 550 555 560
Tyr Leu Gly Lys Pro Leu Ile Ser Cys Ile Ile Ala Ile Lys Arg Ser
565 570 575
Leu Arg Tyr Asn His Leu Phe Ser Arg Arg Tyr Val Lys Ser Val His
580 585 590
Ile Lys Met Lys Glu Arg Ile Gly Thr Phe Gly Arg Asn Tyr Phe Glu
595 600 605
Asn Tyr Gly Ile Ile Arg Asp Met Val Gln Asn His Gly Leu Gln Leu
610 615 620
Leu Ser Leu Ile Ala Met Asp Lys Pro Asp Arg Leu Asn Asp Ser Val
625 630 635 640
Ala Asp Glu Lys Arg Lys Val Leu Arg Ala Val Arg Thr Val Arg Leu
645 650 655
Glu Asp Thr Ile Ile Gly Gln Tyr Thr Glu Ser Glu Asp Gly Ser Glu
660 665 670
Cys Ala Tyr Leu Gln Glu Lys Gly Thr Pro Pro Asp Ser Leu Cys Ser
675 680 685
Thr Phe Cys Ser Met Val Met Tyr Val Asp Asn Asp Lys Trp Gln Gly
690 695 700
Val Pro Phe Val Ile Thr Thr Gly Lys Gly Leu Asp Glu His Leu Cys
705 710 715 720
Glu Val Arg Leu Asn Met Lys Ser Asn Phe Ala Ala Glu Phe Gly Ala
725 730 735
Asp Phe Glu Ala Arg Asn Leu Val Phe Arg Val Gln Pro Asn Pro Thr
740 745 750
Val Phe Trp Ser Val Asp Ser Arg His Phe Asp Thr Gln Gln Glu Ser
755 760 765
Glu Lys Leu Val Phe Thr Ser Asp His Asp Thr Asp Asn Met Gly Leu
770 775 780
Lys Val Glu Glu Arg Ile Leu Arg Asp Val Asp Tyr Asn Leu Thr Lys
785 790 795 800
Arg Pro Val Val Gly Cys Tyr Glu Ile Leu Leu Tyr His Ala Phe Ser
805 810 815
Gly Lys Arg Asn Tyr Phe Pro Ser Leu Glu Glu Val Ser Glu Cys Trp
820 825 830
Arg Ile Leu Thr Pro Val Leu His Glu Ile Asp Asp Lys Arg Val Arg
835 840 845
Pro Ile Leu Tyr Pro Arg Gly Ser Ser Gly Pro Lys Glu Gln Glu Arg
850 855 860
His Leu Gln Leu Leu Asn Ser Ile
865 870
<210> 15
<211> 1391
<212> DNA
<213> 人工序列()
<400> 15
gcttgatgtc ttggtattga gtctacggtg tacaatggtg tactgcgtgt agcggccccc 60
agtatgacca agatctaaag ggcattattt tttaggccag attgtttaga ttgaattaga 120
ctagccatgt ataggtggat attattcagt atcgcctgac tatgatatga taaccacatt 180
aggtttatat aatgacgttt tatgtattat acacgcatgg ggagtgatca ctgttatcaa 240
aatcactgag aaagagctaa gactattggt tcaaacccca gtacagtttc atgcaaagtt 300
ctagatggcg ggatctaggt tcccaacaag gcagaaatat tgaattgaat tttttctaaa 360
aagaataaac aatggtaaaa aaattccaaa gagatgatta attgaaatta aaagagaaaa 420
taaaaccatg acaagcgcgt gatcctcatt tgcctaccgg tgaatctttt caggaatcag 480
gagagcagaa gaggcttaga gagatcgcta agcccatgat atgagtgtcg agagggaaga 540
ggggcagtgg accatagttg atccggtccg atatctcggc ccggaaacgg aaaggtcaca 600
ccgagtgccc ctcatttttc cattgcttcc atccattaag cttgggtggg atgctgtggt 660
ctgtagtgtt agtctgtatg gccagattgt aattacatca tgcccctcta tggggatgcc 720
