CN111235191B - 一种微生物合成乙酰氨基酚的方法 - Google Patents

一种微生物合成乙酰氨基酚的方法 Download PDF

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CN111235191B
CN111235191B CN202010065334.2A CN202010065334A CN111235191B CN 111235191 B CN111235191 B CN 111235191B CN 202010065334 A CN202010065334 A CN 202010065334A CN 111235191 B CN111235191 B CN 111235191B
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郭道义
潘虹
孔思佳
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Abstract

本发明公开了一种在微生物合成乙酰氨基酚的方法，以单糖或甘油为原料产乙酰氨基酚，其包括如下步骤：将表达aroGfbr pabA、pabB、pabC和Mnx1基因的微生物以单糖和/或甘油作为底物进行发酵得到含有对氨基苯酚的发酵产物；所述含有对氨基苯酚的发酵产物在表达NAT基因的微生物的作用下与乙酰辅酶A反应合成乙酰氨基酚。本发明可实现由单糖或甘油到乙酰氨基酚的微生物转化，为大规模的生物合成乙酰氨基酚提供了一个可行道路。

Description

一种微生物合成乙酰氨基酚的方法
技术领域
本发明属于微生物代谢工程领域，具体涉及改造微生物代谢途径，从单糖或甘油发酵生物合成乙酰氨基酚的方法。
背景技术
乙酰氨基酚是使用最为广泛的镇痛和退热一线药物，又名扑热息痛(paracetamol)是非那西丁(phenacetin)的体内代谢产物，属于苯胺类。通过抑制下丘脑体温调节中枢***素合成酶，减少***素PGE1的合成和释放，导致外周血管扩张、出汗而达到解热的作用，其解热作用强度与阿司匹林相似，但无明显的抗炎作用；通过抑制***素PGE1、缓激肽和组胺等的合成和释放，提高痛阈而起到镇痛作用，属于外周性镇痛药，作用较阿司匹林弱，仅对轻、中度疼痛有效。
目前已商品化的乙酰氨基酚均为通过化学法合成。化学法合成生长素需涉及到多个反应步骤导致合成成本较高。通过生物技术手段，实现微生物发酵单糖或甘油合成乙酰氨基酚，可有效的降低成本。
发明内容
本发明的目的在于提供一种微生物体合成乙酰氨基酚的方法。
为实现上述目的，本发明提供了在大肠杆菌合成乙酰氨基酚的方法，生物合成路径图如图1所示，主要步骤为：
(1)构建产对氨基苯酚工程菌GDY1
将来自大肠杆菌的经人工合成解反馈抑制aroGfbr(3-脱氧-2-***庚酮糖-7-磷酸合成酶,3-deoxy-D-arabinoheptulosonate-7-phosphate synthase)基因重组到载体pBBR1MCS1上，得到载体pDY001。将来自大肠杆菌的pabA(氨基脱氧分支酸合酶II,aminodeoxychorismate synthase,subunit II)、pabB(氨基脱氧分支酸合酶I,aminodeoxychorismate synthase,subunit I)、pabC(氨基脱氧分支酸裂解酶,4-amino-4-deoxychorismate lyase component of para-aminobenzoate synthase)及来自Candidaparapsilosis菌的经密码子人工合成Mnx1(黄素蛋白单加氧酶,flavoproteinmonooxygenase)基因，重组到载体pET28a(+)上，得到载体pDY002。将载体pDY001和pDY002转入到大肠杆菌BL21(DE3)中，实现上述基因在大肠杆菌中的高量表达，得到大肠杆菌工程菌株GDY1。表达解除反馈抑制的aroGfbr基因用于加强大肠杆菌胞内分支酸的合成。表达pabA、pabB和pabC用于催化分支酸转化为对氨基苯甲酸，表达Mnx1用于催化对氨基苯甲酸转化为对氨基苯酚。上述构建的GDY1工程菌可有效的将单糖或甘油转化为对氨基苯酚。
(2)构建产对乙酰氨基苯甲酸工程菌GDY2
将来自大肠杆菌的pabA、pabB、pabC及来自Pseudomonas aeruginosa菌的经密码子人工合成NAT(芳胺乙酰转移酶,arylamine N-acetyltransferase)基因，重组到载体pET28a(+)上，得到载体pDY003。将载体pDY001和pDY003转入到大肠杆菌BL21(DE3)中，实现上述基因在大肠杆菌中的高量表达，得到大肠杆菌工程菌株GDY2。表达解除反馈抑制的aroGfbr基因用于加强大肠杆菌胞内分支酸的合成。表达pabA、pabB和pabC用于催化分支酸转化为对氨基苯甲酸，表达NAT用于催化对氨基苯甲酸转化为对乙酰氨基苯甲酸。上述构建的GDY2工程菌可有效的将单糖或甘油转化为对乙酰氨基苯甲酸。
(3)构建产乙酰氨基酚工程菌GDY3
将来自Pseudomonas aeruginosa菌的经密码子优化人工合成NAT基因，重组到载体pET28a(+)上，得到载体pDY003。将pDY003转入到大肠杆菌BL21(DE3)中，实现NAT基因在大肠杆菌中的高量表达，得到大肠杆菌工程菌株GDY3。表达NAT基因基因用于催化将GDY1工程菌生成的对氨基苯酚转化乙酰氨基酚。上述构建的GDY3工程菌可有效的将GDY1工程菌发酵生成的对氨基苯酚转化为乙酰氨基酚。
本发明通过两步发酵，最终实现以单糖或甘油为原料合成乙酰氨基酚，不需要通过过多的化学合成反应，减少化学合成过程中有毒物质的释放，也减少了合成成本。同时，由于微生物生长速度快，遗传操作技术成熟，且发酵过程不会对环境造成污染破坏。
附图说明
图1.乙酰氨基酚生物合成路径图
图2.GC-MS分析发酵产物；1号峰，苯甲酸(内标)；2号峰，对氨基苯酚；3号峰，对氨基苯甲酸；4号峰，乙酰氨基酚；5号峰，乙酰氨基苯甲酸
图3.对氨基苯酚GC-MS鉴定。图2中2号峰离子碎片(上)与标准库对氨基苯酚离子碎片(下)比对。
图4.对乙酰氨基苯甲酸GC-MS鉴定。图2中5号峰离子碎片(上)与标准库对乙酰氨基苯甲酸离子碎片(下)比对。
图5.乙酰氨基酚GC-MS鉴定。图2中4号峰离子碎片(上)与标准库乙酰氨基酚离子碎片(下)比对。
具体实施例
以下实施例用于进一步说明本发明，但不应理解为对本发明的限制。
实施例1构建产对氨基苯酚大肠杆菌工程菌
1、载体pDY001的构建
利用引物aroGfbr-XbaI(GTATCTAGAAAGAGGAGATATAATGAATTATCAGAACGACGATTTACGC)和aroGfbr-SpeI-BamHI(TATGGATCCACTAGTTTACCCGCGA CGCGCTTTTA)PCR扩增人工合成的大肠杆菌aroGfbr基因，随后将扩增的片段以XbaI和BamHI***到pET28a(+)中得到载体pDY01。利用XbaI和NheI双酶切pDY01得到aroGfbr表达框，将其***以XbaI和XhoI双酶切的载体pBBR1MCS1上，得到载体pDY001。所述aroGfbr基因具有SEQ ID NO：1所示的核苷酸序列；所述aroGfbr基因具有与SEQ ID NO：2所示的氨基酸序列；
