CN101886045A - 基因修饰微生物及以此微生物生产衣康酸的方法 - Google Patents
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Abstract
本发明系提供一种基因修饰微生物,其可大量生产衣康酸(itaconicacid),以及此微生物的使用。
Description
技术领域
本发明系有关于一种基因修饰之大肠杆菌菌株,其衣康酸的产量高于野生型之大肠杆菌菌株。
背景技术
衣康酸,为许多产物(如,聚丙烯纤维、橡胶、人工钻石及镜片)之必需前体,其广泛地使用于化学工业上。传统上,衣康酸是由土曲霉(Aspergillusterreus)中所分离出来。然而,土曲霉(A.terreus)生长缓慢,且在产孢时期并无法形成衣康酸。因此,业界亟需一种可大量生产衣康酸的方法。
发明内容
在本发明之一范畴中,基因修饰微生物之特征包括(i)突变之内源性icd基因,其柠檬酸脱氢酶的表达量低于其野生型的表达量,以及(ii)外源性核酸序列,其编码顺式乌头酸脱羧酶(CAD),且连接至启动子(可在微生物中进行基因转录)。经修饰的微生物可为黑曲霉(Aspergillus niger)、土曲霉(Aspergillus terreus)、大肠杆菌(Escherichia coli)、担子菌类酵母菌(Pseudozyma Antarctica)、解脂耶氏酵母菌(Yarrowia lipolytica)或酿酒酵母(Saccharomyces cerevisiae)。此微生物可还包括至少外源性核酸序列,其编码下列3种酶其中之一:(a)可将磷酸烯醇丙酮酸转变为草酰乙酸的酶(例如,磷酸烯醇丙酮酸羧化酶以及磷酸烯醇丙酮酸羧激酶),(b)可将草酰乙酸转变为柠檬酸的酶(例如,柠檬酸合成酶、2-甲基柠檬酸合成酶以及柠檬酸裂解酶),以及(c)可将柠檬酸或异柠檬酸转变为顺式乌头酸的酶(例如,乌头酸酶及2-甲基柠檬酸脱氢酶)。此3个外源性核酸序列连接至适当的启动子。
在本发明另一实施样态中,基因修饰微生物之特征包括(i)第一外源性核酸序列,其编码顺式乌头酸脱羧酶(CAD),(ii)第二外源性核酸序列,其编码上述酶(a)或酶(b),以及选择性(iii)第三外源性核酸序列,其编码上述酶(c)。此外,此微生物包括(i)第一外源性核酸序列,其编码顺式乌头酸脱羧酶(CAD),(ii)第二外源性核酸序列,其编码上述酶(a),(iii)第三外源性核酸序列,其编码上述酶(b),以及选择性(iv)第四外源性核酸序列,其编码上述酶(c)。各个外源性核酸序列连接至适当的启动子,使上述酶于微生物内表达。
在本发明之范畴中,还包括一种以上述任何基因修饰微生物表达衣康酸的方法。本发明之方法包括将此基因修饰微生物培养于培养基中以产生衣康酸,收集培养基并纯化此衣康酸产物。在一实施例中,培养基包括以葡萄糖作为基质以生产衣康酸,葡萄糖的浓度介于5-80g/L,较佳约10-40g/L。在另一实施例中,此培养基包括以柠檬酸作为基质以生产衣康酸,柠檬酸的浓度介于5-80g/L,较佳约10-40g/L。。当以柠檬酸作为基质时,基因修饰微生物较易经透化(permeabilize)处理。
为了让本发明之上述和其它目的、特征、和优点能更明显易懂,下文特举较佳实施例,并配合所附图示,作详细说明如下:
实施方式
本发明系提供一种基因修饰之微生物,其可高度表达顺式乌头酸脱羧酶(cis-aconitic acid decarboxylase,CAD),此酶可将顺式乌头酸转变为衣康酸。微物生包括,但不限于,曲霉(Aspergillus)、柠檬酸杆菌(Citrobacter)、棒状杆菌(Corynebacterium)、德克酵母菌(Dekkera)、肠杆菌(Enterobacter)、肠球菌(Enterococcus)、埃希氏菌(Escherichia)、欧文氏菌(Erwinia)、克雷伯氏菌(Klebsiella)、克鲁维酵母(Kluyveromyces)、乳杆菌(Lactobacillus)、乳球菌(Lactococcus)、摩根氏菌(Morganella)、团泛菌(Pantoea)、果胶杆菌(Pectobacterium)、青霉菌(Penicillum)、毕赤酵母菌(Pichia)、变形杆菌(Proteus)、假单胞菌(Pseudomonas)、二形性酵母菌(Pseudozyma)、红酵母菌(Rhodotorula)、沙门氏菌(Salmonella)、沙雷氏菌(Serratia)、志贺氏菌(Shigella)、酵母菌(Saccharomyces)、黑穗菌(Ustilago)、及耶氏酵母菌(Yarrowia)。
本发明所述之“顺式乌头酸脱羧酶”或“CAD”系指任何天然的顺式乌头酸脱羧酶(例如,土曲霉(A.terreus)之CAD,相关内容可参照Dwiarti etal.,J.Bioscience and Bioengineering,94(1):29-33,2002以及WO2009/014437),以及具相同功能的物质。下列核酸及氨基酸序列为土曲霉(A.terreus)核酸序列(序列识别号:1)及氨基酸序列(序列识别号:2)之一实施例。
A.terreus顺式乌头酸脱羧酶
atgaccaagcagtctgctgattccaacgcgaagtctggtgtgacctctgagatctgtcac
M T K Q S A D S N A K S G V T S E I C H
tgggcgtctaatctcgccactgatgatatcccgagcgacgttctggagcgtgcaaaatac
W A S N L A T D D I P S D V L E R A K Y
ctgatcctggatggtatcgcgtgcgcgtgggtaggtgctcgtgtcccatggtctgaaaaa
L I L D G I A C A W V G A R V P W S E K
tacgttcaagcgaccatgtctttcgaacctccgggtgcgtgtcgtgtcatcggttacggc
Y V Q A T M S F E P P G A C R V I G Y G
cagaaactgggtccggtagcggctgccatgacgaactctgcatttattcaggcgaccgaa
Q K L G P V A A A M T N S A F I Q A T E
ctcgatgactatcactctgaagcgccgctgcattccgcgtctatcgttctcccggcagtt
L D D Y H S E A P L H S A S I V L P A V
ttcgcggcgagcgaagtactggccgaacagggtaaaaccatctctggtattgacgtgatt
F A A S E V L A E Q G K T I S G I D V I
ctggctgcgatcgttggtttcgagagcggtcctcgcatcggcaaagcgatctacggttct
L A A I V G F E S G P R I G K A I Y G S
gacctcctgaacaacggctggcactgcggtgcggtatatggcgcaccggctggtgcgctc
D L L N N G W H C G A V Y G A P A G A L
gcaactggtaagctcctgggcctcacgccggacagcatggaagatgcactgggtattgcc
A T G K L L G L T P D S M E D A L G I A
tgcacgcaagcatgcggcctcatgtccgcgcagtatggtggcatggttaaacgtgttcag
C T Q A C G L M S A Q Y G G M V K R V Q
cacggtttcgcagcgcgtaatggtctcctcggtggcctcctggctcacggcggctacgag
H G F A A R N G L L G G L L A H G G Y E
gcgatgaaaggtgttctcgagcgttcttacggtggcttcctgaagatgttcaccaagggc
A M K G V L E R S Y G G F L K M F T K G
aacggtcgtgaaccgccgtacaaagaagaagaggttgtggctggtctgggtagcttctgg
N G R E P P Y K E E E V V A G L G S F W
cacaccttcaccattcgtatcaaactgtacgcgtgctgcggtctcgtacacggtcctgtt
H T F T I R I K L Y A C C G L V H G P V
gaagccattgaaaacctccagggtcgttacccggaactgctcaatcgtgctaacctgtct
E A I E N L Q G R Y P E L L N R A N L S
aacatccgccacgttcacgtacaactctctaccgcgagcaactcccactgtggttggatc
N I R H V H V Q L S T A S N S H C G W I
ccagaagagcgcccaatctcttctatcgcgggtcaaatgtctgtcgcatatatcctcgcc
P E E R P I S S I A G Q M S V A Y I L A
gttcagctcgttgaccaacagtgtctgctcagccagttctccgagtttgacgataatctg
V Q L V D Q Q C L L S Q F S E F D D N L
gaacgcccggaagtgtgggacctggcacgtaaggttaccagctctcaatctgaggagttc
E R P E V W D L A R K V T S S Q S E E F
gaccaggacggtaactgtctctctgccggtcgcgtccgtattgagttcaacgacggctcc
D Q D G N C L S A G R V R I E F N D G S
tccatcaccgaatccgttgagaagccgctcggtgtaaaggaaccaatgccaaatgaacgc
S I T E S V E K P L G V K E P M P N E R
atcctgcacaaataccgtaccctggcgggttctgtaacggacgaaagccgtgttaaggag
I L H K Y R T L A G S V T D E S R V K E
atcgaggatctcgtgctcggcctggaccgtctgaccgatattagcccgctcctcgagctg
I E D L V L G L D R L T D I S P L L E L
Ctgaattgtccggttaaatccccactggtttaa (SEQ ID NO:1)
L N C P V K S P L V - (SEQ ID NO:2)
本发明所述之与顺式乌头酸脱羧酶(例如,土曲霉(A.terreus)的CAD或本文中所述之任何酶)具相同功能的物质为多肽,其具有与顺式乌头酸脱羧酶至少60%(例如,85%、90%或95%)的相似度,且具有与顺式乌头酸脱羧酶相同的酶活性。
两个氨基酸的相似性可藉由Karlin及Altschul Proc.Natl.Acad.Sci.USA87:2264-68,1990算法,以及Karlin及Altschul Proc.Natl.Acad.Sci.USA90:5873-77,1993算法来判断。此算法已写入Altschul,et al.J.Mol.Biol.215:403-10,1990的BLASTN及BLASTX程序(2.0版)中。BLAST蛋白质搜寻可使用BLASTX程序,分数(score)为50,字符串长度(wordlength)为3,以获得与本发明同源的氨基酸序列。可使用Gapped BLAST(Altschul et al.,Nucleic Acids Res.25:3389-3402,1997)来确定两序列中的差异(gap)。当使用BLAST及Gapped BLAST程序时,也可使用各自程序(例如,BLASTX及BLASTN)的默认参数。
上述之基因修饰微生物可具有突变的内源性icd基因(编码异柠檬酸脱氢酶),使其相对于野生型的微生物,具有较低的异柠檬酸脱氢酶表达量。Icd基因存在于各种的微生物中,包括土曲霉(Aspergillus terreus)(基因银行编号:XM_001210553及XP_001210553)、克氏柠檬酸杆菌(Citrobacterkoseri)(基因银行编号:YP_001453397)、发酵乳酸杆菌(Lactobacillusfermentum)(基因银行编号:NC_009792及YP_001843755)、酿酒酵母菌(Saccharomyces cerevisiae)(基因银行编号:NC_001146及NP_014361)、解脂耶氏酵母菌(Yarrowia lipolytica)(基因银行编号:XM_503571及XP_503571)以及大肠杆菌(Escherichia coli)(基因银行编号:NC_000913及NP_415654)。例如,大肠杆菌其中一编码区域如下所示:
大肠杆菌icd基因的核酸及氨基酸序列
atggaaagtaaagtagttgttccggcacaaggcaagaagatcaccctgcaaaacggcaaa
M E S K V V V P A Q G K K I T L Q N G K
ctcaacgttcctgaaaatccgattatcccttacattgaaggtgatggaatcggtgtagat
L N V P E N P I I P Y I E G D G I G V D
gtaaccccagccatgctgaaagtggtcgacgctgcagtcgagaaagcctataaaggcgag
V T P A M L K V V D A A V E K A Y K G E
cgtaaaatctcctggatggaaatttacaccggtgaaaaatccacacaggtttatggtcag
R K I S W M E I Y T G E K S T Q V Y G Q
gacgtctggctgcctgctgaaactcttgatctgattcgtgaatatcgcgttgccattaaa
D V W L P A E T L D L I R E Y R V A I K
ggtccgctgaccactccggttggtggcggtattcgctctctgaacgttgccctgcgccag
G P L T T P V G G G I R S L N V A L R Q
gaactggatctctacatctgcctgcgtccggtacgttactatcagggcactccaagcccg
E L D L Y I C L R P V R Y Y Q G T P S P
gttaaacaccctgaactgaccgatatggttatcttccgtgaaaactcggaagacatttat
V K H P E L T D M V I F R E N S E D I Y
gcgggtatcgaatggaaagcagactctgccgacgccgagaaagtgattaaattcctgcgt
A G I E W K A D S A D A E K V I K F L R
gaagagatgggggtgaagaaaattcgcttcccggaacattgtggtatcggtattaagccg
E E M G V K K I R F P E H C G I G I K P
tgttcggaagaaggcaccaaacgtctggttcgtgcagcgatcgaatacgcaattgctaac
C S E E G T K R L V R A A I E Y A I A N
gatcgtgactctgtgactctggtgcacaaaggcaacatcatgaagttcaccgaaggagcg
D R D S V T L V H K G N I M K F T E G A
tttaaagactggggctaccagctggcgcgtgaagagtttggcggtgaactgatcgacggt
F K D W G Y Q L A R E E F G G E L I D G
ggcccgtggctgaaagttaaaaacccgaacactggcaaagagatcgtcattaaagacgtg
G P W L K V K N P N T G K E I V I K D V
attgctgatgcattcctgcaacagatcctgctgcgtccggctgaatatgatgttatcgcc
I A D A F L Q Q I L L R P A E Y D V I A
tgtatgaacctgaacggtgactacatttctgacgccctggcagcgcaggttggcggtatc
C M N L N G D Y I S D A L A A Q V G G I
ggtatcgcccctggtgcaaacatcggtgacgaatgcgccctgtttgaagccacccacggt
G I A P G A N I G D E C A L F E A T H G
actgcgccgaaatatgccggtcaggacaaagtaaatcctggctctattattctctccgct
T A P K Y A G Q D K V N P G S I I L S A
gagatgatgctgcgccacatgggttggaccgaagcggctgacttaattgttaaaggtatg
E M M L R H M G W T E A A D L I V K G M
gaaggcgcaatcaacgcgaaaaccgtaacctatgacttcgagcgtctgatggatggcgct
E G A I N A K T V T Y D F E R L M D G A
Aaactgctgaaatgttcagagtttggtgacgcgatcatcgaaaacatgtaa (SEQ ID NO:3)
K L L K C S E F G D A I I E N M - (SEQ ID NO:4)
制备经内源性icd基因突变之微生物的方法已为此技术领域人士所习知。例如,以同源性重组造成icd基因的突变(如,***、删除、位置突变(sitemutation))。
此外,基因修饰之微生物也可过度表达下列一个或多个酶:(a)可将磷酸烯醇丙酮酸转变为草酰乙酸的酶(例如,磷酸烯醇丙酮酸羧化酶/羧激酶,其包括3种亚型EC4.1.1.32,EC4.1.1.38及EC4.1.1.49),(b)可将草酰乙酸转变为柠檬酸的酶(例如,柠檬酸合成酶、2-甲基柠檬酸合成酶以及柠檬酸裂解酶),以及(c)可将柠檬酸或异柠檬酸转变为顺式乌头酸的酶(例如,乌头酸酶及2-甲基柠檬酸脱氢酶)。
本发明所述之“磷酸烯醇丙酮酸羧化酶/羧激酶”、“柠檬酸合成酶”、“2-甲基柠檬酸合成酶”、“柠檬酸裂解酶”、“乌头酸酶”、以及“2-甲基柠檬酸脱氢酶”系指具有上述酶活性的酶,包括天然的酶及具相同功能的物质。大肠杆菌的磷酸烯醇丙酮酸羧化酶(ppc基因)、柠檬酸合成酶(gltA基因)、乌头酸酶A(acnA基因)、以及乌头酸酶B(acnB基因)如下所示:
大肠杆菌的磷酸烯醇丙酮酸羧化酶
atgaacgaacaatattccgcattgcgtagtaatgtcagtatgctcggcaaagtgctggga
M N E Q Y S A L R S N V S M L G K V L G
gaaaccatcaaggatgcgttgggagaacacattcttgaacgcgtagaaactatccgtaag
E T I K D A L G E H I L E R V E T I R K
ttgtcgaaatcttcacgcgctggcaatgatgctaaccgccaggagttgctcaccacctta