tcaggtatgg gaccccaggg tatcatttcc ccctcaattg cttgaactac ggaacaaagg 780
acaaaaagat agagtaatag ccgggatcgt cttcctcgta gcctaggtag tactgccccc 840
tcgattccga aaaactggca aaagattcac gagatggtag gattgagtac ccggcatgct 900
ggatttgagg cacgctcatt ggccagaccg gtagctgccg aggagaggca gagtcccaaa 960
tatcgtgagt ctcctgcttt gcccggtgta tgaaaccgga aagggctgct gggagctggg 1020
gagcggcgca agccgggaaa acagctgaca aggacccatt tcactctgga tcttgaggag 1080
agctgtagct tttgccccgt ctgtccaccc ggtgactgga ttagtgacct ggtcgttgcg 1140
tcagtcaaca ttgctctttt tttatctccc cctcccccgc cgtccgactt ttctcccctt 1200
ttctactctc ttcgtatact caccactgca atcaccttat ccctttgtct tcttacttaa 1260
agtgagtcgt ctcccgccca tcattccctt tggatctcat aaagcagagc cactttcaag 1320
tgcctaccgt ttccctttcc acacagattg actgacagct accccgccac accaacagac 1380
acatctaaac a 1391
<210> 16
<211> 1032
<212> DNA
<213> 人工序列()
<400> 16
tcattcatcc agaacaagag acatctacat tcattcaacc tacagtgctc gacatctatc 60
attagcacca agctgcctcg acatcaatct ggaacatcct gacttagact aaacatactt 120
aataataccc aagaggttaa gcagtgttcg cttgatcctc atcacattca tcattggttc 180
gtggaaggat gactatatat catacaggat gcccatactg atccctcgtc ttctagtcac 240
aatggacgct acatacacca tggcaccaac ggtgcaagga caaccatcat ttgcatacta 300
ccccactgct gattcacaaa gacaacaata cacaagccac ccggctgagc cccagccata 360
ctatggacag attcaggcct tccctcagca acactgcctc ccagagcaac aaccggtcta 420
caatgctcaa cccatgatga acatgcatca gatggctacc accaatgcct tccgcggagc 480
aatgaacatg actcccatcg cctctcccca accatctcac ctgaagccca caatcgtcgt 540
ccagcagggt tccccagccc tcatgccttt ggacacaagg ttcgtcggtg actactatag 600
cttcccttct acccctccac tctccaccgc gggaagttct atcagcagcc cgccatccag 660
cagtggcacc ttgcacaccc caatcaacga ctgcttcttc tcattcgaaa aggtggaggg 720
tgtaaaggag ggttgtgaaa gtgacgtcca tgccgagctt ttggccagtg ccgactggac 780
tcggtcagca acttcgcctc ccatgactcc tggtatgttt tcttctcatc agttgagtag 840
ggggttggaa ccagtctaac ttcttctagt cttcatcaac gccaattctc tcaccgccag 900
tcagagctca gacttcctct ctgcccacgg gtcttgcccc tctctttctc cttcgccttc 960
tcctgtatct tccttgttta ctccgtctca gtcggctttc cctgttgagc aggccacctc 1020
cgacttctgt ga 1032
<210> 17
<211> 1024
<212> DNA
<213> 人工序列()
<400> 17
gaacaacgca tctgaacggt atgataatcg cgacgcgagt accatgggtg ctgtgctcta 60
tgaagctgcc cacgctgcgg cgaccaagtc gacgactagc gaatcatccg aagatggtgt 120
ttctgacgca ccctctatcg atcgtcggtc cgccaagaag cgcaagcgtg acgagcacgt 180
ttagttgccc ccactgccgt ttgcaacttc acgacgcgat gtgaccagga gctcccccct 240
tactacttaa tacactatca taattactcc accattgcac tggttggtct ggcttttgtc 300