2、载体pDY002的构建
利用引物pabA-XbaI(AGCTCTAGATTTAAGAAGGAGATATAATGATCCTGCTTATAGATAACTACGATTCTTT)和pabA-SpeI-BamHI(ACAGGATCCACTAGTTCAG CGATGCAGGAAATTAGCC)PCR扩增来自大肠杆菌的pabA基因，随后将扩增的片段以XbaI和BamHI***到pET28a(+)中得到载体pDY02。利用引物pabB-XbaI(ATCTCTAGATTTAAGAAGGAGATATAATGAAGACGTTATCTCCCGCTGTG)和pabB-SpeI-BamHI(ACAGGATCCACTAGTTTACTTCTCCAGT TGCTTCAGGATACG)PCR扩增来自大肠杆菌的pabB基因，利用XbaI和BamHI***到pET28a(+)中得到载体pDY03。利用引物pabC-XbaI(AGCTCTAGATTTAAGAAGGAGATATAATGTTCTTAATTAACGGTCATAAGCAGG)和pabC-SpeI-BamHI(ACAGGATCCACTAGTCTAATTCGGGCGCTCACAAAGT)PCR扩增来自大肠杆菌的pabC基因，随后将扩增的片段以XbaI和BamHI***到pET28a(+)中得到载体pDY04。利用引物Mnx1-XbaI(ATCTCTAG ATTTAAGAAGGAGATATAATGGCCGTGCAAGCCCC)和Mnx1-SpeI-BamHI(TCA GGATCCACTAGTTTAGCCGCTCGCGCTCAGTG)PCR扩增来密码子优化人工合成的Mnx1基因，随后将扩增的片段以XbaI和BamHI***到pET28a(+)中得到载体pDY05。利用XbaI和XhoI双酶切pDY03得到pabB表达框，将其***以SpeI和XhoI双酶切的载体pDY02上，得到载体pDY06。利用XbaI和XhoI双酶切pDY04得到pabC表达框，将其***以SpeI和XhoI双酶切的载体pDY06上，得到载体pDY07。利用XbaI和XhoI双酶切pDY05得到Mnx1表达框，将其***以SpeI和XhoI双酶切的载体pDY07上，得到载体pDY002。构建的pDY002可实现pabA、pabB、pabC和Mnx1基因的共表达。所述pabA基因具有SEQ ID NO：3所示的核苷酸序列编码；所述pabA基因具有SEQ ID NO：4所示的氨基酸序列；所述pabB基因具有SEQ ID NO：5所示的核苷酸序列编码；所述pabB基因具有SEQ ID NO：6所示的氨基酸序列；所述pabC基因具有SEQ ID NO：7所示的核苷酸序列编码；所述pabC基因具有SEQ ID NO：8所示的氨基酸序列；所述Mnx1基因具有SEQ ID NO：9所示的核苷酸序列编码；所述Mnx1基因具有SEQ ID NO：10所示的氨基酸序列；
3.将重组载体pDY001和pDY002同时导入大肠杆菌中，得到GDY1工程菌。该工程菌可实现aroGfbr、pabA、pabB、pabC和Mnx1基因的高表达，可有效的将单糖或甘油转化为对氨基苯酚。
实施例2构建产对乙酰氨基苯甲酸大肠杆菌工程菌
1、载体pDY003的构建
利用引物NAT-XbaI(AACTCTAGATTTAAGAAGGAGATATAATGACGCCGCTGAC CCCA)和NAT-SpeI-BamHI(ATAGGATCCACTAGTTTACGCACTAATCAGACC CGCCA)PCR扩增经密码子优化人工合成的Pseudomonas aeruginosa NAT基因，随后将扩增的片段以XbaI和BamHI***到pET28a(+)中得到载体pDY003。所述NAT基因具有SEQ ID NO：11所示的核苷酸序列编码；所述NAT基因编码的蛋白具有SEQ ID NO：12所示的氨基酸序列；
2、载体pDY004的构建
利用XbaI和XhoI双酶切pDY003得到NAT表达框，将其***以SpeI和XhoI双酶切的载体pDY07上，得到载体pDY004。
3.将重组载体pDY003和pDY004同时导入大肠杆菌中，得到GDY2工程菌。该工程菌可实现aroGfbr、pabA、pabB、pabC和NAT基因的高表达，可有效的将单糖或甘油转化为对乙酰氨基苯甲酸。
实施例3构建以对氨基苯酚为底物合成乙酰氨基酚大肠杆菌工程菌
1、由于NAT具有催化具有催化对氨基苯甲酸转化为对乙酰氨基苯甲酸和催化对氨基苯酚转化为乙酰氨基酚的能力。为避免副产物对乙酰氨基苯甲酸的生成，我采用两步发酵法合成乙酰氨基酚。
2.将重组载体pDY003同时导入大肠杆菌中，得到GDY3工程菌。该工程菌可实现NAT基因的高表达，可有效的将GDY1发酵生成的对氨基苯酚转化为乙酰氨基酚。
实施例4大肠杆菌工程菌GDY1合成对氨基苯酚的发酵实验
1、将37度LB中过夜培养的大肠杆菌工程菌GDY1，接种到50ml M9发酵培养基中(包含2％葡萄糖)，进行30度摇瓶发酵，待菌体OD600为0.8左右，加入0.1mM IPTG进行目的基因的诱导表达。
2、发酵培养24小时后收集菌体，超声波破细胞，利用乙酸乙酯萃取产物。
3、利用旋转蒸发仪将溶剂乙酸乙酯旋干，再用一定量的乙酸乙酯溶解产物。
4、GC-MS(气相色谱-质谱联用仪)检测样品。
实验结果：大肠杆菌工程菌利用葡萄糖合成对氨基苯酚的GC-MS检测结果如图2B。图2A为对照空白菌株GC-MS。对氨基苯酚GC-MS鉴定如图3所示。
实施例5大肠杆菌工程菌GDY2合成对乙酰氨基苯甲酸的发酵实验
5、将37度LB中过夜培养的大肠杆菌工程菌GDY2，接种到50ml M9发酵培养基中(包含2％葡萄糖)，进行30度摇瓶发酵，待菌体OD600为0.8左右，加入0.1mM IPTG进行目的基因的诱导表达。
6、发酵培养24小时后收集菌体，超声波破细胞，利用乙酸乙酯萃取产物。
7、利用旋转蒸发仪将溶剂乙酸乙酯旋干，再用一定量的乙酸乙酯溶解产物。
8、GC-MS(气相色谱-质谱联用仪)检测样品。
实验结果：大肠杆菌工程菌利用葡萄糖合成对乙酰氨基苯甲酸的GC-MS检测结果如图2C。乙酰氨基苯甲酸GC-MS鉴定如图4所示。
实施例6大肠杆菌工程菌GDY3以工程菌GDY1发酵合成的对氨基苯酚为前体底物合成乙酰氨基酚的发酵实验
1、乙酸乙酯萃取工程菌GDY1发酵合成的对氨基苯酚。利用旋转蒸发仪将溶剂乙酸乙酯旋干，随后用一定量的蒸馏水重新溶解对氨基苯酚。
2、将37度LB中过夜培养的大肠杆菌工程菌GDY3，接种到50ml M9发酵培养基中(包含2％葡萄糖)，进行30度摇瓶发酵，待菌体OD600为0.8左右，加入0.1mM IPTG进行目的基因的诱导表达。随后加入乙酸乙酯萃取工程菌GDY1发酵合成的对氨基苯酚。
3、发酵培养8小时后收集菌体，超声波破细胞，利用乙酸乙酯萃取产物。