L S K S S R A G N D A N R Q E L L T T L
caaaatttgtcgaacgacgagctgctgcccgttgcgcgtgcgtttagtcagttcctgaac
Q N L S N D E L L P V A R A F S Q F L N
ctggccaacaccgccgagcaataccacagcatttcgccgaaaggcgaagctgccagcaac
L A N T A E Q Y H S I S P K G E A A S N
ccggaagtgatcgcccgcaccctgcgtaaactgaaaaaccagccggaactgagcgaagac
P E V I A R T L R K L K N Q P E L S E D
accatcaaaaaagcagtggaatcgctgtcgctggaactggtcctcacggctcacccaacc
T I K K A V E S L S L E L V L T A H P T
gaaattacccgtcgtacactgatccacaaaatggtggaagtgaacgcctgtttaaaacag
E I T R R T L I H K M V E V N A C L K Q
ctcgataacaaagatatcgctgactacgaacacaaccagctgatgcgtcgcctgcgccag
L D N K D I A D Y E H N Q L M R R L R Q
ttgatcgcccagtcatggcataccgatgaaatccgtaagctgcgtccaagcccggtagat
L I A Q S W H T D E I R K L R P S P V D
gaagccaaatggggctttgccgtagtggaaaacagcctgtggcaaggcgtaccaaattac
E A K W G F A V V E N S L W Q G V P N Y
ctgcgcgaactgaacgaacaactggaagagaacctcggctacaaactgcccgtcgaattt
L R E L N E Q L E E N L G Y K L P V E F
gttccggtccgttttacttcgtggatgggcggcgaccgcgacggcaacccgaacgtcact
V P V R F T S W M G G D R D G N P N V T
gccgatatcacccgccacgtcctgctactcagccgctggaaagccaccgatttgttcctg
A D I T R H V L L L S R W K A T D L F L
aaagatattcaggtgctggtttctgaactgtcgatggttgaagcgacccctgaactgctg
K D I Q V L V S E L S M V E A T P E L L
gcgctggttggcgaagaaggtgccgcagaaccgtatcgctatctgatgaaaaacctgcgt
A L V G E E G A A E P Y R Y L M K N L R
tctcgcctgatggcgacacaggcatggctggaagcgcgcctgaaaggcgaagaactgcca
S R L M A T Q A W L E A R L K G E E L P
aaaccagaaggcctgctgacacaaaacgaagaactgtgggaaccgctctacgcttgctac
K P E G L L T Q N E E L W E P L Y A C Y
cagtcacttcaggcgtgtggcatgggtattatcgccaacggcgatctgctcgacaccctg
Q S L Q A C G M G I I A N G D L L D T L
cgccgcgtgaaatgtttcggcgtaccgctggtccgtattgatatccgtcaggagagcacg
R R V K C F G V P L V R I D I R Q E S T
cgtcataccgaagcgctgggcgagctgacccgctacctcggtatcggcgactacgaaagc
R H T E A L G E L T R Y L G I G D Y E S
tggtcagaggccgacaaacaggcgttcctgatccgcgaactgaactccaaacgtccgctt
W S E A D K Q A F L I R E L N S K R P L
ctgccgcgcaactggcaaccaagcgccgaaacgcgcgaagtgctcgatacctgccaggtg
L P R N W Q P S A E T R E V L D T C Q V
attgccgaagcaccgcaaggctccattgccgcctacgtgatctcgatggcgaaaacgccg
I A E A P Q G S I A A Y V I S M A K T P
tccgacgtactggctgtccacctgctgctgaaagaagcgggtatcgggtttgcgatgccg
S D V L A V H L L L K E A G I G F A M P
gttgctccgctgtttgaaaccctcgatgatctgaacaacgccaacgatgtcatgacccag
V A P L F E T L D D L N N A N D V M T Q
ctgctcaatattgactggtatcgtggcctgattcagggcaaacagatggtgatgattggc
L L N I D W Y R G L I Q G K Q M V M I G
tattccgactcagcaaaagatgcgggagtgatggcagcttcctgggcgcaatatcaggca
Y S D S A K D A G V M A A S W A Q Y Q A
caggatgcattaatcaaaacctgcgaaaaagcgggtattgagctgacgttgttccacggt
Q D A L I K T C E K A G I E L T L F H G
cgcggcggttccattggtcgcggcggcgcacctgctcatgcggcgctgctgtcacaaccg
R G G S I G R G G A P A H A A L L S Q P
ccaggaagcctgaaaggcggcctgcgcgtaaccgaacagggcgagatgatccgctttaaa
P G S L K G G L R V T E Q G E M I R F K
tatggtctgccagaaatcaccgtcagcagcctgtcgctttataccggggcgattctggaa
Y G L P E I T V S S L S L Y T G A I L E
gccaacctgctgccaccgccggagccgaaagagagctggcgtcgcattatggatgaactg
A N L L P P P E P K E S W R R I M D E L
tcagtcatctcctgcgatgtctaccgcggctacgtacgtgaaaacaaagattttgtgcct
S V I S C D V Y R G Y V R E N K D F V P
tacttccgctccgctacgccggaacaagaactgggcaaactgccgttgggttcacgtccg
Y F R S A T P E Q E L G K L P L G S R P
gcgaaacgtcgcccaaccggcggcgtcgagtcactacgcgccattccgtggatcttcgcc
A K R R P T G G V E S L R A I P W I F A
tggacgcaaaaccgtctgatgctccccgcctggctgggtgcaggtacggcgctgcaaaaa
W T Q N R L M L P A W L G A G T A L Q K
gtggtcgaagacggcaaacagagcgagctggaggctatgtgccgcgattggccattcttc
V V E D G K Q S E L E A M C R D W P F F
tcgacgcgtctcggcatgctggagatggtcttcgccaaagcagacctgtggctggcggaa
S T R L G M L E M V F A K A D L W L A E
tactatgaccaacgcctggtagacaaagcactgtggccgttaggtaaagagttacgcaac
Y Y D Q R L V D K A L W P L G K E L R N
ctgcaagaagaagacatcaaagtggtgctggcgattgccaacgattcccatctgatggcc
L Q E E D I K V V L A I A N D S H L M A
gatctgccgtggattgcagagtctattcagctacggaatatttacaccgacccgctgaac
D L P W I A E S I Q L R N I Y T D P L N
gtattgcaggccgagttgctgcaccgctcccgccaggcagaaaaagaaggccaggaaccg
V L Q A E L L H R S R Q A E K E G Q E P
gatcctcgcgtcgaacaagcgttaatggtcactattgccgggattgcggcaggtatgcgt
D P R V E Q A L M V T I A G I A A G M R
aataccggctaa (SEQ ID NO:5)
N T G - (SEQ ID NO:6)
大肠杆菌的柠檬酸合成酶
atggctgatacaaaagcaaaactcaccctcaacggggatacagctgttgaactggatgtg
M A D T K A K L T L N G D T A V E L D V
ctgaaaggcacgctgggtcaagatgttattgatatccgtactctcggttcaaaaggtgtg
L K G T L G Q D V I D I R T L G S K G V
ttcacctttgacccaggcttcacttcaaccgcatcctgcgaatctaaaattacttttatt
F T F D P G F T S T A S C E S K I T F I
gatggtgatgaaggtattttgctgcaccgcggtttcccgatcgatcagctggcgaccgat
D G D E G I L L H R G F P I D Q L A T D
tctaactacctggaagtttgttacatcctgctgaatggtgaaaaaccgactcaggaacag
S N Y L E V C Y I L L N G E K P T Q E Q
tatgacgaatttaaaactacggtgacccgtcataccatgatccacgagcagattacccgt
Y D E F K T T V T R H T M I H E Q I T R
ctgttccatgctttccgtcgcgactcgcatccaatggcagtcatgtgtggtattaccggc
L F H A F R R D S H P M A V M C G I T G
gcgctggcggcgttctatcacgactcgctggatgttaacaatcctcgtcaccgtgaaatt
A L A A F Y H D S L D V N N P R H R E I
gccgcgttccgcctgctgtcgaaaatgccgaccatggccgcgatgtgttacaagtattcc
A A F R L L S K M P T M A A M C Y K Y S
attggtcagccatttgtttacccgcgcaacgatctctcctacgccggtaacttcctgaat
I G Q P F V Y P R N D L S Y A G N F L N
atgatgttctccacgccgtgcgaaccgtatgaagttaatccgattctggaacgtgctatg
M M F S T P C E P Y E V N P I L E R A M
gaccgtattctgatcctgcacgctgaccatgaacagaacgcctctacctccaccgtgcgt
D R I L I L H A D H E Q N A S T S T V R
accgctggctcttcgggtgcgaacccgtttgcctgtatcgcagcaggtattgcttcactg
T A G S S G A N P F A C I A A G I A S L
tggggacctgcgcacggcggtgctaacgaagcggcgctgaaaatgctggaagaaatcagc
W G P A H G G A N E A A L K M L E E I S
tccgttaaacacattccggaatttgttcgtcgtgcgaaagacaaaaatgattctttccgc
S V K H I P E F V R R A K D K N D S F R
ctgatgggcttcggtcaccgcgtgtacaaaaattacgacccgcgcgccaccgtaatgcgt
L M G F G H R V Y K N Y D P R A T V M R
gaaacctgccatgaagtgctgaaagagctgggcacgaaggatgacctgctggaagtggct
E T C H E V L K E L G T K D D L L E V A
atggagctggaaaacatcgcgctgaacgacccgtactttatcgagaagaaactgtacccg
M E L E N I A L N D P Y F I E K K L Y P
aacgtcgatttctactctggtatcatcctgaaagcgatgggtattccgtcttccatgttc
N V D F Y S G I I L K A M G I P S S M F
accgtcattttcgcaatggcacgtaccgttggctggatcgcccactggagcgaaatgcac
T V I F A M A R T V G W I A H W S E M H
agtgacggtatgaagattgcccgtccgcgtcagctgtatacaggatatgaaaaacgcgac
S D G M K I A R P R Q L Y T G Y E K R D
Tttaaaagcgatatcaagcgttaa (SEQ ID NO:7)
F K S D I K R - (SEQ ID NO:8)
大肠杆菌的乌头酸酶A
atgtcgtcaaccctacgagaagccagtaaggacacgttgcaggccaaagataaaacttac
M S S T L R E A S K D T L Q A K D K T Y
cactactacagcctgccgcttgctgctaaatcactgggcgatatcacccgtctacccaag
H Y Y S L P L A A K S L G D I T R L P K
tcactcaaagttttgctcgaaaacctgctgcgctggcaggatggtaactcggttaccgaa
S L K V L L E N L L R W Q D G N S V T E
gaggatatccacgcgctggcaggatggctgaaaaatgcccatgctgaccgtgaaattgcc
E D I H A L A G W L K N A H A D R E I A
taccgcccggcaagggtgctgatgcaggactttaccggcgtacctgccgttgttgatctg
Y R P A R V L M Q D F T G V P A V V D L
gcggcaatgcgcgaagcggttaaacgcctcggcggcgatactgcaaaggttaacccgctc
A A M R E A V K R L G G D T A K V N P L
tcaccggtcgacctggtcattgaccactcggtgaccgtcgatcgttttggtgatgatgag
S P V D L V I D H S V T V D R F G D D E
gcatttgaagaaaacgtacgcctggaaatggagcgcaaccacgaacgttatgtgttcctg
A F E E N V R L E M E R N H E R Y V F L
aaatggggaaagcaagcgttcagtcggtttagcgtcgtgccgccaggcacaggcatttgc
K W G K Q A F S R F S V V P P G T G I C
catcaggttaacctcgaatatctcggcaaagcagtgtggagtgaattgcaggacggtgaa
H Q V N L E Y L G K A V W S E L Q D G E
tggattgcttatccggatacactcgttggtactgactcgcacaccaccatgatcaacggc
W I A Y P D T L V G T D S H T T M I N G
cttggcgtgctggggtggggcgttggtgggatcgaagcagaagccgcaatgttaggccag
L G V L G W G V G G I E A E A A M L G Q
ccggtttccatgcttatcccggatgtagtgggcttcaaacttaccggaaaattacgtgaa
P V S M L I P D V V G F K L T G K L R E
ggtattaccgccacagacctggttctcactgttacccaaatgctgcgcaaacatggcgtg
G I T A T D L V L T V T Q M L R K H G V
gtggggaaattcgtcgaattttatggtgatggtctggattcactaccgttggcggatcgc
V G K F V E F Y G D G L D S L P L A D R
gccaccattgccaatatgtcgccagaatatggtgccacctgtggcttcttcccaatcgat
A T I A N M S P E Y G A T C G F F P I D
gctgtaaccctcgattacatgcgtttaagcgggcgcagcgaagatcaggtcgagttggtc
A V T L D Y M R L S G R S E D Q V E L V
gaaaaatatgccaaagcgcagggcatgtggcgtaacccgggcgatgaaccaatttttacc
E K Y A K A Q G M W R N P G D E P I F T
agtacgttagaactggatatgaatgacgttgaagcgagcctggcagggcctaaacgccca
S T L E L D M N D V E A S L A G P K R P
caggatcgcgttgcactgcccgatgtaccaaaagcatttgccgccagtaacgaactggaa
Q D R V A L P D V P K A F A A S N E L E
gtgaatgccacgcataaagatcgccagccggtcgattatgttatgaacggacatcagtat
V N A T H K D R Q P V D Y V M N G H Q Y