tttgcggttt tggcgtctgg gcatttacat tggcggtgtc aggtgggagg ctaaggtgtc 360
tggcaaaagg atttcgtttc tcacagggga aaagagaaac ataatatagc acatggtctt 420
cacaaaggaa gatttacatt ggaggcatta gaccggttgt ctgggcggca acggattcgg 480
cgcactggga gtccacggtt tggactaaac ttagagcgca tgacgccttc tttgaggcac 540
aggtggtgat ttgggatttt tctttttttc tcgtcgcatt ttctggcaaa gatgtttttg 600
cctcgatcaa cagaatggct cgagtcattc ttcactcttc ttgtacccat ttttcttttc 660
tttttttttt tttttccctc tttttctctt ctctatactt ctcgttcatc ttgcattctg 720
tgtccctatt tgatgtaata tttgatttga ccttgacccg ggttgcaccc tcaccacgtc 780
tttcctgtgc tggccacatg tatacttcaa cttgtcccca atcattgcct catacccctg 840
tgcgcatcat cctgttttca ttttgtttgc atccataata ttcccccgtt gttaattcaa 900
gcgcccattt gatttgtggc acagttagtg ataatcaaag attcatcaag aaataccttt 960
ccaagtatgt agagtagtag agtagtatgt tctgtcaacc catggtcagt ggtttggctt 1020
ccgt 1024

Claims (6)

1.一株重组米曲霉（Aspergillus oryzae）菌株，来自米曲霉自源基因重组米曲霉菌株，保藏号CGMCC18557，保藏机构:中国微生物菌种保藏管理委员会普通微生物中心CGMCC。
2.权利要求1所述重组米曲霉菌株的构建方法，其特征在于：将保藏号为CGMCC3.042的米曲霉菌株自体的次级代谢产物全局调控因子laeA 基因、曲酸合成基因簇内kojA, kojR,kojT基因、葡萄糖-6-磷酸脱氢酶gdh基因分别整合到米曲霉基因组上pyrG基因、ku70基因、msn2基因得到的重组米曲霉菌株CGMCC18557；所述次级代谢产物全局调控因子laeA 基因受PsodM启动子调控，所述曲酸合成基因簇内kojA, kojR, kojT基因受PglaA启动子调控，所述葡萄糖-6-磷酸脱氢酶gdh基因受PgpdA启动子调控；
所述次级代谢产物全局调控因子laeA 基因核苷酸序列如SEQ ID NO.1，其氨基酸序列如SEQ ID NO.2所示；所述PsodM启动子为诱导型启动子，该启动子的核苷酸序列如SEQ IDNO.3所示；
所述曲酸合成基因簇内kojA, kojR, kojT基因核苷酸序列如SEQ ID NO.6，其氨基酸序列如SEQ ID NO.7所示；
所述葡萄糖-6-磷酸脱氢酶gdh基因核苷酸序列如SEQ ID NO.13所示，其氨基酸序列如SEQ ID NO.14所示；所述PgpdA启动子为组成型启动子，该启动子的核苷酸序列如SEQ IDNO.16所示。
3.权利要求1所述的重组米曲霉菌株在提高曲酸产量方面的应用。
4.一种次级代谢产物全局调控因子laeA 基因表达框，其特征在于所述表达框包含pyrGL左臂、PsodM启动子、次级代谢产物全局调控因子laeA基因、pyrGR右臂、按照pyrGL-PsodM-laeA-pyrGR的顺序排布；所述pyrGL左臂的核苷酸序列如SEQ ID NO.4所示；所述pyrGR右臂的核苷酸序列如SEQ ID NO.5所示。
5.一种曲酸合成基因簇内kojA, kojR, kojT基因表达框，其特征在于所述表达框包含ku70左臂、PglaA启动子、曲酸合成基因簇内kojA, kojR, kojT基因、pyrG基因、曲酸合成基因簇kojT基因3’部分终止子、ku70R、按照ku70L-PglaA-kojA,KojR,kojT-pyrG-kojT3’-ku70R的顺序排布；所述ku70L左臂的核苷酸序列如SEQ ID NO.9所示；所述PglaA启动子的核苷酸序列如SEQ ID NO.8所示；所述曲酸合成基因簇内kojA, kojR, kojT基因的核苷酸序列如SEQ ID NO.6所示；所述pyrG的核苷酸序列如SEQ ID NO.10；所述kojT终止子的核苷酸序列如SEQ ID NO.11；所述ku70R右臂的核苷酸序列如SEQ ID NO.12所示。
6.一种葡萄糖-6-磷酸脱氢酶gdh基因表达框，其特征在于所述表达框包含msn2L左臂、PgpdA启动子、葡萄糖-6-磷酸脱氢酶gdh基因、pyrG、msn2R右臂按照msn2L-PgpdA-gdh-pyrG-msn2R的顺序排布；所述msn2L左臂的核苷酸序列如SEQ ID NO.16所示；所述PgpdA启动子的核苷酸序列如SEQ ID NO.15所示；所述葡萄糖-6-磷酸脱氢酶gdh基因的序列如SEQID NO.13所示；所述pyrG的核苷酸序列如SEQ ID NO.10；所述msn2R右臂的核苷酸序列如SEQ ID NO.17所示。
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