4、利用旋转蒸发仪将溶剂乙酸乙酯旋干，再用一定量的乙酸乙酯溶解产物。
5、GC-MS(气相色谱-质谱联用仪)检测样品。
实验结果：大肠杆菌工程菌GDY3以工程菌GDY1发酵合成的对氨基苯酚为前体底物合成乙酰氨基酚的发酵实验的GC-MS检测结果如图2D所示。乙酰氨基酚GC-MS鉴定如图5所示。
以上所述仅是本发明的优选实施方案，应当指出，对于本技术领域的技术人员，在不脱离本发明原理的前提下，还可以做出适当的改进，这些改进也应视为本发明的保护范围。
序列表
<110> 赣南师范大学
<120> 一种微生物合成乙酰氨基酚的方法
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1053
<212> DNA
<213> 未知(Unknown)
<400> 1
atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60
gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120
aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180
tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240
gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300
acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360
gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420
gcaggtgagt ttctcaatat gatcacccca caatatctcg ctgacctgat gagctggggc 480
gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540
tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600
aatgccgccg gtgcgccgca ctgcttcctg tccgtaacga aatgggggca ttcggcgatt 660
gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720
tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780
caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840
gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900
gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960
ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020
ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053
<210> 2
<211> 350
<212> PRT
<213> 未知(Unknown)
<400> 2
Met Asn Tyr Gln Asn Asp Asp Leu Arg Ile Lys Glu Ile Lys Glu Leu
1 5 10 15
Leu Pro Pro Val Ala Leu Leu Glu Lys Phe Pro Ala Thr Glu Asn Ala
20 25 30
Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys
35 40 45
Gly Asn Asp Asp Arg Leu Leu Val Val Ile Gly Pro Cys Ser Ile His
50 55 60
Asp Pro Val Ala Ala Lys Glu Tyr Ala Thr Arg Leu Leu Ala Leu Arg
65 70 75 80
Glu Glu Leu Lys Asp Glu Leu Glu Ile Val Met Arg Val Tyr Phe Glu
85 90 95
Lys Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro His
100 105 110
Met Asp Asn Ser Phe Gln Ile Asn Asp Gly Leu Arg Ile Ala Arg Lys
115 120 125
Leu Leu Leu Asp Ile Asn Asp Ser Gly Leu Pro Ala Ala Gly Glu Phe
130 135 140
Leu Asn Met Ile Thr Pro Gln Tyr Leu Ala Asp Leu Met Ser Trp Gly
145 150 155 160
Ala Ile Gly Ala Arg Thr Thr Glu Ser Gln Val His Arg Glu Leu Ala
165 170 175
Ser Gly Leu Ser Cys Pro Val Gly Phe Lys Asn Gly Thr Asp Gly Thr
180 185 190
Ile Lys Val Ala Ile Asp Ala Ile Asn Ala Ala Gly Ala Pro His Cys
195 200 205
Phe Leu Ser Val Thr Lys Trp Gly His Ser Ala Ile Val Asn Thr Ser
210 215 220
Gly Asn Gly Asp Cys His Ile Ile Leu Arg Gly Gly Lys Glu Pro Asn
225 230 235 240
Tyr Ser Ala Lys His Val Ala Glu Val Lys Glu Gly Leu Asn Lys Ala
245 250 255
Gly Leu Pro Ala Gln Val Met Ile Asp Phe Ser His Ala Asn Ser Ser
260 265 270
Lys Gln Phe Lys Lys Gln Met Asp Val Cys Ala Asp Val Cys Gln Gln
275 280 285
Ile Ala Gly Gly Glu Lys Ala Ile Ile Gly Val Met Val Glu Ser His
290 295 300
Leu Val Glu Gly Asn Gln Ser Leu Glu Ser Gly Glu Pro Leu Ala Tyr
305 310 315 320