cagttacctgatggcgctgtggtcattgctgcgataacctcgtgcaccaacacctctaac
Q L P D G A V V I A A I T S C T N T S N
ccaagtgtgctgatggccgcaggcttgctggcgaaaaaagccgtaactctgggcctcaag
P S V L M A A G L L A K K A V T L G L K
cggcaaccatgggtcaaagcgtcgctggcaccgggttcgaaagtcgtttctgattatctg
R Q P W V K A S L A P G S K V V S D Y L
gcaaaagcgaaactgacaccgtatctcgacgaactggggtttaaccttgtgggatacggt
A K A K L T P Y L D E L G F N L V G Y G
tgtaccacctgtattggtaactctgggccgctgcccgatcctatcgaaacggcaatcaaa
C T T C I G N S G P L P D P I E T A I K
aaaagcgatttaaccgtcggtgcggtgctgtccggcaaccgtaactttgaaggccgtatc
K S D L T V G A V L S G N R N F E G R I
catccgctggttaaaactaactggctggcctcgccgccgctggtggttgcctatgcgctg
H P L V K T N W L A S P P L V V A Y A L
gcgggaaatatgaatatcaacctggcttctgagcctatcggccatgatcgcaaaggcgat
A G N M N I N L A S E P I G H D R K G D
ccggtttatctgaaagatatctggccatcggcacaagaaattgcccgtgcggtagaacaa
P V Y L K D I W P S A Q E I A R A V E Q
gtctccacagaaatgttccgcaaagagtacgcagaagtttttgaaggcacagcagagtgg
V S T E M F R K E Y A E V F E G T A E W
aagggaattaacgtcacacgatccgatacctacggttggcaggaggactcaacctatatt
K G I N V T R S D T Y G W Q E D S T Y I
cgcttatcgcctttctttgatgaaatgcaggcaacaccagcaccagtggaagatattcac
R L S P F F D E M Q A T P A P V E D I H
ggtgcgcggatcctcgcaatgctgggggattcagtcaccactgaccatatctctccggcg
G A R I L A M L G D S V T T D H I S P A
ggcagtattaagcccgacagcccagcgggtcgatatctacaaggtcggggtgttgagcga
G S I K P D S P A G R Y L Q G R G V E R
aaagactttaactcctacggttcgcggcgtggtaaccatgaagtgatgatgcgcggcacc
K D F N S Y G S R R G N H E V M M R G T
ttcgccaatattcgcatccgtaatgaaatggtgcctggcgttgaaggggggatgacgcgg
F A N I R I R N E M V P G V E G G M T R
catttacctgacagcgacgtagtctctatttatgatgctgcgatgcgctataagcaggag
H L P D S D V V S I Y D A A M R Y K Q E
caaacgccgctggcggtgattgccgggaaagagtatggatcaggctccagtcgtgactgg
Q T P L A V I A G K E Y G S G S S R D W
gcggcaaaaggtccgcgtctgcttggtattcgtgtggtgattgccgaatcgtttgaacga
A A K G P R L L G I R V V I A E S F E R
attcaccgttcgaatttaattggcatgggcatcctgccgctggaatttccgcaaggcgta
I H R S N L I G M G I L P L E F P Q G V
acgcgtaaaacgttagggctaaccggggaagagaagattgatattggcgatctgcaaaac
T R K T L G L T G E E K I D I G D L Q N
ctacaacccggcgcgacggttccggtgacgcttacgcgcgcggatggtagccaggaagtc
L Q P G A T V P V T L T R A D G S Q E V
gtaccctgccgttgtcgtatcgacaccgcgacggagttgacctactaccagaacgacggc
V P C R C R I D T A T E L T Y Y Q N D G
Attttgcattatgtcattcgtaatatgttgaagtaa (SEQ ID NO:9)
I L H Y V I R N M L K - (SEQ ID NO:10)
大肠杆菌的乌头酸酶B
atgctagaagaataccgtaagcacgtagctgagcgtgccgctgaggggattgcgcccaaa
M L E E Y R K H V A E R A A E G I A P K
cccctggatgcaaaccaaatggccgcacttgtagagctgctgaaaaacccgcccgcgggc
P L D A N Q M A A L V E L L K N P P A G
gaagaagaattcctgttagatctgttaaccaaccgtgttcccccaggcgtcgatgaagcc
E E E F L L D L L T N R V P P G V D E A
gcctatgtcaaagcaggcttcctggctgctatcgcgaaaggcgaagccaaatcccctctg
A Y V K A G F L A A I A K G E A K S P L
ctgactccggaaaaagccatcgaactgctgggcaccatgcagggtggttacaacattcat
L T P E K A I E L L G T M Q G G Y N I H
ccgctgatcgacgcgctggatgatgccaaactggcacctattgctgccaaagcactttct
P L I D A L D D A K L A P I A A K A L S
cacacgctgctgatgttcgataacttctatgacgtagaagagaaagcgaaagcaggcaac
H T L L M F D N F Y D V E E K A K A G N
gaatatgcgaagcaggttatgcagtcctgggcggatgccgaatggttcctgaatcgcccg
E Y A K Q V M Q S W A D A E W F L N R P
gcgctggctgaaaaactgaccgttactgtcttcaaagtcactggcgaaactaacaccgat
A L A E K L T V T V F K V T G E T N T D
gacctttctccggcaccggatgcgtggtcacgcccggatatcccactgcacgcgctggcg
D L S P A P D A W S R P D I P L H A L A
atgctgaaaaacgcccgtgaaggtattgagccagaccagcctggtgttgttggtccgatc
M L K N A R E G I E P D Q P G V V G P I
aagcaaatcgaagctctgcaacagaaaggtttcccgctggcgtacgtcggtgacgttgtg
K Q I E A L Q Q K G F P L A Y V G D V V
ggtacgggttcttcgcgtaaatccgccactaactccgttctgtggtttatgggcgatgat
G T G S S R K S A T N S V L W F M G D D
attccacatgtgccgaacaaacgcggcggtggtttgtgcctcggcggtaaaattgcaccc
I P H V P N K R G G G L C L G G K I A P
atcttctttaacacgatggaagacgcgggtgcactgccaatcgaagtcgacgtctctaac
I F F N T M E D A G A L P I E V D V S N
ctgaacatgggcgacgtgattgacgtttacccgtacaaaggtgaagtgcgtaaccacgaa
L N M G D V I D V Y P Y K G E V R N H E
accggcgaactgctggcgaccttcgaactgaaaaccgacgtgctgattgatgaagtgcgt
T G E L L A T F E L K T D V L I D E V R
gctggtggccgtattccgctgattatcgggcgtggcctgaccaccaaagcgcgtgaagca
A G G R I P L I I G R G L T T K A R E A
cttggtctgccgcacagtgatgtgttccgtcaggcgaaagatgtcgctgagagcgatcgc
L G L P H S D V F R Q A K D V A E S D R
ggcttctcgctggcgcaaaaaatggtaggccgtgcctgtggcgtgaaaggcattcgtccg
G F S L A Q K M V G R A C G V K G I R P
ggcgcgtactgtgaaccgaaaatgacttctgtaggttcccaggacaccaccggcccgatg
G A Y C E P K M T S V G S Q D T T G P M
acccgtgatgaactgaaagacctggcgtgcctgggcttctcggctgacctggtgatgcag
T R D E L K D L A C L G F S A D L V M Q
tctttctgccacaccgcggcgtatccgaagccagttgacgtgaacacgcaccacacgctg
S F C H T A A Y P K P V D V N T H H T L
ccggacttcattatgaaccgtggcggtgtgtcgctgcgtccgggtgacggcgtcattcac
P D F I M N R G G V S L R P G D G V I H
tcctggctgaaccgtatgctgctgccggataccgtcggtaccggtggtgactcccatacc
S W L N R M L L P D T V G T G G D S H T
cgtttcccgatcggtatctctttcccggcgggttctggtctggtggcgtttgctgccgca
R F F I G I S F P A G S G L V A F A A A
actggcgtaatgccgcttgatatgccggaatccgttctggtgcgcttcaaaggcaaaatg
T G V M P L D M P E S V L V R F K G K M
cagccgggcatcaccctgcgcgatctggtacacgctattccgctgtatgcgatcaaacaa
Q P G I T L R D L V H A I P L Y A I K Q
ggtctgctgaccgttgagaagaaaggcaagaaaaacatcttctctggccgcatcctggaa
G L L T V E K K G K K N I F S G R I L E
attgaaggtctgccggatctgaaagttgagcaggcctttgagctaaccgatgcgtccgcc
I E G L P D L K V E Q A F E L T D A S A
gagcgttctgccgctggttgtaccatcaagctgaacaaagaaccgatcatcgaatacctg
E R S A A G C T I K L N K E P I I E Y L
aactctaacatcgtcctgctgaagtggatgatcgcggaaggttacggcgatcgtcgtacc
N S N I V L L K W M I A E G Y G D R R T
ctggaacgtcgtattcagggcatggaaaaatggctggcgaatcctgagctgctggaagcc
L E R R I Q G M E K W L A N P E L L E A
gatgcagatgcggaatacgcggcagtgatcgacatcgatctggcggatattaaagagcca
D A D A E Y A A V I D I D L A D I K E P
atcctgtgtgctccgaacgacccggatgacgcgcgtccgctgtctgcggtacagggtgag
I L C A P N D P D D A R P L S A V Q G E
aagatcgacgaagtgtttatcggttcctgcatgaccaacatcggtcacttccgtgctgcg
K I D E V F I G S C M T N I G H F R A A
ggtaaactgctggatgcgcataaaggtcagttgccgacccgcctgtgggtggcaccgcca
G K L L D A H K G Q L P T R L W V A P P
acccgtatggacgccgcacagttgaccgaagaaggctactacagcgtcttcggtaagagt
T R M D A A Q L T E E G Y Y S V F G K S
ggtgcgcgtatcgagatccctggctgttccctgtgtatgggtaaccaggcgcgtgtggcg
G A R I E I P G C S L C M G N Q A R V A
gacggtgcaacggtggtttccacctctacccgtaacttcccgaaccgtctgggtactggc
D G A T V V S T S T R N F P N R L G T G
gcgaatgtcttcctggcttctgcggaactggcggctgttgcggcgctgattggcaaactg
A N V F L A S A E L A A V A A L I G K L
ccgacgccggaagagtaccagacctacgtggcgcaggtagataaaacagccgttgatact
P T P E E Y Q T Y V A Q V D K T A V D T
taccgttatctgaacttcaaccagctttctcagtacaccgagaaagccgatggggtgatt
Y R Y L N F N Q L S Q Y T E K A D G V I
ttccagactgcggtttaa (SEQ ID NO:11)
F Q T A V - (SEQ ID NO:12)
下列表一显示磷酸烯醇丙酮酸羧化酶/羧激酶、柠檬酸合成酶及乌头酸酶的其它例子,以及2-甲基柠檬酸合成酶、柠檬酸裂解酶与2-甲基柠檬酸脱氢酶的例子。
酶 | 基因银行编号 |
磷酸烯醇丙酮酸羧化酶/羧激酶 | NP_417862(E.coli,EC4.1.1.49);AAB07805(S.aureus,EC4.1.1.32);CAC32156(M.leprae),XP_645396(D.discoideum);EDN6000(S.cerevisiae);及XP_001210573(A.terreus);PC2168(E.coli,EC4.1.1.38),NP_850372(A.thaliana);CAA35251(S.bicolor);CAB95920(S.coelicolor);XP_001391222(A.niger) |
柠檬酸合成酶 | AAC73814(E.coli);NP_001080194(X.laevix);CAB66275(S.coelicolor);NP_080720(M.musculus);ABP36423(C.phaeovibrioides);XP_001827205(A.oryzae);及EDN61138(S.cerevisiae) |
2-甲基柠檬酸合成酶 | ABN63514(S.baltica);ABI57944(A.ehrlichei);AP_000985(E.coli);XP_001209805(A.terreus);P45858(B.subtilis);Q56063(S.typhimurium) |
柠檬酸裂解酶 | YP-_662283(P.atlantica);ABH11558(L.helveticus);AAL50820(R.erythropolis);AP_001263(E.coli);XP_750953(A. |
fumigatus);及NP_669690(Y.pestis) | |
乌头酸酶 | CAA90177(B.taurus);CAQ017353(C.michiganesis);CAC37548(S.coelicolor);AAC46192(M.avium);1L5JB(E.coli);EDN59216(S.cerevisiae);AAC61778(A.terreus);YP_910600(C.phaeobacteroides) |
酶 | 基因银行编号 |
2-甲基柠檬酸脱氢酶 | ZP_03698315(L.nitroferrum);YP_002029406(S.maltophilia);AP_000986(E.coli);EDV11211(S.cerevisiae);XP_01209777(A.terreus);YP_001860514(B.phymatum) |
上述之基因修饰微生物可以习知重组方法构筑完成,可参照,例如Sambrook et al.,Molecular Cloning:A Laboratory Manual,Cold Spring HarborLaboratory Press。
特别是,基因修饰微生物可过度表达上述一或多个酶。可利用聚合酶链锁反应(PCR)由其天然来源(可来自基因银行)获得编码上述一或多个酶的DNA片段。此DNA片段接着连接至适当的启动子以形成表达框架(expression cassette)。在一实施例中,表达框架包括连接启动子的编码序列。在另一实施例中,表达框架包括多个编码序列,其皆连接至启动子。在此例子中,各编码序列的5’端较佳具有核糖体结合位。若有需要,此编码序列为宿主微生物的最佳化编码序列。
