CN111808871A - 3-氯邻苯二酚降解基因簇的优化重组与应用 - Google Patents

3-氯邻苯二酚降解基因簇的优化重组与应用 Download PDF

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CN111808871A
CN111808871A CN202010741963.2A CN202010741963A CN111808871A CN 111808871 A CN111808871 A CN 111808871A CN 202010741963 A CN202010741963 A CN 202010741963A CN 111808871 A CN111808871 A CN 111808871A
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王波
姚泉洪
彭日荷
田永生
高建杰
许晶
付晓燕
韩红娟
李振军
王丽娟
张福建
邓永东
张文慧
黄悠楠
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Shanghai Academy of Agricultural Sciences
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Abstract

本发明公开了一种优化后适用于大肠杆菌表达的3‑氯邻苯二酚降解基因簇及其应用。该基因簇包含四个相关基因，每个基因都由独立的T7启动子和终止子控制，其核苷酸序列分别如SEQ ID No 1、SEQ ID No 2、SEQ ID No 3和SEQ ID No 4所示，其编码的蛋白质的氨基酸序列分别如SEQ ID No 5、SEQ ID No 6、SEQ ID No 7和SEQ ID No 8所示。本发明优化合成的基因能够在大肠杆菌中成功表达，发现阳性株系不仅能够降解3‑氯邻苯二酚，还可以降解邻苯二酚和4‑氯邻苯二酚，因此本发明所优化的基因簇可用于制备降解3‑氯邻苯二酚、邻苯二酚和4‑氯邻苯二酚的微生物。