Gly Lys Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Asp Thr Asp Ala
325 330 335
Leu Leu Arg Gln Leu Ala Asn Ala Val Lys Ala Arg Arg Gly
340 345 350
<210> 3
<211> 564
<212> DNA
<213> 未知(Unknown)
<400> 3
atgatcctgc ttatagataa ctacgattct tttacctgga acctctacca gtacttttgt 60
gaactggggg cggatgtgct ggttaagcgc aacgatgcgt tgacgctggc ggatatcgac 120
gcccttaaac cacaaaaaat tgtcatctca cctggcccct gtacgccaga tgaagccggg 180
atctcccttg acgttattcg ccactatgcc gggcgcttgc cgattcttgg cgtctgcctc 240
ggtcatcagg caatggcgca ggcatttggc ggtaaagttg tgcgcgccgc aaaggtcatg 300
cacggcaaaa cctcgccgat tacacataac ggtgagggcg tatttcgggg gctggcaaat 360
ccacttaccg tgacacgcta ccattcgctg gtggtggaac ctgactcatt accagcgtgc 420
tttgacgtga cggcctggag cgaaacccga gagattatgg ggattcgcca tcgccagtgg 480
gatctggaag gtgtgcagtt ccatccagaa agtattctta gcgaacaagg acatcaactg 540
ctggctaatt tcctgcatcg ctga 564
<210> 4
<211> 187
<212> PRT
<213> 未知(Unknown)
<400> 4
Met Ile Leu Leu Ile Asp Asn Tyr Asp Ser Phe Thr Trp Asn Leu Tyr
1 5 10 15
Gln Tyr Phe Cys Glu Leu Gly Ala Asp Val Leu Val Lys Arg Asn Asp
20 25 30
Ala Leu Thr Leu Ala Asp Ile Asp Ala Leu Lys Pro Gln Lys Ile Val
35 40 45
Ile Ser Pro Gly Pro Cys Thr Pro Asp Glu Ala Gly Ile Ser Leu Asp
50 55 60
Val Ile Arg His Tyr Ala Gly Arg Leu Pro Ile Leu Gly Val Cys Leu
65 70 75 80
Gly His Gln Ala Met Ala Gln Ala Phe Gly Gly Lys Val Val Arg Ala
85 90 95
Ala Lys Val Met His Gly Lys Thr Ser Pro Ile Thr His Asn Gly Glu
100 105 110
Gly Val Phe Arg Gly Leu Ala Asn Pro Leu Thr Val Thr Arg Tyr His
115 120 125
Ser Leu Val Val Glu Pro Asp Ser Leu Pro Ala Cys Phe Asp Val Thr
130 135 140
Ala Trp Ser Glu Thr Arg Glu Ile Met Gly Ile Arg His Arg Gln Trp
145 150 155 160
Asp Leu Glu Gly Val Gln Phe His Pro Glu Ser Ile Leu Ser Glu Gln
165 170 175
Gly His Gln Leu Leu Ala Asn Phe Leu His Arg
180 185
<210> 5
<211> 1362
<212> DNA
<213> 未知(Unknown)
<400> 5
atgaagacgt tatctcccgc tgtgattact ttactctggc gtcaggacgc cgctgaattt 60
tatttctccc gcttaagcca cctgccgtgg gcgatgcttt tacactccgg ctatgccgat 120
catccgtata gccgctttga tattgtggtc gccgagccga tttgcacttt aaccactttc 180
ggtaaagaaa ccgttgttag tgaaagcgaa aaacgcacaa cgaccactga tgacccgcta 240
caggtgctcc agcaggtgct ggatcgcgca gacattcgcc caacgcataa cgaagatttg 300
ccatttcagg gcggcgcact ggggttgttt ggctacgatc tgggccgccg ttttgagtca 360
ctgccagaaa ttgcggaaca agatatcgtt ctgccggata tggcagtggg tatctacgat 420
tgggcgctca ttgtcgacca ccagcgtcat acagtttctt tgctgagtca taatgatgtc 480
aatgcccgtc gggcctggct ggaaagccag caattctcgc cgcaggaaga tttcacgctc 540
acttccgact ggcaatccaa tatgacccgc gagcagtacg gcgaaaaatt tcgccaggta 600
caggaatatc tgcacagcgg tgattgctat caggtgaatc tcgcccaacg ttttcatgcg 660
acctattctg gcgatgaatg gcaggcattc cttcagctta atcaggccaa ccgcgcgcca 720
tttagcgctt ttttacgtct tgaacagggt gcaattttaa gcctttcgcc agagcggttt 780
attctttgtg ataatagtga aatccagacc cgcccgatta aaggcacgct accacgcctg 840
cccgatcctc aggaagatag caaacaagca gtaaaactgg cgaactcagc gaaagatcgt 900
gccgaaaatc tgatgattgt cgatttaatg cgtaatgata tcggtcgtgt tgccgtagca 960
ggttcggtaa aagtaccaga gctgttcgtg gtggaaccct tccctgccgt gcatcatctg 1020