本发明中所述之“启动子”系指核酸序列,在宿主微生物中,此核酸序列的一部分可开启连接核酸序列的转录。启动子至少包括RNA聚合酶结合位。启动子可还包括一或多个调控部分,其可控制启动子的开启/停止状态。当以大肠杆菌作为宿主微生物时,代表性的大肠杆菌启动子包括,但不限于,β-内酰胺酶及乳糖启动子***(Chang et al.,Nature 275:615-624,1978),SP6、T3、T5及T7RNAs聚合酶启动子(Studier et al.,Meth.Enzymol.185:60-89,1990),lambda启动子(Elvin et al.,Gene 87:123-126,1990),trp启动子(Nichols and Yanofsky,Meth.In Enzymology 101:155-164,1983)以及Tac及Trc启动子(Russell et al.,Gene 20:231-243,1982)。当以酵母菌作为宿主微生物时,代表性的酵母菌启动子包括,但不限于,3-磷酸甘油酸激酶启动子、甘油醛3-磷酸脱氢酶(GAPDH)启动子、半乳糖激酶(GAL1)启动子、半乳糖表异构酶(galactoepimerase)启动子、以及醇脱氢酶(ADH)启动子。在其它微生物中,适合用于表达基因的启动子亦为熟悉此技术领域人士所习知。
接着,将上述之表达框架转染至适合的微生物中,以获得上述之基因修饰微生物,筛选转殖微生物,并证明一或多个酶过度表达的方法为熟悉此技术领域人士所习知,例如,免疫墨点或酶活性分析。将此基因修饰微生物培养于适当的培养基中以产生衣康酸。此培养基较佳具有葡萄糖或柠檬酸,其为产生衣康酸的前体。在充分的培养后,收集此培养基,并分离分泌于培养基中的衣康酸。
【实施例】
1.质粒及基因修饰大肠杆菌菌株
本实施例中使用大肠杆菌BW25113(rrnBT14ΔlacZWJ16hsdR514ΔaraBADAH33ΔrhaBADLD78)及BL21(dcm ompT hsdS(rB-mB-)gal),请参照Datsehko et al.,Proc.Natl.Acad.Sci.USA,97:6640-6645,2000,以重组技术进行基因修饰。简言之,将BW25113的内源性icd基因以FLP-FRT重组技术破坏,以获得BW25113突变株(即PCI400)。相较于BW25113,BW25113突变株的异柠檬酸脱氢酶表达量较低。
将一些质粒转染至母菌株及PCI400中,此质粒如表二所述,其以重组技术构筑完成。
表二、质粒的表达
质粒名称 | 基因型 |
pZE12-1uc | ColE1 ori;AmpR;PLlacO1::luc(VF) |
pPC 1 | ColE1 ori;KanR;PLlacO1::cad(AT) |
pPC 2 | pZE12,PLlacO1::acnA(EC) |
pPC 3 | pZE12,PLlacO1::acnB(EC) |
pPC 4 | ColE1 ori;SpeR;PLlacO1::gltA(EC) |
pPC 5 | ColE1 ori;SpeR;PLlacO1::ppc(EC) |
pPC 6 | ColE1 ori;SpeR;PLlacO1::ppc(EC)-gltA(EC) |
pPC 104 | pZE12,PLlacO1::cad(AT)-acnA(EC) |
pPC 107 | pA15 ori;AmpR;PLlacO1::cad(AT)-acnA(EC) |
pPC 171 | pA15 ori;SpeR;PLlacO1::ppc(EC)-gltA(EC) |
*luc(VF):V.fischeri萤光素酶(luciferase)。
*cad(AT):A.terreus顺式乌头酸脱羧酶基因。
*acnA(EC):大肠杆菌乌头酸酶A基因。
*acnB(EC):大肠杆菌乌头酸酶B基因。
*gltA(EC):大肠杆菌柠檬酸合成酶基因。
*ppc(EC):大肠杆菌磷酸烯醇丙酮酸羧化酶基因。
将表二中所述之一或多个质粒转染至BW25113、PCI400、或BL21以获得各种修饰的大肠杆菌菌株,如表三所示。
表三、基因修饰之大肠杆菌菌株
名称 | 母菌株 | 转染的质粒 |
PCI 010 | BW25113 | pPC1 |
PCI 011 | BW25113 | pPC1及pPC5 |
PCI 012 | BW25113 | pPC1及pPC4 |
PCI 013 | BW25113 | pPC1及pPC3 |
PCI 014 | BW25113 | pPC1、pPC3及pPC5 |
PCI 015 | BW25113 | pPC1、pPC3及pPC4 |
PCI 016 | BW25113 | pPC1、pPC3及pPC6 |
PCI 017 | BW25113 | pPC1及pPC6 |
PCI 019 | BW25113 | pPC1、pPC2及pPC6 |
PCI 213 | BL21 | pPC1 |
PCI 510 | PCI400 | pPC1 |
PCI 511 | PCI400 | pPC1及pPC5 |
PCI 512 | PCI400 | pPC1及pPC4 |
PCI 513 | PCI400 | pPC1及pPC3 |
PCI 514 | PCI400 | pPC1、pPC3及pPC5 |
PCI 515 | PCI400 | pPC1、pPC3及pPC4 |
PCI 516 | PCI400 | pPC1、pPC3及pPC6 |
PCI 517 | PCI400 | pPC1及pPC6 |
PCI 519 | PCI400 | pPC1、pPC2及pPC6 |
PCI 543 | PCI400 | pPC6及pPC107 |
名称 | 母菌株 | 转染的质粒 |
PCI 545 | PCI400 | pPC104及pPC171 |
此外,关于质粒和修饰菌株的构建参考:
(1)Atsumi,S.,Hanai,T.,Liao,J.C.,2008.Engineering syntheticnon-ferment ative pathways for production of branched-chain higheralcohols as biofuels.Nature 451,86-89;
(2)C.R.Shen,J.C.Liao.2008.Metabolic engineering of Esche richiacoli for 1-butanol and 1-propanol production via the keto-acidpathways.Metabolic Engineering 10(2008)312-320。
2.以PCI 213表达衣康酸
PCI 213菌株(BL21过度表达土曲霉(A.terreus)之CAD)于37℃下培养于LB(Luria-Bertani)培养基隔夜,再接种(1%)至基础培养基,其包括80g/L的葡萄糖、氯化钠、及磷酸盐缓冲液,并于30℃下培养适当的期间,直到OD600达0.2-0.6。接着,添加异丙基-β-D-硫代半乳糖苷(IPTG,0.5mM)至培养基中以诱导CAD的表达。24小时后,收集此培养基。培养基中衣康酸的浓度为约100mg/l。
3.以PCI010表达衣康酸
PCI010菌株(BW25133过度表达土曲霉(A.terreus)之CAD)于37℃下培养于基础培养基隔夜,基础培养基包括40g/L的葡萄糖、氯化钠、及磷酸盐缓冲液。将此隔夜培养基接种至M9培养基中,其包括20或40g/L的葡萄糖、氯化钠、及磷酸盐缓冲液,另含有或不含有1mM的麸氨酸(Vetsin),于30℃下培养。当OD600达0.2-0.4时,加入0.5mM的异丙基-β-D-硫代半乳糖苷(IPTG)至培养基中以诱导土曲霉(A.terreus)之CAD的表达。培养24小时后,测定衣康酸的含量。此结果如表四所示。
表四、PCI010的衣康酸表达
4.以葡萄糖为基质,于各种修饰的大肠杆菌菌株表达衣康酸
将表五所述之修饰大肠杆菌菌株在37℃下,于旋转震荡器(250rpm)中,培养于LB(Luria-Bertani)培养基隔夜,再接种至M9培养基中,其包括20g/L的葡萄糖、1g/L的酵母萃取物、氯化钠、及磷酸盐缓冲液,于37℃下培养。当OD600达0.2-0.4时,加入0.5mM的异丙基-β-D-硫代半乳糖苷(IPTG)至培养基中以诱导外源性基因表达。分别于不同的时间点收集培养基,并测定培养基中衣康酸的含量,如表五所示。
表五、基因修饰大肠杆菌菌株中衣康酸的表达
大肠杆菌菌株 | 衣康酸含量(g/L) | 培养时间(小时) |
PCI 014 | 0.005 | 48 |
PCI 013 | 0.021 | 48 |
PCI 015 | 0.032 | 48 |
PCI 016 | 0.034 | 48 |
PCI 010 | 0.054 | 48 |
PCI 010 | 0.057 | 70 |
PCI 514 | 0.074 | 49 |
PCI 510 | 0.280 | 49 |
PCI 511 | 0.288 | 49 |
PCI 513 | 0.346 | 49 |
PCI 512 | 0.404 | 49 |
PCI 515 | 0.598 | 49 |
PCI 516 | 2.106 | 49 |
PCI 516 | 4.02 | 72 |
PCI 519 | 4.158 | 73 |
如表五所示,PCI 519(BW25133;Δicd,gltA,ppc,cad及acnA)及PCI516(Δicd,gltA,ppc,cad及acnB)菌株在培养72小时后,培养基中具有超过4g/L的衣康酸。PCI 519与PCI 516的葡萄糖-衣康酸转换率分别为,每公克的葡萄糖可转换0.52g衣康酸及0.68g衣康酸。
5.以柠檬酸为基质,于各种修饰的大肠杆菌菌株表达衣康酸
将PCI 513(BW25133;Δicd,cad及acnB)、PCI 516及PCI 519于37℃下培养于LB培养基16小时。接着,将此大肠杆菌菌株接种至含0.5mMIPTG的新鲜LB培养基中培养16小时。将上述细胞以Triton X-100处理后,并重新悬浮于含50mg/ml柠檬酸的磷酸缓冲液中。于48或72小时后,检测培养基的衣康酸含量。
PCI 513在培养48小时后可产生1.27g/L的衣康酸。此菌株的葡萄糖-衣康酸转换率为每公克的柠檬酸转换0.24g衣康酸。
PCI 519及PCI 516在培养72小时后可分别产生6g/L及5g/L的衣康酸。PCI 519及PCI 516菌株的葡萄糖-衣康酸转换率分别为,每公克的柠檬酸转换0.61g衣康酸及0.34g衣康酸。
6.以甘油为基质,于各种修饰的大肠杆菌菌株表达衣康酸
将PCI 519及PCI 543的E.coli菌株在37℃下,于旋转震荡器(250rpm)中,培养于LB(Luria-Bertani)培养基隔夜,再接种至TB培养基中,其包括20-50g/L的甘油、24g/L的酵母萃取物、12g/L胰蛋白胨(tryptone)、及磷酸盐缓冲液,于37℃下培养。当OD600达0.2-0.4时,加入0.5mM的异丙基-β-D-硫代半乳糖苷(IPTG)至培养基中以诱导外源性基因表达。于24小时后收集培养基,并测定培养基中衣康酸的含量,如表六所示。
表六、基因修饰大肠杆菌菌株中衣康酸的表达
大肠杆菌菌株 | 衣康酸含量(g/L) | 培养时间(小时) |
PCI 519 | 1.77 | 24 |
PCI 543 | 3.05~8 | 24 |
PCI 545 | 8.98 | 24 |
虽然本发明已以较佳实施例揭露如上,然其并非用以限定本发明,任何熟习此技艺者,在不脱离本发明之精神和范围内,当可作些许之更动与润饰,因此本发明之保护范围当视后附之中请专利范围所界定者为准。
序列表
<110>财团法人工业技术研究院
<120>基因修饰微生物及以此微生物生产衣康酸的方法
<130>P54980051CN
<150>12/463,677
<151>2009-05-11
<160>12
<170>PatentIn version 3.3
<210>1
<211>1473
<212>DNA
<213>土曲菌(Aspergillus terreus)
<400>1
atgaccaagc agtctgctga ttccaacgcg aagtctggtg tgacctctga gatctgtcac 60
tgggcgtcta atctcgccac tgatgatatc ccgagcgacg ttctggagcg tgcaaaatac 120
ctgatcctgg atggtatcgc gtgcgcgtgg gtaggtgctc gtgtcccatg gtctgaaaaa 180
tacgttcaag cgaccatgtc tttcgaacct ccgggtgcgt gtcgtgtcat cggttacggc 240
cagaaactgg gtccggtagc ggctgccatg acgaactctg catttattca ggcgaccgaa 300
ctcgatgact atcactctga agcgccgctg cattccgcgt ctatcgttct cccggcagtt 360
ttcgcggcga gcgaagtact ggccgaacag ggtaaaacca tctctggtat tgacgtgatt 420
ctggctgcga tcgttggttt cgagagcggt cctcgcatcg gcaaagcgat ctacggttct 480
gacctcctga acaacggctg gcactgcggt gcggtatatg gcgcaccggc tggtgcgctc 540
gcaactggta agctcctggg cctcacgccg gacagcatgg aagatgcact gggtattgcc 600
tgcacgcaag catgcggcct catgtccgcg cagtatggtg gcatggttaa acgtgttcag 660
cacggtttcg cagcgcgtaa tggtctcctc ggtggcctcc tggctcacgg cggctacgag 720
gcgatgaaag gtgttctcga gcgttcttac ggtggcttcc tgaagatgtt caccaagggc 780
aacggtcgtg aaccgccgta caaagaagaa gaggttgtgg ctggtctggg tagcttctgg 840
cacaccttca ccattcgtat caaactgtac gcgtgctgcg gtctcgtaca cggtcctgtt 900
gaagccattg aaaacctcca gggtcgttac ccggaactgc tcaatcgtgc taacctgtct 960
aacatccgcc acgttcacgt acaactctct accgcgagca actcccactg tggttggatc 1020
ccagaagagc gcccaatctc ttctatcgcg ggtcaaatgt ctgtcgcata tatcctcgcc 1080
gttcagctcg ttgaccaaca gtgtctgctc agccagttct ccgagtttga cgataatctg 1140
gaacgcccgg aagtgtggga cctggcacgt aaggttacca gctctcaatc tgaggagttc 1200
gaccaggacg gtaactgtct ctctgccggt cgcgtccgta ttgagttcaa cgacggctcc 1260
tccatcaccg aatccgttga gaagccgctc ggtgtaaagg aaccaatgcc aaatgaacgc 1320
atcctgcaca aataccgtac cctggcgggt tctgtaacgg acgaaagccg tgttaaggag 1380
atcgaggatc tcgtgctcgg cctggaccgt ctgaccgata ttagcccgct cctcgagctg 1440
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Glu Ile Cys His Trp Ala Ser Asn Leu Ala Thr Asp Asp Ile Pro Ser
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Asp Val Leu Glu Arg Ala Lys Tyr Leu Ile Leu Asp Gly Ile Ala Cys
35 40 45
Ala Trp Val Gly Ala Arg Val Pro Trp Ser Glu Lys Tyr Val Gln Ala
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Thr Met Ser Phe Glu Pro Pro Gly Ala Cys Arg Val Ile Gly Tyr Gly
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Gln Lys Leu Gly Pro Val Ala Ala Ala Met Thr Asn Ser Ala Phe Ile
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Gln Ala Thr Glu Leu Asp Asp Tyr His Ser Glu Ala Pro Leu His Ser
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Ala Ser Ile Val Leu Pro Ala Val Phe Ala Ala Ser Glu Val Leu Ala
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Glu Gln Gly Lys Thr Ile Ser Gly Ile Asp Val Ile Leu Ala Ala Ile
130 135 140
Val Gly Phe Glu Ser Gly Pro Arg Ile Gly Lys Ala Ile Tyr Gly Ser
145 150 155 160
Asp Leu Leu Asn Asn Gly Trp His Cys Gly Ala Val Tyr Gly Ala Pro
165 170 175
Ala Gly Ala Leu Ala Thr Gly Lys Leu Leu Gly Leu Thr Pro Asp Ser
180 185 190
Met Glu Asp Ala Leu Gly Ile Ala Cys Thr Gln Ala Cys Gly Leu Met
195 200 205
Ser Ala Gln Tyr Gly Gly Met Val Lys Arg Val Gln His Gly Phe Ala
210 215 220
Ala Arg Asn Gly Leu Leu Gly Gly Leu Leu Ala His Gly Gly Tyr Glu
225 230 235 240
Ala Met Lys Gly Val Leu Glu Arg Ser Tyr Gly Gly Phe Leu Lys Met
245 250 255
Phe Thr Lys Gly Asn Gly Arg Glu Pro Pro Tyr Lys Glu Glu Glu Val
260 265 270
Val Ala Gly Leu Gly Ser Phe Trp His Thr Phe Thr Ile Arg Ile Lys
275 280 285
Leu Tyr Ala Cys Cys Gly Leu Val His Gly Pro Val Glu Ala Ile Glu
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Asn Leu Gln Gly Arg Tyr Pro Glu Leu Leu Asn Arg Ala Asn Leu Ser
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Asn Ile Arg His Val His Val Gln Leu Ser Thr Ala Ser Asn Ser His
