CN112195110A - 一株重组米曲霉菌株及其曲酸发酵方法与应用 - Google Patents

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(Aspergillusoryzae)，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SEQID 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SEQ IDNO.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SEQ IDNO.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：

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6 h。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3 mL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 (10)

1.一株重组米曲霉菌株，其分类为Aspergillus oryzae，来自米曲霉自源基因重组米曲霉菌株，保藏号CGMCC No. 18557，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。

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：将米曲霉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(Aspergillus oryzae 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。

3.一种米曲霉曲酸培养基,其特征在于可利用碳源种类有：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；所述的米曲霉高产曲酸发酵培养基，具体步骤如下:

1）按碳源添加量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。

4.权利要求1所述的重组米曲霉菌株在大幅提高曲酸产量方面的应用。

5.一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。

6.一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基因的氨基酸序列如SEQ ID NO.7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。

7.权利要求2所述的构建方法，其特征在于所述葡萄糖-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。

8.权利要求2所述的构建方法，其特征在于所述PgpdA启动子为组成型启动子，该启动子的核苷酸序列如SEQ ID NO.16所示。

9.一种葡萄糖-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。

10.权利要求1所述的重组米曲霉菌，其特征在于所述重组米曲霉菌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-koj(kojA, kojR, kojT)-pyrG-kojT终止子-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。

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