gtcagcacca taacggcgca actaccagaa cagttacacg ccagcgatct gctgcgcgca 1080
gcttttcctg gtggctcaat aaccggggct ccgaaagtac gggctatgga aattatcgac 1140
gaactggaac cgcagcgacg caatgcctgg tgcggcagca ttggctattt gagcttttgc 1200
ggcaacatgg ataccagtat tactatccgc acgctgactg ccattaacgg acaaattttc 1260
tgctctgcgg gcggtggaat tgtcgccgat agccaggaag aagcggaata tcaggaaact 1320
tttgataaag ttaatcgtat cctgaagcaa ctggagaagt aa 1362
<210> 6
<211> 453
<212> PRT
<213> 未知(Unknown)
<400> 6
Met Lys Thr Leu Ser Pro Ala Val Ile Thr Leu Leu Trp Arg Gln Asp
1 5 10 15
Ala Ala Glu Phe Tyr Phe Ser Arg Leu Ser His Leu Pro Trp Ala Met
20 25 30
Leu Leu His Ser Gly Tyr Ala Asp His Pro Tyr Ser Arg Phe Asp Ile
35 40 45
Val Val Ala Glu Pro Ile Cys Thr Leu Thr Thr Phe Gly Lys Glu Thr
50 55 60
Val Val Ser Glu Ser Glu Lys Arg Thr Thr Thr Thr Asp Asp Pro Leu
65 70 75 80
Gln Val Leu Gln Gln Val Leu Asp Arg Ala Asp Ile Arg Pro Thr His
85 90 95
Asn Glu Asp Leu Pro Phe Gln Gly Gly Ala Leu Gly Leu Phe Gly Tyr
100 105 110
Asp Leu Gly Arg Arg Phe Glu Ser Leu Pro Glu Ile Ala Glu Gln Asp
115 120 125
Ile Val Leu Pro Asp Met Ala Val Gly Ile Tyr Asp Trp Ala Leu Ile
130 135 140
Val Asp His Gln Arg His Thr Val Ser Leu Leu Ser His Asn Asp Val
145 150 155 160
Asn Ala Arg Arg Ala Trp Leu Glu Ser Gln Gln Phe Ser Pro Gln Glu
165 170 175
Asp Phe Thr Leu Thr Ser Asp Trp Gln Ser Asn Met Thr Arg Glu Gln
180 185 190
Tyr Gly Glu Lys Phe Arg Gln Val Gln Glu Tyr Leu His Ser Gly Asp
195 200 205
Cys Tyr Gln Val Asn Leu Ala Gln Arg Phe His Ala Thr Tyr Ser Gly
210 215 220
Asp Glu Trp Gln Ala Phe Leu Gln Leu Asn Gln Ala Asn Arg Ala Pro
225 230 235 240
Phe Ser Ala Phe Leu Arg Leu Glu Gln Gly Ala Ile Leu Ser Leu Ser
245 250 255
Pro Glu Arg Phe Ile Leu Cys Asp Asn Ser Glu Ile Gln Thr Arg Pro
260 265 270
Ile Lys Gly Thr Leu Pro Arg Leu Pro Asp Pro Gln Glu Asp Ser Lys
275 280 285
Gln Ala Val Lys Leu Ala Asn Ser Ala Lys Asp Arg Ala Glu Asn Leu
290 295 300
Met Ile Val Asp Leu Met Arg Asn Asp Ile Gly Arg Val Ala Val Ala
305 310 315 320
Gly Ser Val Lys Val Pro Glu Leu Phe Val Val Glu Pro Phe Pro Ala
325 330 335
Val His His Leu Val Ser Thr Ile Thr Ala Gln Leu Pro Glu Gln Leu
340 345 350
His Ala Ser Asp Leu Leu Arg Ala Ala Phe Pro Gly Gly Ser Ile Thr
355 360 365
Gly Ala Pro Lys Val Arg Ala Met Glu Ile Ile Asp Glu Leu Glu Pro
370 375 380
Gln Arg Arg Asn Ala Trp Cys Gly Ser Ile Gly Tyr Leu Ser Phe Cys
385 390 395 400
Gly Asn Met Asp Thr Ser Ile Thr Ile Arg Thr Leu Thr Ala Ile Asn
405 410 415
Gly Gln Ile Phe Cys Ser Ala Gly Gly Gly Ile Val Ala Asp Ser Gln
420 425 430
Glu Glu Ala Glu Tyr Gln Glu Thr Phe Asp Lys Val Asn Arg Ile Leu
435 440 445
Lys Gln Leu Glu Lys
450
<210> 7
<211> 810
<212> DNA
<213> 未知(Unknown)
<400> 7
atgttcttaa ttaacggtca taagcaggaa tcgctggcag taagcgatcg ggcaacgcag 60
tttggtgatg gttgttttac caccgccaga gttatcgacg gtaaagtcag tttgttatcg 120
gcgcatatcc agcgactaca ggatgcttgt cagcggttga tgatttcctg tgacttctgg 180
cctcagcttg aacaagagat gaaaacgctg gcagcagaac agcaaaatgg tgtgctgaaa 240
gtcgtgatca gtcgcggtag tggcgggcga gggtacagca cattgaacag cggaccggca 300