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Cys Gly Trp Ile Pro Glu Glu Arg Pro Ile Ser Ser Ile Ala Gly Gln
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Met Ser Val Ala Tyr Ile Leu Ala Val Gln Leu Val Asp Gln Gln Cys
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Asn Asp Gly Ser Ser Ile Thr Glu Ser Val Glu Lys Pro Leu Gly Val
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<210>3
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<213>大肠杆菌(Escherichia coli)
<400>3
atggaaagta aagtagttgt tccggcacaa ggcaagaaga tcaccctgca aaacggcaaa 60
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gtaaccccag ccatgctgaa agtggtcgac gctgcagtcg agaaagccta taaaggcgag 180
cgtaaaatct cctggatgga aatttacacc ggtgaaaaat ccacacaggt ttatggtcag 240
gacgtctggc tgcctgctga aactcttgat ctgattcgtg aatatcgcgt tgccattaaa 300
ggtccgctga ccactccggt tggtggcggt attcgctctc tgaacgttgc cctgcgccag 360
gaactggatc tctacatctg cctgcgtccg gtacgttact atcagggcac tccaagcccg 420
gttaaacacc ctgaactgac cgatatggtt atcttccgtg aaaactcgga agacatttat 480
gcgggtatcg aatggaaagc agactctgcc gacgccgaga aagtgattaa attcctgcgt 540
gaagagatgg gggtgaagaa aattcgcttc ccggaacatt gtggtatcgg tattaagccg 600
tgttcggaag aaggcaccaa acgtctggtt cgtgcagcga tcgaatacgc aattgctaac 660
gatcgtgact ctgtgactct ggtgcacaaa ggcaacatca tgaagttcac cgaaggagcg 720
tttaaagact ggggctacca gctggcgcgt gaagagtttg gcggtgaact gatcgacggt 780
ggcccgtggc tgaaagttaa aaacccgaac actggcaaag agatcgtcat taaagacgtg 840
attgctgatg cattcctgca acagatcctg ctgcgtccgg ctgaatatga tgttatcgcc 900
tgtatgaacc tgaacggtga ctacatttct gacgccctgg cagcgcaggt tggcggtatc 960
ggtatcgccc ctggtgcaaa catcggtgac gaatgcgccc tgtttgaagc cacccacggt 1020
actgcgccga aatatgccgg tcaggacaaa gtaaatcctg gctctattat tctctccgct 1080
gagatgatgc tgcgccacat gggttggacc gaagcggctg acttaattgt taaaggtatg 1140
gaaggcgcaa tcaacgcgaa aaccgtaacc tatgacttcg agcgtctgat ggatggcgct 1200
aaactgctga aatgttcaga gtttggtgac gcgatcatcg aaaacatgta a 1251
<210>4
<211>416
<212>PRT
<213>大肠杆菌(Escherichia coli)
<400>4
Met Glu Ser Lys Val Val Val Pro Ala Gln Gly Lys Lys Ile Thr Leu
1 5 10 15
Gln Asn Gly Lys Leu Asn Val Pro Glu Asn Pro Ile Ile Pro Tyr Ile
20 25 30
Glu Gly Asp Gly Ile Gly Val Asp Val Thr Pro Ala Met Leu Lys Val
35 40 45
Val Asp Ala Ala Val Glu Lys Ala Tyr Lys Gly Glu Arg Lys Ile Ser
50 55 60
Trp Met Glu Ile Tyr Thr Gly Glu Lys Ser Thr Gln Val Tyr Gly Gln
65 70 75 80
Asp Val Trp Leu Pro Ala Glu Thr Leu Asp Leu Ile Arg Glu Tyr Arg
85 90 95
Val Ala Ile Lys Gly Pro Leu Thr Thr Pro Val Gly Gly Gly Ile Arg
100 105 110
Ser Leu Asn Val Ala Leu Arg Gln Glu Leu Asp Leu Tyr Ile Cys Leu
115 120 125
Arg Pro Val Arg Tyr Tyr Gln Gly Thr Pro Ser Pro Val Lys His Pro
130 135 140
Glu Leu Thr Asp Met Val Ile Phe Arg Glu Asn Ser Glu Asp Ile Tyr
145 150 155 160
Ala Gly Ile Glu Trp Lys Ala Asp Ser Ala Asp Ala Glu Lys Val Ile
165 170 175
Lys Phe Leu Arg Glu Glu Met Gly Val Lys Lys Ile Arg Phe Pro Glu
180 185 190
His Cys Gly Ile Gly Ile Lys Pro Cys Ser Glu Glu Gly Thr Lys Arg
195 200 205
Leu Val Arg Ala Ala Ile Glu Tyr Ala Ile Ala Asn Asp Arg Asp Ser
210 215 220
Val Thr Leu Val His Lys Gly Asn Ile Met Lys Phe Thr Glu Gly Ala
225 230 235 240
Phe Lys Asp Trp Gly Tyr Gln Leu Ala Arg Glu Glu Phe Gly Gly Glu
245 250 255
Leu Ile Asp Gly Gly Pro Trp Leu Lys Val Lys Asn Pro Asn Thr Gly
260 265 270
Lys Glu Ile Val Ile Lys Asp Val Ile Ala Asp Ala Phe Leu Gln Gln
275 280 285
Ile Leu Leu Arg Pro Ala Glu Tyr Asp Val Ile Ala Cys Met Asn Leu
290 295 300
Asn Gly Asp Tyr Ile Ser Asp Ala Leu Ala Ala Gln Val Gly Gly Ile
305 310 315 320
Gly Ile Ala Pro Gly Ala Asn Ile Gly Asp Glu Cys Ala Leu Phe Glu
325 330 335
Ala Thr His Gly Thr Ala Pro Lys Tyr Ala Gly Gln Asp Lys Val Asn
340 345 350
Pro Gly Ser Ile Ile Leu Ser Ala Glu Met Met Leu Arg His Met Gly
355 360 365
Trp Thr Glu Ala Ala Asp Leu Ile Val Lys Gly Met Glu Gly Ala Ile
370 375 380
Asn Ala Lys Thr Val Thr Tyr Asp Phe Glu Arg Leu Met Asp Gly Ala
385 390 395 400
Lys Leu Leu Lys Cys Ser Glu Phe Gly Asp Ala Ile Ile Glu Asn Met
405 410 415
<210>5
<211>2652
<212>DNA
<213>大肠杆菌(Escherichia coli)
<400>5
atgaacgaac aatattccgc attgcgtagt aatgtcagta tgctcggcaa agtgctggga 60
gaaaccatca aggatgcgtt gggagaacac attcttgaac gcgtagaaac tatccgtaag 120
ttgtcgaaat cttcacgcgc tggcaatgat gctaaccgcc aggagttgct caccacctta 180
caaaatttgt cgaacgacga gctgctgccc gttgcgcgtg cgtttagtca gttcctgaac 240
ctggccaaca ccgccgagca ataccacagc atttcgccga aaggcgaagc tgccagcaac 300
ccggaagtga tcgcccgcac cctgcgtaaa ctgaaaaacc agccggaact gagcgaagac 360
accatcaaaa aagcagtgga atcgctgtcg ctggaactgg tcctcacggc tcacccaacc 420
gaaattaccc gtcgtacact gatccacaaa atggtggaag tgaacgcctg tttaaaacag 480
ctcgataaca aagatatcgc tgactacgaa cacaaccagc tgatgcgtcg cctgcgccag 540
ttgatcgccc agtcatggca taccgatgaa atccgtaagc tgcgtccaag cccggtagat 600
gaagccaaat ggggctttgc cgtagtggaa aacagcctgt ggcaaggcgt accaaattac 660
ctgcgcgaac tgaacgaaca actggaagag aacctcggct acaaactgcc cgtcgaattt 720
gttccggtcc gttttacttc gtggatgggc ggcgaccgcg acggcaaccc gaacgtcact 780
gccgatatca cccgccacgt cctgctactc agccgctgga aagccaccga tttgttcctg 840
aaagatattc aggtgctggt ttctgaactg tcgatggttg aagcgacccc tgaactgctg 900
gcgctggttg gcgaagaagg tgccgcagaa ccgtatcgct atctgatgaa aaacctgcgt 960
tctcgcctga tggcgacaca ggcatggctg gaagcgcgcc tgaaaggcga agaactgcca 1020
aaaccagaag gcctgctgac acaaaacgaa gaactgtggg aaccgctcta cgcttgctac 1080
cagtcacttc aggcgtgtgg catgggtatt atcgccaacg gcgatctgct cgacaccctg 1140
cgccgcgtga aatgtttcgg cgtaccgctg gtccgtattg atatccgtca ggagagcacg 1200
cgtcataccg aagcgctggg cgagctgacc cgctacctcg gtatcggcga ctacgaaagc 1260
tggtcagagg ccgacaaaca ggcgttcctg atccgcgaac tgaactccaa acgtccgctt 1320
ctgccgcgca actggcaacc aagcgccgaa acgcgcgaag tgctcgatac ctgccaggtg 1380
attgccgaag caccgcaagg ctccattgcc gcctacgtga tctcgatggc gaaaacgccg 1440
tccgacgtac tggctgtcca cctgctgctg aaagaagcgg gtatcgggtt tgcgatgccg 1500
gttgctccgc tgtttgaaac cctcgatgat ctgaacaacg ccaacgatgt catgacccag 1560
ctgctcaata ttgactggta tcgtggcctg attcagggca aacagatggt gatgattggc 1620
tattccgact cagcaaaaga tgcgggagtg atggcagctt cctgggcgca atatcaggca 1680
caggatgcat taatcaaaac ctgcgaaaaa gcgggtattg agctgacgtt gttccacggt 1740
cgcggcggtt ccattggtcg cggcggcgca cctgctcatg cggcgctgct gtcacaaccg 1800
ccaggaagcc tgaaaggcgg cctgcgcgta accgaacagg gcgagatgat ccgctttaaa 1860
tatggtctgc cagaaatcac cgtcagcagc ctgtcgcttt ataccggggc gattctggaa 1920
gccaacctgc tgccaccgcc ggagccgaaa gagagctggc gtcgcattat ggatgaactg 1980
tcagtcatct cctgcgatgt ctaccgcggc tacgtacgtg aaaacaaaga ttttgtgcct 2040
tacttccgct ccgctacgcc ggaacaagaa ctgggcaaac tgccgttggg ttcacgtccg 2100
gcgaaacgtc gcccaaccgg cggcgtcgag tcactacgcg ccattccgtg gatcttcgcc 2160
tggacgcaaa accgtctgat gctccccgcc tggctgggtg caggtacggc gctgcaaaaa 2220
gtggtcgaag acggcaaaca gagcgagctg gaggctatgt gccgcgattg gccattcttc 2280
tcgacgcgtc tcggcatgct ggagatggtc ttcgccaaag cagacctgtg gctggcggaa 2340
tactatgacc aacgcctggt agacaaagca ctgtggccgt taggtaaaga gttacgcaac 2400
ctgcaagaag aagacatcaa agtggtgctg gcgattgcca acgattccca tctgatggcc 2460
gatctgccgt ggattgcaga gtctattcag ctacggaata tttacaccga cccgctgaac 2520
gtattgcagg ccgagttgct gcaccgctcc cgccaggcag aaaaagaagg ccaggaaccg 2580
gatcctcgcg tcgaacaagc gttaatggtc actattgccg ggattgcggc aggtatgcgt 2640
aataccggct aa 2652
<210>6
<211>883
<212>PRT
<213>大肠杆菌(Escherichia coli)
<400>6
Met Asn Glu Gln Tyr Ser Ala Leu Arg Ser Asn Val Ser Met Leu Gly
1 5 10 15
Lys Val Leu Gly Glu Thr Ile Lys Asp Ala Leu Gly Glu His Ile Leu
20 25 30
Glu Arg Val Glu Thr Ile Arg Lys Leu Ser Lys Ser Ser Arg Ala Gly
35 40 45
Asn Asp Ala Asn Arg Gln Glu Leu Leu Thr Thr Leu Gln Asn Leu Ser
50 55 60
Asn Asp Glu Leu Leu Pro Val Ala Arg Ala Phe Ser Gln Phe Leu Asn
65 70 75 80
Leu Ala Asn Thr Ala Glu Gln Tyr His Ser Ile Ser Pro Lys Gly Glu
85 90 95
Ala Ala Ser Asn Pro Glu Val Ile Ala Arg Thr Leu Arg Lys Leu Lys
100 105 110
Asn Gln Pro Glu Leu Ser Glu Asp Thr Ile Lys Lys Ala Val Glu Ser
115 120 125
Leu Ser Leu Glu Leu Val Leu Thr Ala His Pro Thr Glu Ile Thr Arg
130 135 140
Arg Thr Leu Ile His Lys Met Val Glu Val Asn Ala Cys Leu Lys Gln
145 150 155 160
Leu Asp Asn Lys Asp Ile Ala Asp Tyr Glu His Asn Gln Leu Met Arg
165 170 175
Arg Leu Arg Gln Leu Ile Ala Gln Ser Trp His Thr Asp Glu Ile Arg
180 185 190
Lys Leu Arg Pro Ser Pro Val Asp Glu Ala Lys Trp Gly Phe Ala Val
195 200 205
Val Glu Asn Ser Leu Trp Gln Gly Val Pro Asn Tyr Leu Arg Glu Leu
210 215 220
Asn Glu Gln Leu Glu Glu Asn Leu Gly Tyr Lys Leu Pro Val Glu Phe
225 230 235 240
Val Pro Val Arg Phe Thr Ser Trp Met Gly Gly Asp Arg Asp Gly Asn
245 250 255
Pro Asn Val Thr Ala Asp Ile Thr Arg His Val Leu Leu Leu Ser Arg
260 265 270