acgcggattc tctccgttac ggcttatcct gcacattacg accgtttgcg taacgagggg 360
attacgttgg cgctaagccc ggtgcggctg gggcgcaatc ctcatcttgc aggtattaaa 420
catctcaatc gtcttgagca agtattgatt cgctctcatc ttgagcagac aaacgctgat 480
gaggcgctgg tccttgacag cgaagggtgg gttacggaat gctgtgcggc taatttgttc 540
tggcggaagg gcaacgtagt ttatacgccg cgactggatc aggcaggtgt taacggcatt 600
atgcgacaat tctgtatccg tttgctggca caatcctctt atcagcttgt cgaagtgcaa 660
gcctctctgg aagagtcgtt gcaggcagat gagatggtta tttgtaatgc gttaatgcca 720
gtgatgcccg tatgtgcctg tggcgatgtc tccttttcgt cagcaacgtt atatgaatat 780
ttagccccac tttgtgagcg cccgaattag 810
<210> 8
<211> 269
<212> PRT
<213> 未知(Unknown)
<400> 8
Met Phe Leu Ile Asn Gly His Lys Gln Glu Ser Leu Ala Val Ser Asp
1 5 10 15
Arg Ala Thr Gln Phe Gly Asp Gly Cys Phe Thr Thr Ala Arg Val Ile
20 25 30
Asp Gly Lys Val Ser Leu Leu Ser Ala His Ile Gln Arg Leu Gln Asp
35 40 45
Ala Cys Gln Arg Leu Met Ile Ser Cys Asp Phe Trp Pro Gln Leu Glu
50 55 60
Gln Glu Met Lys Thr Leu Ala Ala Glu Gln Gln Asn Gly Val Leu Lys
65 70 75 80
Val Val Ile Ser Arg Gly Ser Gly Gly Arg Gly Tyr Ser Thr Leu Asn
85 90 95
Ser Gly Pro Ala Thr Arg Ile Leu Ser Val Thr Ala Tyr Pro Ala His
100 105 110
Tyr Asp Arg Leu Arg Asn Glu Gly Ile Thr Leu Ala Leu Ser Pro Val
115 120 125
Arg Leu Gly Arg Asn Pro His Leu Ala Gly Ile Lys His Leu Asn Arg
130 135 140
Leu Glu Gln Val Leu Ile Arg Ser His Leu Glu Gln Thr Asn Ala Asp
145 150 155 160
Glu Ala Leu Val Leu Asp Ser Glu Gly Trp Val Thr Glu Cys Cys Ala
165 170 175
Ala Asn Leu Phe Trp Arg Lys Gly Asn Val Val Tyr Thr Pro Arg Leu
180 185 190
Asp Gln Ala Gly Val Asn Gly Ile Met Arg Gln Phe Cys Ile Arg Leu
195 200 205
Leu Ala Gln Ser Ser Tyr Gln Leu Val Glu Val Gln Ala Ser Leu Glu
210 215 220
Glu Ser Leu Gln Ala Asp Glu Met Val Ile Cys Asn Ala Leu Met Pro
225 230 235 240
Val Met Pro Val Cys Ala Cys Gly Asp Val Ser Phe Ser Ser Ala Thr
245 250 255
Leu Tyr Glu Tyr Leu Ala Pro Leu Cys Glu Arg Pro Asn
260 265
<210> 9
<211> 1440
<212> DNA
<213> 未知(Unknown)
<400> 9
atggccgtgc aagccccgag taaaacctac ggcttccaga aagccccgat tcagctgacg 60
tttgtggttg ttggtgcggg tctgggtggt gttgcggcca gtatctgtct gcgcctcgcg 120
ggtcaccgcg tgattctgct ggaggcggcg accgaactgg gtgaagtggg tgcgggcatc 180
cagatcccgc caccaagcac caagattctg aaggcgatcg gcgttctcga tgcggtggac 240
aaagtgagca tccacccgca tgacattctg gtgaagaaat ataagggcga gctgctgagt 300
acccagaacc tcgtgccgta cgttagcgag aagtacgacg gcatgtacct ccacatccac 360
cgtgcggatt atcacaaagt gctcgtggat cgcgcggaag aactcggcgt ggaaatccac 420
acgaacagcc gcgttgttga catcgacttt gagaaggcga ccgttacgac ggccaccggt 480
aaacagtaca gtggtgacgt gatcgtgggc tacgatggcg ttcgcagtca gacccgtgcg 540
ctgctgacgg gtgatagtag cggtgcgtac gataccggtg atctggccta ccgcgcgctg 600
atcaaggtgg aggatatgaa gaaagtgccg ggtctggaaa agttttacgc caatccgaac 660
atcaattttt ggtggggccc gacgatgcac atcgtgatgt actttctgca cgagggcgaa 720
atctgtaacg ttgttgcgct gtgtccagat acgctcccga aaggtgtgct gaaacaagat 780
gccagccaag aagaactgct cgatctcgtg aaaggttggg accaagatct gacgaccgtg 840
tttaaactga tcaccagcgt gagtaaatgg cgtctgcaag atagtcgcga gctcaaaacg 900