Trp Lys Ala Thr Asp Leu Phe Leu Lys Asp Ile Gln Val Leu Val Ser
275 280 285
Glu Leu Ser Met Val Glu Ala Thr Pro Glu Leu Leu Ala Leu Val Gly
290 295 300
Glu Glu Gly Ala Ala Glu Pro Tyr Arg Tyr Leu Met Lys Asn Leu Arg
305 310 315 320
Ser Arg Leu Met Ala Thr Gln Ala Trp Leu Glu Ala Arg Leu Lys Gly
325 330 335
Glu Glu Leu Pro Lys Pro Glu Gly Leu Leu Thr Gln Asn Glu Glu Leu
340 345 350
Trp Glu Pro Leu Tyr Ala Cys Tyr Gln Ser Leu Gln Ala Cys Gly Met
355 360 365
Gly Ile Ile Ala Asn Gly Asp Leu Leu Asp Thr Leu Arg Arg Val Lys
370 375 380
Cys Phe Gly Val Pro Leu Val Arg Ile Asp Ile Arg Gln Glu Ser Thr
385 390 395 400
Arg His Thr Glu Ala Leu Gly Glu Leu Thr Arg Tyr Leu Gly Ile Gly
405 410 415
Asp Tyr Glu Ser Trp Ser Glu Ala Asp Lys Gln Ala Phe Leu Ile Arg
420 425 430
Glu Leu Asn Ser Lys Arg Pro Leu Leu Pro Arg Asn Trp Gln Pro Ser
435 440 445
Ala Glu Thr Arg Glu Val Leu Asp Thr Cys Gln Val Ile Ala Glu Ala
450 455 460
Pro Gln Gly Ser Ile Ala Ala Tyr Val Ile Ser Met Ala Lys Thr Pro
465 470 475 480
Ser Asp Val Leu Ala Val His Leu Leu Leu Lys Glu Ala Gly Ile Gly
485 490 495
Phe Ala Met Pro Val Ala Pro Leu Phe Glu Thr Leu Asp Asp Leu Asn
500 505 510
Asn Ala Asn Asp Val Met Thr Gln Leu Leu Asn Ile Asp Trp Tyr Arg
515 520 525
Gly Leu Ile Gln Gly Lys Gln Met Val Met Ile Gly Tyr Ser Asp Ser
530 535 540
Ala Lys Asp Ala Gly Val Met Ala Ala Ser Trp Ala Gln Tyr Gln Ala
545 550 555 560
Gln Asp Ala Leu Ile Lys Thr Cys Glu Lys Ala Gly Ile Glu Leu Thr
565 570 575
Leu Phe His Gly Arg Gly Gly Ser Ile Gly Arg Gly Gly Ala Pro Ala
580 585 590
His Ala Ala Leu Leu Ser Gln Pro Pro Gly Ser Leu Lys Gly Gly Leu
595 600 605
Arg Val Thr Glu Gln Gly Glu Met Ile Arg Phe Lys Tyr Gly Leu Pro
610 615 620
Glu Ile Thr Val Ser Ser Leu Ser Leu Tyr Thr Gly Ala Ile Leu Glu
625 630 635 640
Ala Asn Leu Leu Pro Pro Pro Glu Pro Lys Glu Ser Trp Arg Arg Ile
645 650 655
Met Asp Glu Leu Ser Val Ile Ser Cys Asp Val Tyr Arg Gly Tyr Val
660 665 670
Arg Glu Asn Lys Asp Phe Val Pro Tyr Phe Arg Ser Ala Thr Pro Glu
675 680 685
Gln Glu Leu Gly Lys Leu Pro Leu Gly Ser Arg Pro Ala Lys Arg Arg
690 695 700
Pro Thr Gly Gly Val Glu Ser Leu Arg Ala Ile Pro Trp Ile Phe Ala
705 710 715 720
Trp Thr Gln Asn Arg Leu Met Leu Pro Ala Trp Leu Gly Ala Gly Thr
725 730 735
Ala Leu Gln Lys Val Val Glu Asp Gly Lys Gln Ser Glu Leu Glu Ala
740 745 750
Met Cys Arg Asp Trp Pro Phe Phe Ser Thr Arg Leu Gly Met Leu Glu
755 760 765
Met Val Phe Ala Lys Ala Asp Leu Trp Leu Ala Glu Tyr Tyr Asp Gln
770 775 780
Arg Leu Val Asp Lys Ala Leu Trp Pro Leu Gly Lys Glu Leu Arg Asn
785 790 795 800
Leu Gln Glu Glu Asp Ile Lys Val Val Leu Ala Ile Ala Asn Asp Ser
805 810 815
His Leu Met Ala Asp Leu Pro Trp Ile Ala Glu Ser Ile Gln Leu Arg
820 825 830
Asn Ile Tyr Thr Asp Pro Leu Asn Val Leu Gln Ala Glu Leu Leu His
835 840 845
Arg Ser Arg Gln Ala Glu Lys Glu Gly Gln Glu Pro Asp Pro Arg Val
850 855 860
Glu Gln Ala Leu Met Val Thr Ile Ala Gly Ile Ala Ala Gly Met Arg
865 870 875 880
Asn Thr Gly
<210>7
<211>1284
<212>DNA
<213>大肠杆菌(Escherichia coli)
<400>7
atggctgata caaaagcaaa actcaccctc aacggggata cagctgttga actggatgtg 60
ctgaaaggca cgctgggtca agatgttatt gatatccgta ctctcggttc aaaaggtgtg 120
ttcacctttg acccaggctt cacttcaacc gcatcctgcg aatctaaaat tacttttatt 180
gatggtgatg aaggtatttt gctgcaccgc ggtttcccga tcgatcagct ggcgaccgat 240
tctaactacc tggaagtttg ttacatcctg ctgaatggtg aaaaaccgac tcaggaacag 300
tatgacgaat ttaaaactac ggtgacccgt cataccatga tccacgagca gattacccgt 360
ctgttccatg ctttccgtcg cgactcgcat ccaatggcag tcatgtgtgg tattaccggc 420
gcgctggcgg cgttctatca cgactcgctg gatgttaaca atcctcgtca ccgtgaaatt 480
gccgcgttcc gcctgctgtc gaaaatgccg accatggccg cgatgtgtta caagtattcc 540
attggtcagc catttgttta cccgcgcaac gatctctcct acgccggtaa cttcctgaat 600
atgatgttct ccacgccgtg cgaaccgtat gaagttaatc cgattctgga acgtgctatg 660
gaccgtattc tgatcctgca cgctgaccat gaacagaacg cctctacctc caccgtgcgt 720
accgctggct cttcgggtgc gaacccgttt gcctgtatcg cagcaggtat tgcttcactg 780
tggggacctg cgcacggcgg tgctaacgaa gcggcgctga aaatgctgga agaaatcagc 840
tccgttaaac acattccgga atttgttcgt cgtgcgaaag acaaaaatga ttctttccgc 900
ctgatgggct tcggtcaccg cgtgtacaaa aattacgacc cgcgcgccac cgtaatgcgt 960
gaaacctgcc atgaagtgct gaaagagctg ggcacgaagg atgacctgct ggaagtggct 1020
atggagctgg aaaacatcgc gctgaacgac ccgtacttta tcgagaagaa actgtacccg 1080
aacgtcgatt tctactctgg tatcatcctg aaagcgatgg gtattccgtc ttccatgttc 1140
accgtcattt tcgcaatggc acgtaccgtt ggctggatcg cccactggag cgaaatgcac 1200
agtgacggta tgaagattgc ccgtccgcgt cagctgtata caggatatga aaaacgcgac 1260
tttaaaagcg atatcaagcg ttaa 1284
<210>8
<211>427
<212>PRT
<213>大肠杆菌(Escherichia coli)
<400>8
Met Ala Asp Thr Lys Ala Lys Leu Thr Leu Asn Gly Asp Thr Ala Val
1 5 10 15
Glu Leu Asp Val Leu Lys Gly Thr Leu Gly Gln Asp Val Ile Asp Ile
20 25 30
Arg Thr Leu Gly Ser Lys Gly Val Phe Thr Phe Asp Pro Gly Phe Thr
35 40 45
Ser Thr Ala Ser Cys Glu Ser Lys Ile Thr Phe Ile Asp Gly Asp Glu
50 55 60
Gly Ile Leu Leu His Arg Gly Phe Pro Ile Asp Gln Leu Ala Thr Asp
65 70 75 80
Ser Asn Tyr Leu Glu Val Cys Tyr Ile Leu Leu Asn Gly Glu Lys Pro
85 90 95
Thr Gln Glu Gln Tyr Asp Glu Phe Lys Thr Thr Val Thr Arg His Thr
100 105 110
Met Ile His Glu Gln Ile Thr Arg Leu Phe His Ala Phe Arg Arg Asp
115 120 125
Ser His Pro Met Ala Val Met Cys Gly Ile Thr Gly Ala Leu Ala Ala
1301 35 140
Phe Tyr His Asp Ser Leu Asp Val Asn Asn Pro Arg His Arg Glu Ile
145 150 155 160
Ala Ala Phe Arg Leu Leu Ser Lys Met Pro Thr Met Ala Ala Met Cys
165 170 175
Tyr Lys Tyr Ser Ile Gly Gln Pro Phe Val Tyr Pro Arg Asn Asp Leu
180 185 190
Ser Tyr Ala Gly Asn Phe Leu Asn Met Met Phe Ser Thr Pro Cys Glu
195 200 205
Pro Tyr Glu Val Asn Pro Ile Leu Glu Arg Ala Met Asp Arg Ile Leu
210 215 220
Ile Leu His Ala Asp His Glu Gln Asn Ala Ser Thr Ser Thr Val Arg
225 230 235 240
Thr Ala Gly Ser Ser Gly Ala Asn Pro Phe Ala Cys Ile Ala Ala Gly
245 250 255
Ile Ala Ser Leu Trp Gly Pro Ala His Gly Gly Ala Asn Glu Ala Ala
260 265 270
Leu Lys Met Leu Glu Glu Ile Ser Ser Val Lys His Ile Pro Glu Phe
275 280 285
Val Arg Arg Ala Lys Asp Lys Asn Asp Ser Phe Arg Leu Met Gly Phe
290 295 300
Gly His Arg Val Tyr Lys Asn Tyr Asp Pro Arg Ala Thr Val Met Arg
305 310 315 320
Glu Thr Cys His Glu Val Leu Lys Glu Leu Gly Thr Lys Asp Asp Leu
325 330 335
Leu Glu Val Ala Met Glu Leu Glu Asn Ile Ala Leu Asn Asp Pro Tyr
340 345 350
Phe Ile Glu Lys Lys Leu Tyr Pro Asn Val Asp Phe Tyr Ser Gly Ile
355 360 365
Ile Leu Lys Ala Met Gly Ile Pro Ser Ser Met Phe Thr Val Ile Phe
370 375 380
Ala Met Ala Arg Thr Val Gly Trp Ile Ala His Trp Ser Glu Met His
385 390 395 400
Ser Asp Gly Met Lys Ile Ala Arg Pro Arg Gln Leu Tyr Thr Gly Tyr
405 410 415
Glu Lys Arg Asp Phe Lys Ser Asp Ile Lys Arg
420 425
<210>9
<211>2676
<212>DNA
<213>大肠杆菌(Escherichia coli)
<400>9
atgtcgtcaa ccctacgaga agccagtaag gacacgttgc aggccaaaga taaaacttac 60
cactactaca gcctgccgct tgctgctaaa tcactgggcg atatcacccg tctacccaag 120
tcactcaaag ttttgctcga aaacctgctg cgctggcagg atggtaactc ggttaccgaa 180
gaggatatcc acgcgctggc aggatggctg aaaaatgccc atgctgaccg tgaaattgcc 240
taccgcccgg caagggtgct gatgcaggac tttaccggcg tacctgccgt tgttgatctg 300
gcggcaatgc gcgaagcggt taaacgcctc ggcggcgata ctgcaaaggt taacccgctc 360
tcaccggtcg acctggtcat tgaccactcg gtgaccgtcg atcgttttgg tgatgatgag 420
gcatttgaag aaaacgtacg cctggaaatg gagcgcaacc acgaacgtta tgtgttcctg 480
aaatggggaa agcaagcgtt cagtcggttt agcgtcgtgc cgccaggcac aggcatttgc 540
catcaggtta acctcgaata tctcggcaaa gcagtgtgga gtgaattgca ggacggtgaa 600
tggattgctt atccggatac actcgttggt actgactcgc acaccaccat gatcaacggc 660
cttggcgtgc tggggtgggg cgttggtggg atcgaagcag aagccgcaat gttaggccag 720
ccggtttcca tgcttatccc ggatgtagtg ggcttcaaac ttaccggaaa attacgtgaa 780
ggtattaccg ccacagacct ggttctcact gttacccaaa tgctgcgcaa acatggcgtg 840
gtggggaaat tcgtcgaatt ttatggtgat ggtctggatt cactaccgtt ggcggatcgc 900
gccaccattg ccaatatgtc gccagaatat ggtgccacct gtggcttctt cccaatcgat 960
gctgtaaccc tcgattacat gcgtttaagc gggcgcagcg aagatcaggt cgagttggtc 1020
gaaaaatatg ccaaagcgca gggcatgtgg cgtaacccgg gcgatgaacc aatttttacc 1080
agtacgttag aactggatat gaatgacgtt gaagcgagcc tggcagggcc taaacgccca 1140
caggatcgcg ttgcactgcc cgatgtacca aaagcatttg ccgccagtaa cgaactggaa 1200
gtgaatgcca cgcataaaga tcgccagccg gtcgattatg ttatgaacgg acatcagtat 1260
cagttacctg atggcgctgt ggtcattgct gcgataacct cgtgcaccaa cacctctaac 1320
ccaagtgtgc tgatggccgc aggcttgctg gcgaaaaaag ccgtaactct gggcctcaag 1380
cggcaaccat gggtcaaagc gtcgctggca ccgggttcga aagtcgtttc tgattatctg 1440
gcaaaagcga aactgacacc gtatctcgac gaactggggt ttaaccttgt gggatacggt 1500
tgtaccacct gtattggtaa ctctgggccg ctgcccgatc ctatcgaaac ggcaatcaaa 1560
aaaagcgatt taaccgtcgg tgcggtgctg tccggcaacc gtaactttga aggccgtatc 1620
catccgctgg ttaaaactaa ctggctggcc tcgccgccgc tggtggttgc ctatgcgctg 1680