tgggtgaaca gcaagaccgg caactttatt atcctcggcg acgcgagtca cagcaccctc 960
ccatatctcg ccagcggcgc cagccaagcg gttgaagatg gcgccgttct ggccggtctc 1020
ttcagcaaga ttgagagccg cgatcagatc ccacaactgc tgcagatgac cgagaatctg 1080
cgcaaatggc gcagcagcca agttgttcgt ggcagccatc agtgccaaga tatctaccac 1140
ctcccggacg gcgaactcca agaaatccgt gacagctacc tctacgacaa gcaaccggag 1200
ctcggttgtc cgaatcgctt tgccgatccg gttttccaag attttctgtg gggctacaac 1260
gcgtttgacg aagttgagcg tgcgtggaaa gagttcaagg ccggcggtaa cccgacgtac 1320
acctacccga acctctacaa accgaagagc agcggcgaaa aggatgttag tggcggtggt 1380
gccgcggcca ccctcgcggc cggcaatacc ccagcggccc cactgagcgc gagcggctaa 1440
<210> 10
<211> 479
<212> PRT
<213> 未知(Unknown)
<400> 10
Met Ala Val Gln Ala Pro Ser Lys Thr Tyr Gly Phe Gln Lys Ala Pro
1 5 10 15
Ile Gln Leu Thr Phe Val Val Val Gly Ala Gly Leu Gly Gly Val Ala
20 25 30
Ala Ser Ile Cys Leu Arg Leu Ala Gly His Arg Val Ile Leu Leu Glu
35 40 45
Ala Ala Thr Glu Leu Gly Glu Val Gly Ala Gly Ile Gln Ile Pro Pro
50 55 60
Pro Ser Thr Lys Ile Leu Lys Ala Ile Gly Val Leu Asp Ala Val Asp
65 70 75 80
Lys Val Ser Ile His Pro His Asp Ile Leu Val Lys Lys Tyr Lys Gly
85 90 95
Glu Leu Leu Ser Thr Gln Asn Leu Val Pro Tyr Val Ser Glu Lys Tyr
100 105 110
Asp Gly Met Tyr Leu His Ile His Arg Ala Asp Tyr His Lys Val Leu
115 120 125
Val Asp Arg Ala Glu Glu Leu Gly Val Glu Ile His Thr Asn Ser Arg
130 135 140
Val Val Asp Ile Asp Phe Glu Lys Ala Thr Val Thr Thr Ala Thr Gly
145 150 155 160
Lys Gln Tyr Ser Gly Asp Val Ile Val Gly Tyr Asp Gly Val Arg Ser
165 170 175
Gln Thr Arg Ala Leu Leu Thr Gly Asp Ser Ser Gly Ala Tyr Asp Thr
180 185 190
Gly Asp Leu Ala Tyr Arg Ala Leu Ile Lys Val Glu Asp Met Lys Lys
195 200 205
Val Pro Gly Leu Glu Lys Phe Tyr Ala Asn Pro Asn Ile Asn Phe Trp
210 215 220
Trp Gly Pro Thr Met His Ile Val Met Tyr Phe Leu His Glu Gly Glu
225 230 235 240
Ile Cys Asn Val Val Ala Leu Cys Pro Asp Thr Leu Pro Lys Gly Val
245 250 255
Leu Lys Gln Asp Ala Ser Gln Glu Glu Leu Leu Asp Leu Val Lys Gly
260 265 270
Trp Asp Gln Asp Leu Thr Thr Val Phe Lys Leu Ile Thr Ser Val Ser
275 280 285
Lys Trp Arg Leu Gln Asp Ser Arg Glu Leu Lys Thr Trp Val Asn Ser
290 295 300
Lys Thr Gly Asn Phe Ile Ile Leu Gly Asp Ala Ser His Ser Thr Leu
305 310 315 320
Pro Tyr Leu Ala Ser Gly Ala Ser Gln Ala Val Glu Asp Gly Ala Val
325 330 335
Leu Ala Gly Leu Phe Ser Lys Ile Glu Ser Arg Asp Gln Ile Pro Gln
340 345 350
Leu Leu Gln Met Thr Glu Asn Leu Arg Lys Trp Arg Ser Ser Gln Val
355 360 365
Val Arg Gly Ser His Gln Cys Gln Asp Ile Tyr His Leu Pro Asp Gly
370 375 380
Glu Leu Gln Glu Ile Arg Asp Ser Tyr Leu Tyr Asp Lys Gln Pro Glu
385 390 395 400
Leu Gly Cys Pro Asn Arg Phe Ala Asp Pro Val Phe Gln Asp Phe Leu
405 410 415
Trp Gly Tyr Asn Ala Phe Asp Glu Val Glu Arg Ala Trp Lys Glu Phe
420 425 430
Lys Ala Gly Gly Asn Pro Thr Tyr Thr Tyr Pro Asn Leu Tyr Lys Pro
435 440 445
Lys Ser Ser Gly Glu Lys Asp Val Ser Gly Gly Gly Ala Ala Ala Thr
450 455 460
Leu Ala Ala Gly Asn Thr Pro Ala Ala Pro Leu Ser Ala Ser Gly
465 470 475
<210> 11
<211> 840
<212> DNA
<213> 未知(Unknown)
<400> 11
atgacgccgc tgaccccaga acagacccat gcctatctgc accacatcgg tatcgacgac 60
ccgggcccac cgagtctggc gaatctggac cgtctgattg atgcgcatct gcgccgcgtt 120
gcctttgaaa atctggacgt tctgctggat cgtccgatcg agatcgacgc ggataaagtg 180
ttcgccaagg ttgtggaagg cagtcgcggc ggctactgct tcgagctcaa tagtctgttt 240
gcgcgtctgc tgctggcgct gggttatgaa ctcgaactgc tggttgcccg tgttcgctgg 300
ggtctgccag aagatgcgcc actgacgcag caaagccatc tgatgctgcg tctgtatctg 360
gccgagggcg aatttctggt ggatgttggc ttcggtagtg cgaacccacc acgtgcgctg 420
ccactgccgg gcgacgaagc cgatgcgggt caagttcatt gcgttcgtct ggttgatccg 480
cacgccggtc tgtatgaaag tgccgttcgc ggtcgtagtg gctggctgcc actgtaccgt 540
tttgatctgc gcccacaact gtggatcgac tatatcccgc gcaactggta caccagcacc 600
cacccgcata gcgtttttcg ccaaggtctg aaagcggcca tcacggaagg tgatctgcgt 660
ctgacgctgg cggatggtct gtttggccaa cgtgcgggta acggtgaaac gctgcagcgt 720
cagctgcgcg acgttgagga gctgctggat attctgcaaa cccgtttccg tctgcgtctc 780
gatccggcca gtgaagttcc agcgctggcg cgtcgtctgg cgggtctgat tagtgcgtaa 840
<210> 12
<211> 279
<212> PRT
<213> 未知(Unknown)
<400> 12
Met Thr Pro Leu Thr Pro Glu Gln Thr His Ala Tyr Leu His His Ile
1 5 10 15
Gly Ile Asp Asp Pro Gly Pro Pro Ser Leu Ala Asn Leu Asp Arg Leu
20 25 30
Ile Asp Ala His Leu Arg Arg Val Ala Phe Glu Asn Leu Asp Val Leu
35 40 45
Leu Asp Arg Pro Ile Glu Ile Asp Ala Asp Lys Val Phe Ala Lys Val
50 55 60
Val Glu Gly Ser Arg Gly Gly Tyr Cys Phe Glu Leu Asn Ser Leu Phe
65 70 75 80
Ala Arg Leu Leu Leu Ala Leu Gly Tyr Glu Leu Glu Leu Leu Val Ala
85 90 95
Arg Val Arg Trp Gly Leu Pro Glu Asp Ala Pro Leu Thr Gln Gln Ser
100 105 110
His Leu Met Leu Arg Leu Tyr Leu Ala Glu Gly Glu Phe Leu Val Asp
115 120 125
Val Gly Phe Gly Ser Ala Asn Pro Pro Arg Ala Leu Pro Leu Pro Gly
130 135 140
Asp Glu Ala Asp Ala Gly Gln Val His Cys Val Arg Leu Val Asp Pro
145 150 155 160
His Ala Gly Leu Tyr Glu Ser Ala Val Arg Gly Arg Ser Gly Trp Leu
165 170 175
Pro Leu Tyr Arg Phe Asp Leu Arg Pro Gln Leu Trp Ile Asp Tyr Ile
180 185 190
Pro Arg Asn Trp Tyr Thr Ser Thr His Pro His Ser Val Phe Arg Gln
195 200 205
Gly Leu Lys Ala Ala Ile Thr Glu Gly Asp Leu Arg Leu Thr Leu Ala
210 215 220
Asp Gly Leu Phe Gly Gln Arg Ala Gly Asn Gly Glu Thr Leu Gln Arg
225 230 235 240
Gln Leu Arg Asp Val Glu Glu Leu Leu Asp Ile Leu Gln Thr Arg Phe
245 250 255
Arg Leu Arg Leu Asp Pro Ala Ser Glu Val Pro Ala Leu Ala Arg Arg
260 265 270
Leu Ala Gly Leu Ile Ser Ala
275

Claims (1)

1.一种在微生物合成乙酰氨基酚的方法，其特征在于以单糖为原料产乙酰氨基酚，其包括如下步骤：
将表达aroGfbr 、pabA、pabB、pabC和Mnx1基因的微生物以单糖和/或甘油作为底物进行发酵得到含有对氨基苯酚的发酵产物；
所述含有对氨基苯酚的发酵产物在表达NAT基因的微生物的作用下与乙酰辅酶A反应合成乙酰氨基酚；所述微生物为真菌或细菌，所述单糖为葡萄糖，半乳糖，果糖或木糖中的一种或者多种；
所述aroGfbr基因为SEQ ID NO：1所示的核苷酸序列或者所述aroGfbr基因为编码SEQ IDNO：2所示的氨基酸序列的核苷酸序列；
所述pabA基因为SEQ ID NO：3所示的核苷酸序列编码或者所述pabA基因为编码SEQ IDNO：4所示的氨基酸序列的核苷酸序列；
所述pabB基因为SEQ ID NO：5所示的核苷酸序列编码或者所述pabB基因为编码SEQ IDNO：6所示的氨基酸序列的核苷酸序列；
所述pabC基因为SEQ ID NO：7所示的核苷酸序列编码或者所述pabC基因为编码SEQ IDNO：8所示的氨基酸序列的核苷酸序列；
所述Mnx1基因为SEQ ID NO：9所示的核苷酸序列编码或者所述Mnx1基因为编码SEQ IDNO：10所示的氨基酸序列的核苷酸序列；
所述NAT基因为SEQ ID NO：11所示的核苷酸序列编码或者所述NAT基因为编码SEQ IDNO：12所示的氨基酸序列的核苷酸序列。
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