gcgggaaata tgaatatcaa cctggcttct gagcctatcg gccatgatcg caaaggcgat 1740
ccggtttatc tgaaagatat ctggccatcg gcacaagaaa ttgcccgtgc ggtagaacaa 1800
gtctccacag aaatgttccg caaagagtac gcagaagttt ttgaaggcac agcagagtgg 1860
aagggaatta acgtcacacg atccgatacc tacggttggc aggaggactc aacctatatt 1920
cgcttatcgc ctttctttga tgaaatgcag gcaacaccag caccagtgga agatattcac 1980
ggtgcgcgga tcctcgcaat gctgggggat tcagtcacca ctgaccatat ctctccggcg 2040
ggcagtatta agcccgacag cccagcgggt cgatatctac aaggtcgggg tgttgagcga 2100
aaagacttta actcctacgg ttcgcggcgt ggtaaccatg aagtgatgat gcgcggcacc 2160
ttcgccaata ttcgcatccg taatgaaatg gtgcctggcg ttgaaggggg gatgacgcgg 2220
catttacctg acagcgacgt agtctctatt tatgatgctg cgatgcgcta taagcaggag 2280
caaacgccgc tggcggtgat tgccgggaaa gagtatggat caggctccag tcgtgactgg 2340
gcggcaaaag gtccgcgtct gcttggtatt cgtgtggtga ttgccgaatc gtttgaacga 2400
attcaccgtt cgaatttaat tggcatgggc atcctgccgc tggaatttcc gcaaggcgta 2460
acgcgtaaaa cgttagggct aaccggggaa gagaagattg atattggcga tctgcaaaac 2520
ctacaacccg gcgcgacggt tccggtgacg cttacgcgcg cggatggtag ccaggaagtc 2580
gtaccctgcc gttgtcgtat cgacaccgcg acggagttga cctactacca gaacgacggc 2640
attttgcatt atgtcattcg taatatgttg aagtaa 2676
<210>10
<211>891
<212>PRT
<213>大肠杆菌(Escherichia coli)
<400>10
Met Ser Ser Thr Leu Arg Glu Ala Ser Lys Asp Thr Leu Gln Ala Lys
1 5 10 15
Asp Lys Thr Tyr His Tyr Tyr Ser Leu Pro Leu Ala Ala Lys Ser Leu
20 25 30
Gly Asp Ile Thr Arg Leu Pro Lys Ser Leu Lys Val Leu Leu Glu Asn
35 40 45
Leu Leu Arg Trp Gln Asp Gly Asn Ser Val Thr Glu Glu Asp Ile His
50 55 60
Ala Leu Ala Gly Trp Leu Lys Asn Ala His Ala Asp Arg Glu Ile Ala
65 70 75 80
Tyr Arg Pro Ala Arg Val Leu Met Gln Asp Phe Thr Gly Val Pro Ala
85 90 95
Val Val Asp Leu Ala Ala Met Arg Glu Ala Val Lys Arg Leu Gly Gly
100 105 110
Asp Thr Ala Lys Val Asn Pro Leu Ser Pro Val Asp Leu Val Ile Asp
115 120 125
His Ser Val Thr Val Asp Arg Phe Gly Asp Asp Glu Ala Phe Glu Glu
130 135 140
Asn Val Arg Leu Glu Met Glu Arg Asn His Glu Arg Tyr Val Phe Leu
145 150 155 160
Lys Trp Gly Lys Gln Ala Phe Ser Arg Phe Ser Val Val Pro Pro Gly
165 170 175
Thr Gly Ile Cys His Gln Val Asn Leu Glu Tyr Leu Gly Lys Ala Val
180 185 190
Trp Ser Glu Leu Gln Asp Gly Glu Trp Ile Ala Tyr Pro Asp Thr Leu
195 200 205
Val Gly Thr Asp Ser His Thr Thr Met Ile Asn Gly Leu Gly Val Leu
210 215 220
Gly Trp Gly Val Gly Gly Ile Glu Ala Glu Ala Ala Met Leu Gly Gln
225 230 235 240
Pro Val Ser Met Leu Ile Pro Asp Val Val Gly Phe Lys Leu Thr Gly
245 250 255
Lys Leu Arg Glu Gly Ile Thr Ala Thr Asp Leu Val Leu Thr Val Thr
260 265 270
Gln Met Leu Arg Lys His Gly Val Val Gly Lys Phe Val Glu Phe Tyr
275 280 285
Gly Asp Gly Leu Asp Ser Leu Pro Leu Ala Asp Arg Ala Thr Ile Ala
290 295 300
Asn Met Ser Pro Glu Tyr Gly Ala Thr Cys Gly Phe Phe Pro Ile Asp
305 310 315 320
Ala Val Thr Leu Asp Tyr Met Arg Leu Ser Gly Arg Ser Glu Asp Gln
325 330 335
Val Glu Leu Val Glu Lys Tyr Ala Lys Ala Gln Gly Met Trp Arg Asn
340 345 350
Pro Gly Asp Glu Pro Ile Phe Thr Ser Thr Leu Glu Leu Asp Met Asn
355 360 365
Asp Val Glu Ala Ser Leu Ala Gly Pro Lys Arg Pro Gln Asp Arg Val
370 375 380
Ala Leu Pro Asp Val Pro Lys Ala Phe Ala Ala Ser Asn Glu Leu Glu
385 390 395 400
Val Asn Ala Thr His Lys Asp Arg Gln Pro Val Asp Tyr Val Met Asn
405 410 415
Gly His Gln Tyr Gln Leu Pro Asp Gly Ala Val Val Ile Ala Ala Ile
420 425 430
Thr Ser Cys Thr Asn Thr Ser Asn Pro Ser Val Leu Met Ala Ala Gly
435 440 445
Leu Leu Ala Lys Lys Ala Val Thr Leu Gly Leu Lys Arg Gln Pro Trp
450 455 460
Val Lys Ala Ser Leu Ala Pro Gly Ser Lys Val Val Ser Asp Tyr Leu
465 470 475 480
Ala Lys Ala Lys Leu Thr Pro Tyr Leu Asp Glu Leu Gly Phe Asn Leu
485 490 495
Val Gly Tyr Gly Cys Thr Thr Cys Ile Gly Asn Ser Gly Pro Leu Pro
500 505 510
Asp Pro Ile Glu Thr Ala Ile Lys Lys Ser Asp Leu Thr Val Gly Ala
515 520 525
Val Leu Ser Gly Asn Arg Asn Phe Glu Gly Arg Ile His Pro Leu Val
530 535 540
Lys Thr Asn Trp Leu Ala Ser Pro Pro Leu Val Val Ala Tyr Ala Leu
545 550 555 560
Ala Gly Asn Met Asn Ile Asn Leu Ala Ser Glu Pro Ile Gly His Asp
565 570 575
Arg Lys Gly Asp Pro Val Tyr Leu Lys Asp Ile Trp Pro Ser Ala Gln
580 585 590
Glu Ile Ala Arg Ala Val Glu Gln Val Ser Thr Glu Met Phe Arg Lys
595 600 605
Glu Tyr Ala Glu Val Phe Glu Gly Thr Ala Glu Trp Lys Gly Ile Asn
610 615 620
Val Thr Arg Ser Asp Thr Tyr Gly Trp Gln Glu Asp Ser Thr Tyr Ile
625 630 635 640
Arg Leu Ser Pro Phe Phe Asp Glu Met Gln Ala Thr Pro Ala Pro Val
645 650 655
Glu Asp Ile His Gly Ala Arg Ile Leu Ala Met Leu Gly Asp Ser Val
660 665 670
Thr Thr Asp His Ile Ser Pro Ala Gly Ser Ile Lys Pro Asp Ser Pro
675 680 685
Ala Gly Arg Tyr Leu Gln Gly Arg Gly Val Glu Arg Lys Asp Phe Asn
690 695 700
Ser Tyr Gly Ser Arg Arg Gly Asn His Glu Val Met Met Arg Gly Thr
705 710 715 720
Phe Ala Asn Ile Arg Ile Arg Asn Glu Met Val Pro Gly Val Glu Gly
725 730 735
Gly Met Thr Arg His Leu Pro Asp Ser Asp Val Val Ser Ile Tyr Asp
740 745 750
Ala Ala Met Arg Tyr Lys Gln Glu Gln Thr Pro Leu Ala Val Ile Ala
755 760 765
Gly Lys Glu Tyr Gly Ser Gly Ser Ser Arg Asp Trp Ala Ala Lys Gly
770 775 780
Pro Arg Leu Leu Gly Ile Arg Val Val Ile Ala Glu Ser Phe Glu Arg
785 790 795 800
Ile His Arg Ser Asn Leu Ile Gly Met Gly Ile Leu Pro Leu Glu Phe
805 810 815
Pro Gln Gly Val Thr Arg Lys Thr Leu Gly Leu Thr Gly Glu Glu Lys
820 825 830
Ile Asp Ile Gly Asp Leu Gln Asn Leu Gln Pro Gly Ala Thr Val Pro
835 840 845
Val Thr Leu Thr Arg Ala Asp Gly Ser Gln Glu Val Val Pro Cys Arg
850 855 860
Cys Arg Ile Asp Thr Ala Thr Glu Leu Thr Tyr Tyr Gln Asn Asp Gly
865 870 875 880
Ile Leu His Tyr Val Ile Arg Asn Met Leu Lys
885 890
<210>11
<211>2598
<212>DNA
<213>大肠杆菌(Escherichia coli)
<400>11
atgctagaag aataccgtaa gcacgtagct gagcgtgccg ctgaggggat tgcgcccaaa 60
cccctggatg caaaccaaat ggccgcactt gtagagctgc tgaaaaaccc gcccgcgggc 120
gaagaagaat tcctgttaga tctgttaacc aaccgtgttc ccccaggcgt cgatgaagcc 180
gcctatgtca aagcaggctt cctggctgct atcgcgaaag gcgaagccaa atcccctctg 240
ctgactccgg aaaaagccat cgaactgctg ggcaccatgc agggtggtta caacattcat 300
ccgctgatcg acgcgctgga tgatgccaaa ctggcaccta ttgctgccaa agcactttct 360
cacacgctgc tgatgttcga taacttctat gacgtagaag agaaagcgaa agcaggcaac 420
gaatatgcga agcaggttat gcagtcctgg gcggatgccg aatggttcct gaatcgcccg 480
gcgctggctg aaaaactgac cgttactgtc ttcaaagtca ctggcgaaac taacaccgat 540
gacctttctc cggcaccgga tgcgtggtca cgcccggata tcccactgca cgcgctggcg 600
atgctgaaaa acgcccgtga aggtattgag ccagaccagc ctggtgttgt tggtccgatc 660
aagcaaatcg aagctctgca acagaaaggt ttcccgctgg cgtacgtcgg tgacgttgtg 720
ggtacgggtt cttcgcgtaa atccgccact aactccgttc tgtggtttat gggcgatgat 780
attccacatg tgccgaacaa acgcggcggt ggtttgtgcc tcggcggtaa aattgcaccc 840
atcttcttta acacgatgga agacgcgggt gcactgccaa tcgaagtcga cgtctctaac 900
ctgaacatgg gcgacgtgat tgacgtttac ccgtacaaag gtgaagtgcg taaccacgaa 960
accggcgaac tgctggcgac cttcgaactg aaaaccgacg tgctgattga tgaagtgcgt 1020
gctggtggcc gtattccgct gattatcggg cgtggcctga ccaccaaagc gcgtgaagca 1080
cttggtctgc cgcacagtga tgtgttccgt caggcgaaag atgtcgctga gagcgatcgc 1140
ggcttctcgc tggcgcaaaa aatggtaggc cgtgcctgtg gcgtgaaagg cattcgtccg 1200
ggcgcgtact gtgaaccgaa aatgacttct gtaggttccc aggacaccac cggcccgatg 1260
acccgtgatg aactgaaaga cctggcgtgc ctgggcttct cggctgacct ggtgatgcag 1320
tctttctgcc acaccgcggc gtatccgaag ccagttgacg tgaacacgca ccacacgctg 1380
ccggacttca ttatgaaccg tggcggtgtg tcgctgcgtc cgggtgacgg cgtcattcac 1440
tcctggctga accgtatgct gctgccggat accgtcggta ccggtggtga ctcccatacc 1500
cgtttcccga tcggtatctc tttcccggcg ggttctggtc tggtggcgtt tgctgccgca 1560
actggcgtaa tgccgcttga tatgccggaa tccgttctgg tgcgcttcaa aggcaaaatg 1620
cagccgggca tcaccctgcg cgatctggta cacgctattc cgctgtatgc gatcaaacaa 1680
ggtctgctga ccgttgagaa gaaaggcaag aaaaacatct tctctggccg catcctggaa 1740
attgaaggtc tgccggatct gaaagttgag caggcctttg agctaaccga tgcgtccgcc 1800
gagcgttctg ccgctggttg taccatcaag ctgaacaaag aaccgatcat cgaatacctg 1860
aactctaaca tcgtcctgct gaagtggatg atcgcggaag gttacggcga tcgtcgtacc 1920
ctggaacgtc gtattcaggg catggaaaaa tggctggcga atcctgagct gctggaagcc 1980
gatgcagatg cggaatacgc ggcagtgatc gacatcgatc tggcggatat taaagagcca 2040
atcctgtgtg ctccgaacga cccggatgac gcgcgtccgc tgtctgcggt acagggtgag 2100
aagatcgacg aagtgtttat cggttcctgc atgaccaaca tcggtcactt ccgtgctgcg 2160
ggtaaactgc tggatgcgca taaaggtcag ttgccgaccc gcctgtgggt ggcaccgcca 2220
acccgtatgg acgccgcaca gttgaccgaa gaaggctact acagcgtctt cggtaagagt 2280
ggtgcgcgta tcgagatccc tggctgttcc ctgtgtatgg gtaaccaggc gcgtgtggcg 2340
gacggtgcaa cggtggtttc cacctctacc cgtaacttcc cgaaccgtct gggtactggc 2400
gcgaatgtct tcctggcttc tgcggaactg gcggctgttg cggcgctgat tggcaaactg 2460
ccgacgccgg aagagtacca gacctacgtg gcgcaggtag ataaaacagc cgttgatact 2520
taccgttatc tgaacttcaa ccagctttct cagtacaccg agaaagccga tggggtgatt 2580
ttccagactg cggtttaa 2598
<210>12
<211>866
<212>PRT
<213>大肠杆菌(Escherichia coli)
<400>12
Met Val Leu Glu Glu Tyr Arg Lys His Val Ala Glu Arg Ala Ala Glu
1 5 10 15
Gly Ile Ala Pro Lys Pro Leu Asp Ala Asn Gln Met Ala Ala Leu Val
20 25 30
Gln Leu Leu Lys Asn Pro Pro Ala Gly Glu Glu Glu Phe Leu Leu Asp
35 40 45
Leu Leu Thr Asn Arg Val Pro Pro Gly Val Asp Glu Ala Ala Tyr Val
50 55 60
Lys Ala Gly Phe Leu Ala Ala Ile Ala Lys Gly Glu Ala Lys Ser Pro
65 70 75 80
Leu Leu Thr Pro Glu Lys Ala Ile Glu Leu Leu Gly Thr Met Gln Gly
85 90 95
Gly Tyr Asn Ile His Pro Leu Ile Asp Ala Leu Asp Asp Ala Lys Leu
100 105 110
Ala Pro Ile Ala Ala Lys Ala Leu Ser His Thr Leu Leu Met Phe Asp
115 120 125
Asn Phe Tyr Asp Val Glu Glu Lys Ala Lys Ala Gly Asn Glu Tyr Ala
130 135 140
Lys Gln Val Met Gln Ser Trp Ala Asp Ala Glu Trp Phe Leu Asn Arg
145 150 155 160
Pro Ala Leu Ala Glu Lys Leu Thr Val Thr Val Phe Lys Val Thr Gly
165 170 175
Glu Thr Asn Thr Asp Asp Leu Ser Pro Ala Pro Asp Ala Trp Ser Arg
180 185 190
Pro Asp Ile Pro Leu His Ala Leu Ala Met Leu Lys Asn Ala Arg Glu
195 200 205
Gly Ile Glu Pro Asp Gln Pro Gly Val Val Gly Pro Ile Lys Gln Ile
210 215 220
Glu Ala Leu Gln Gln Lys Gly Phe Pro Leu Ala Tyr Val Gly Asp Val
225 230 235 240
Val Gly Thr Gly Ser Ser Arg Lys Ser Ala Thr Asn Ser Val Leu Trp
245 250 255
Phe Met Gly Asp Asp Ile Pro His Val Pro Asn Lys Arg Gly Gly Gly
260 265 270
Leu Cys Leu Gly Gly Lys Ile Ala Pro Ile Phe Phe Asn Thr Met Glu
275 280 285
Asp Ala Gly Ala Leu Pro Ile Glu Val Asp Val Ser Asn Leu Asn Met
290 295 300
Gly Asp Val Ile Asp Val Tyr Pro Tyr Lys Gly Glu Val Arg Asn His
305 310 315 320
Glu Thr Gly Glu Leu Leu Ala Thr Phe Glu Leu Lys Thr Asp Val Leu
325 330 335
Ile Asp Glu Val Arg Ala Gly Gly Arg Ile Pro Leu Ile Ile Gly Arg
340 345 350
Gly Leu Thr Thr Lys Ala Arg Glu Ala Leu Gly Leu Pro His Ser Asp
355 360 365
Val Phe Arg Gln Ala Lys Asp Val Ala Glu Ser Asp Arg Gly Phe Ser
370 375 380
Leu Ala Gln Lys Met Val Gly Arg Ala Cys Gly Val Lys Gly Ile Arg
385 390 395 400
Pro Gly Ala Tyr Cys Glu Pro Lys Met Thr Ser Val Gly Ser Gln Asp
405 410 415
Thr Thr Gly Pro Met Thr Arg Asp Glu Leu Lys Asp Leu Ala Cys Leu
420 425 430
Gly Phe Ser Ala Asp Leu Val Met Gln Ser Phe Cys His Thr Ala Ala
435 440 445
Tyr Pro Lys Pro Val Asp Val Asn Thr His His Thr Leu Pro Asp Phe
450 455 460
Ile Met Asn Arg Gly Gly Val Ser Leu Arg Pro Gly Asp Gly Val Ile
465 470 475 480
His Ser Trp Leu Asn Arg Met Leu Leu Pro Asp Thr Val Gly Thr Gly
485 490 495
Gly Asp Ser His Thr Arg Phe Pro Ile Gly Ile Ser Phe Pro Ala Gly
500 505 510
Ser Gly Leu Val Ala Phe Ala Ala Ala Thr Gly Val Met Pro Leu Asp
515 520 525
Met Pro Glu Ser Val Leu Val Arg Phe Lys Gly Lys Met Gln Pro Gly
530 535 540
Ile Thr Leu Arg Asp Leu Val His Ala Ile Pro Leu Tyr Ala Ile Lys
545 550 555 560
Gln Gly Leu Leu Thr Val Glu Lys Lys Gly Lys Lys Asn Ile Phe Ser
565 570 575
Gly Arg Ile Leu Glu Ile Glu Gly Leu Pro Asp Leu Lys Val Glu Gln
580 585 590
Ala Phe Glu Leu Thr Asp Ala Ser Ala Glu Arg Ser Ala Ala Gly Cys
595 600 605
Thr Ile Lys Leu Asn Lys Glu Pro Ile Ile Glu Tyr Leu Asn Ser Asn
610 615 620
Ile Val Leu Leu Lys Trp Met Ile Ala Glu Gly Tyr Gly Asp Arg Arg
625 630 635 640
Thr Leu Glu Arg Arg Ile Gln Gly Met Glu Lys Trp Leu Ala Asn Pro
645 650 655
Glu Leu Leu Glu Ala Asp Ala Asp Ala Glu Tyr Ala Ala Val Ile Asp
660 665 670
Ile Asp Leu Ala Asp Ile Lys Glu Pro Ile Leu Cys Ala Pro Asn Asp
675 680 685
Pro Asp Asp Ala Arg Pro Leu Ser Ala Val Gln Gly Glu Lys Ile Asp
690 695 700
Glu Val Phe Ile Gly Ser Cys Met Thr Asn Ile Gly His Phe Arg Ala
705 710 715 720
Ala Gly Lys Leu Leu Asp Ala His Lys Gly Gln Leu Pro Thr Arg Leu
725 730 735
Trp Val Ala Pro Pro Thr Arg Met Asp Ala Ala Gln Leu Thr Glu Glu
740 745 785
Gly Tyr Tyr Ser Val Phe Gly Lys Ser Gly Ala Arg Ile Glu Ile Pro
755 760 765
Gly Cys Ser Leu Cys Met Gly Asn Gln Ala Arg Val Ala Asp Gly Ala
770 775 780
Thr Val Val Ser Thr Ser Thr Arg Asn Phe Pro Asn Arg Leu Gly Thr
785 790 795 800
Gly Ala Asn Val Phe Leu Ala Ser Ala GIu Leu Ala Ala Val Ala Ala
805 810 815
Leu Ile Gly Lys Leu Pro Thr Pro Glu Glu Tyr Gln Thr Tyr Val Ala
820 825 830
Gln Val Asp Lys Thr Ala Val Asp Thr Tyr Arg Tyr Leu Asn Phe Asn
835 840 845
Gln Leu Ser Gln Tyr Thr Glu Lys Ala Asp Gly Val Ile Phe Gln Thr
850 855 860
Ala Val
865
Claims (31)
1.一种基因修饰微生物,包括:
第一外源性核酸序列,其编码顺式乌头酸脱羧酶(CAD),该第一外源性核酸序列连接至启动子,以及
突变之内源性icd基因,其编码异柠檬酸脱氢酶,该突变之内源性icd基因的柠檬酸脱氢酶表达量低于其野生型的表达量。
2.根据权利要求1所述之基因修饰微生物,其中该微生物选自下组:曲霉(Aspergillus)、柠檬酸杆菌(Citrobacter)、棒状杆菌(Corynebacterium)、德克酵母菌(Dekkera)、肠杆菌(Enterobacter)、肠球菌(Enterococcus)、埃希氏菌(Escherichia)、欧文氏菌(Erwinia)、克雷伯氏菌(Klebsiella)、克鲁维酵母(Kluyveromyces)、乳杆菌(Lactobacillus)、乳球菌(Lactococcus)、摩根氏菌(Morganella)、团泛菌(Pantoea)、果胶杆菌(Pectobacterium)、青霉菌(Penicillum)、毕赤酵母菌(Pichia)、变形杆菌(Proteus)、假单胞菌(Pseudomonas)、二形性酵母菌(Pseudozyma)、红酵母菌(Rhodotorula)、沙门氏菌(Salmonella)、沙雷氏菌(Serratia)、志贺氏菌(Shigella)、酵母菌(Saccharomyces)、黑穗菌(Ustilago)及耶氏酵母菌(Yarrowia)。
3.根据权利要求2所述之基因修饰微生物,其中该微生物包括黑曲霉(Aspergillus niger)、土曲霉(Aspergillus terreus)、大肠杆菌(Escherichia coli)、担子菌类酵母菌(Pseudozyma Antarctica)、解脂耶氏酵母菌(Yarrowialipolytica)或酿酒酵母(Saccharomyces cerevisiae)。
4.根据权利要求1所述之基因修饰微生物,还包括第二外源性核酸序列,其编码选自磷酸烯醇丙酮酸羧化酶、柠檬酸合成酶、2-甲基柠檬酸合成酶、柠檬酸裂解酶、乌头酸酶及2-甲基柠檬酸脱氢酶中的至少一种多肽,且该第二外源性核酸序列连接至启动子。
5.根据权利要求4所述之基因修饰微生物,其中该微生物为大肠杆菌(Escherichia coli),该第二外源性核酸序列编码磷酸烯醇丙酮酸羧化酶、柠檬酸合成酶或乌头酸酶,且该乌头酸酶为乌头酸酶A或乌头酸酶B。
6.根据权利要求4所述之基因修饰微生物,其中该第二外源性核酸序列编码磷酸烯醇丙酮酸羧化酶,且该微生物还包括第三外源性核酸序列,其编码选自柠檬酸合成酶、2-甲基柠檬酸合成酶、柠檬酸裂解酶、乌头酸酶及2-甲基柠檬酸脱氢酶中的至少一种多肽,该第三外源性核酸序列连接至启动子。
7.根据权利要求6所述之基因修饰微生物,其中该微生物为大肠杆菌(Escherichia coli),该第三外源性核酸序列编码柠檬酸合成酶或乌头酸酶,且该乌头酸酶为乌头酸酶A或乌头酸酶B。
8.根据权利要求4所述之基因修饰微生物,其中该第二外源性核酸序列编码柠檬酸合成酶、2-甲基柠檬酸合成酶或柠檬酸裂解酶,且该微生物还包括第三外源性核酸序列,其编码乌头酸酶或2-甲基柠檬酸脱氢酶,该第三外源性核酸序列连接至启动子。
9.根据权利要求8所述之基因修饰微生物,其中该微生物为大肠杆菌(Escherichia coli),该第二外源性核酸序列编码柠檬酸合成酶,该第三外源性核酸序列编码乌头酸酶,且该乌头酸酶为乌头酸酶A或乌头酸酶B。
10.根据权利要求6所述之基因修饰微生物,其中该第三外源性核酸序列编码柠檬酸合成酶、2-甲基柠檬酸合成酶或柠檬酸裂解酶,且该微生物还包括第四外源性核酸序列,其编码乌头酸酶或2-甲基柠檬酸脱氢酶,该第四外源性核酸序列连接至启动子。
11.根据权利要求10所述之基因修饰微生物,其中该微生物为大肠杆菌(Escherichia coli),该第三外源性核酸序列编码柠檬酸合成酶,该第四外源性核酸序列编码乌头酸酶,且该乌头酸酶为乌头酸酶A或乌头酸酶B。
12.一种基因修饰微生物,包括:
第一外源性核酸序列,其编码顺式乌头酸脱羧酶,以及
第二外源性核酸序列,其编码选自磷酸烯醇丙酮酸羧化酶、柠檬酸合成酶、2-甲基柠檬酸合成酶及柠檬酸裂解酶中的至少一种多肽,
其中该第一及第二外源性核酸序列分别连接至启动子。
13.根据权利要求12所述之基因修饰微生物,其中该微生物选自下组:曲霉(Aspergillus)、柠檬酸杆菌(Citrobacter)、棒状杆菌(Corynebacterium)、德克酵母菌(Dekkera)、肠杆菌(Enterobacter)、肠球菌(Enterococcus)、埃希氏菌(Escherichia)、欧文氏菌(Erwinia)、克雷伯氏菌(Klebsiella)、克鲁维酵母(Kluyveromyces)、乳杆菌(Lactobacillus)、乳球菌(Lactococcus)、摩根氏菌(Morganella)、团泛菌(Pantoea)、果胶杆菌(Pectobacterium)、青霉菌(Penicillum)、毕赤酵母菌(Pichia)、变形杆菌(Proteus)、假单胞菌(Pseudomonas)、二形性酵母菌(Pseudozyma)、红酵母菌(Rhodotorula)、沙门氏菌(Salmonella)、沙雷氏菌(Serratia)、志贺氏菌(Shigella)、酵母菌(Saccharomyces)、黑穗菌(Ustilago)及耶氏酵母菌(Yarrowia)。
14.根据权利要求13所述之基因修饰微生物,其中该微生物为黑曲霉(Aspergillus niger)、土曲霉(Aspergillus terreus)、大肠杆菌(Escherichia coli)、担子菌类酵母菌(Pseudozyma Antarctica)、解脂耶氏酵母菌(Yarrowialipolytica)或酿酒酵母(Saccharomyces cerevisiae)。
15.根据权利要求12所述之基因修饰微生物,其中该第二外源性核酸序列编码磷酸烯醇丙酮酸羧化酶,且该微生物还包括第三外源性核酸序列,其编码选自柠檬酸合成酶、2-甲基柠檬酸合成酶、柠檬酸裂解酶、乌头酸酶、及2-甲基柠檬酸脱氢酶中的至少一种多肽,且该第三外源性核酸序列连接至启动子。
16.根据权利要求12所述之基因修饰微生物,其中该第二外源性核酸序列编码柠檬酸合成酶、2-甲基柠檬酸合成酶或柠檬酸裂解酶,且该微生物还包括第三外源性核酸序列,其编码乌头酸酶或2-甲基柠檬酸脱氢酶,且该第三外源性核酸序列连接至启动子。
17.根据权利要求15所述之基因修饰微生物,其中该第三外源性核酸序列编码柠檬酸合成酶、2-甲基柠檬酸合成酶或柠檬酸裂解酶,且该微生物还包括第四外源性核酸序列,其编码乌头酸酶或2-甲基柠檬酸脱氢酶,该第四外源性核酸序列连接至启动子。
18.根据权利要求14所述之基因修饰微生物,其中该微生物为大肠杆菌(Escherichia coli),该第二外源性核酸序列连接至大肠杆菌启动子,且编码磷酸烯醇丙酮酸羧化酶或柠檬酸合成酶。
19.根据权利要求18所述之基因修饰微生物,其中该第二外源性核酸序列编码磷酸烯醇丙酮酸羧化酶,且该微生物还包括第三外源性核酸序列,其编码柠檬酸合成酶或乌头酸酶,该乌头酸酶为乌头酸酶A或乌头酸酶B,且该第三外源性核酸序列连接至大肠杆菌启动子。
20.根据权利要求18所述之基因修饰微生物,其中该第二外源性核酸序列编码柠檬酸合成酶,且该微生物还包括第三外源性核酸序列,其编码乌头酸酶,该乌头酸酶为乌头酸酶A或乌头酸酶B,且该第三外源性核酸序列连接至大肠杆菌启动子。
21.根据权利要求19所述之基因修饰微生物,其中该第二外源性核酸序列编码磷酸烯醇丙酮酸羧化酶,该第三外源性核酸序列编码柠檬酸合成酶,该微生物还包括第四外源性核酸序列,其编码乌头酸酶,该乌头酸酶为乌头酸酶A或乌头酸酶B,且该第四外源性核酸序列连接至大肠杆菌启动子。
22.一种生产衣康酸的方法,包括:
提供基因修饰微生物,其包括编码顺式乌头酸脱羧酶(CAD)之外源性核酸序列,该外源性核酸序列连接至启动子,以及突变之内源性icd基因,其编码异柠檬酸脱氢酶,其中该突变之内源性icd基因的柠檬酸脱氢酶表达量低于其野生型的表达量;
于培养基中培养该基因修饰微生物;以及
由该培养基中分离衣康酸。
23.根据权利要求22所述之生产衣康酸的方法,其中该培养基包括5-80g/L的葡萄糖。
24.根据权利要求22所述之生产衣康酸的方法,其中该基因修饰微生物被透化,且该培养基包括5-80g/L的柠檬酸。
25.根据权利要求22所述之生产衣康酸的方法,其中该培养基包括5-80g/L的甘油。
26.根据权利要求22所述之生产衣康酸的方法,其中该基因修饰微生物还包括第二外源性核酸序列,其编码选自磷酸烯醇丙酮酸羧化酶、柠檬酸合成酶、2-甲基柠檬酸合成酶、柠檬酸裂解酶、乌头酸酶及2-甲基柠檬酸脱氢酶中的至少一种多肽,且该第二外源性核酸序列连接至启动子。
27.根据权利要求26所述之生产衣康酸的方法,其中该第二外源性核酸序列编码磷酸烯醇丙酮酸羧化酶,且该基因修饰微生物还包括第三外源性核酸序列,其编码选自柠檬酸合成酶、2-甲基柠檬酸合成酶、柠檬酸裂解酶、乌头酸酶及2-甲基柠檬酸脱氢酶中的至少一种多肽,该第三外源性核酸序列连接至启动子。
28.一种生产衣康酸的方法,包括:
提供基因修饰微生物,其包括第一外源性核酸序列,其编码顺式乌头酸脱羧酶,以及第二外源性核酸序列,其编码选自磷酸烯醇丙酮酸羧化酶、柠檬酸合成酶、2-甲基柠檬酸合成酶及柠檬酸裂解酶中的至少一种多肽,其中该第一及第二外源性核酸序列分别连接至启动子;
于培养基中培养该基因修饰微生物;以及
由该培养基中分离衣康酸。
29.根据权利要求28所述之生产衣康酸的方法,其中该培养基包括5-80g/L的葡萄糖。
30.根据权利要求28所述之生产衣康酸的方法,其中该基因修饰微生物被透化,且该培养基包括5-80g/L的柠檬酸。
31.根据权利要求28所述之生产衣康酸的方法,其中该培养基包括5-80g/L的甘油。
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