Description

3-氯邻苯二酚降解基因簇的优化重组与应用
技术领域
本发明属于基因工程领域，具体涉及结构优化的、包含3-氯邻苯二酚降解相关四个基因的基因簇序列。
背景技术
随着社会的不断进步和工农业的飞速发展，有机污染物在土壤中的含量急剧上升，它们大多具有“致癌、致畸、致突变”作用，严重危及到了人类的生存环境。上海地区随着产业结构的升级，工业企业搬迁后腾出的土地是有机污染的重点区域，同时，固体废物的堆放倾倒、有害废水向土壤中的渗透及污水灌溉也是造成土壤有机污染的重要来源。因此，如何有效清除环境中存在的有机污染物已经成为急需解决的问题。
氯代芳香族化合物广泛应用于生产和生活的方方面面。酚类化合物是煤、石油等化工业排放的一类重要污染物，特别是氯代酚类常做为防腐剂、防锈剂、杀菌剂等应用。氯代苯类化合物在农药、制药、染料、溶剂等生产中广泛存在，它们能够通过食物链进入人体，对人的肝脏、肾脏和神经系统造成损害。微生物是生态循环中的分解者，并且具有极强的适应性，其中很多能够降解氯代芳香族化合物并以这些物质为碳源和能源生长。通过研究发现，许多氯代芳香族化合物的降解都能够先通过不同的代谢途径转化为3-氯邻苯二酚或4-氯邻苯二酚，再经过相同或相似的途径打开苯环，最终为微生物所利用。例如，有研究发现，一种红球菌（Rhodococcus opacus 1CP）对2-氯苯酚的降解就是通过先将其转化为3-氯邻苯二酚后再通过开环并最终利用的（Journal of Bacteriology, 184 (2002): 5282-5292）。
生物修复是一种极具潜力污染修复方式，但目前可供利用的微生物还十分有限，且难以适应不同环境条件下的应用需求。因此，如果我们能够通过生物技术手段，有目的的赋予目标生物污染物降解的能力，将极大的拓展可供利用的生物范围、促进生物修复的广泛应用，为生态环境的保护与修复提供技术支持。
发明内容
本发明所要解决的技术问题在于提供能够在大肠杆菌中表达的3-氯邻苯二酚降解相关四个基因：clcA: 氯代邻苯二酚1,2双加氧酶、clcB: 氯代氯粘康酸环异构酶、clcD:编码二烯内酯水解酶、clcE: 编码二烯内酯水解酶，3-氯邻苯二酚经过连续四步酶促反应生成β-酮己二酸。
基于Pseudomonas putida菌原始转座子（AJ617740.2），该转座子全长105032bp，其中包含多个结构基因和大量未知功能的开放阅读框。本发明所涉及的四个基因成簇存在，该段序列还包含了一个转录调节因子基因和一个未知功能的开放阅读框。本发明针对四个结构基因：clcA、clcB、clcD、clcE进行基因结构优化与重组，并分别为每个基因连接上独立的T7启动子和终止子调控其表达。对编码区基因结构的优化遵循以下原则：（一）优化基因密码子，提高基因翻译效率。（二）消除基因内部的常用限制性内切酶的识别位点，便于表达盒构建。（三）消除逆向重复序列、茎环结构和转录终止信号，使基因内部的GC/AT均衡，提高RNA的稳定性。（四）使基因编码蛋白符合N端原则，以提高翻译蛋白的稳定性。（五）优化mRNA二级结构自由能，以提高基因表达效率。
所述经优化的3-氯邻苯二酚降解相关四个基因的核苷酸序列如SEQ ID No 1、SEQID No 2、SEQ ID No 3和SEQ ID No 4所示。每个基因两端分别边上T7启动子与终止子，完整序列两端分别连接EcoRⅠ和HindⅢ酶切位点，全长序列由生工生物工程（上海）有限公司合成。
将合成的基因片段经EcoRⅠ和Hind Ⅲ双酶切后，连入相同酶切的载体pET-28a，得到重组质粒pET-clc（参见图1），并将其转化大肠杆菌BL21(DE3)，得到阳性株系。
阳性菌株在100毫升M9(含1%甘油和50μg/ml卡那霉素)液体培养中37℃摇菌24小时，离心去上清，菌体用10毫升M9(含1%甘油、0.2%阿拉伯糖、50μg/ml卡那霉素和1mM IPTG)液体培养基重悬，向其中添加1mM的3-氯邻苯二酚、邻苯二酚和4-氯邻苯二酚，结果证明阳性菌株能够有效去除培养基中的这三种底物。
有益效果：
本发明优化并合成了包含四个相关基因的3-氯邻苯二酚降解基因簇，并能在大肠杆菌中成功表达。阳性菌株能够有效去除培养基中的3-氯邻苯二酚、邻苯二酚和4-氯邻苯二酚。经本发明的优化后的基因簇可用于制备降解3-氯邻苯二酚、邻苯二酚和4-氯邻苯二酚的微生物，在废水处理和环境修复等领域具有应用潜力。
附图说明：
图1 为用于在大肠杆菌中表达3-氯邻苯二酚降解基因簇的载体示意图。
图2 为阳性株系对不同底物的降解效果。
图3 为培养基中终产物β-酮己二酸的GC-MS检测。
具体实施方式：
下面结合具体实施方式来进一步阐述本发明。实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对发明的技术方案进行修改或者等同替换，而不脱离本发明的技术方案的精神和范围，其均应涵盖在本发明的权利要求范围中。
本发明实施中未注明的实验方法，如连接、转化、相关培养基的配制等参照分子克隆实验指南第三版（黄培堂等译，中国，科学出版社，2002）中方法进行。所用大肠肝菌由上海市农业科学院生物技术研究所植物基因工程研究室保存，各类限制性内切酶、连接酶等购自上海皓嘉公司。未注明的化学药品为分析纯级，购自生工生物工程（上海）股份公司或上海国药集团有限公司。
实施例 1
3-氯邻苯二酚降解相关四个基因clcA、clcB、clcD、clcE的优化设计与合成
基于Pseudomonas putida菌原始转座子中的四个结构基因：clcA、clcB、clcD、clcE按以下原则进行结构优化：（一）优化基因密码子，兼顾大肠杆菌和植物的密码子偏爱，提高基因翻译效率。（二）消除基因内部的常用限制性内切酶的识别位点，便于表达盒构建。（三）消除逆向重复序列、茎环结构和转录终止信号，使基因内部的GC/AT均衡，提高RNA的稳定性。（四）使基因编码蛋白符合N端原则，以提高翻译蛋白的稳定性。（五）优化mRNA二级结构自由能，以提高基因表达效率。每个基因两端分别边上T7启动子与终止子，完整序列两端分别连接EcoRⅠ和HindⅢ酶切位点，全长序列由生工生物工程（上海）有限公司合成。
实施例 2
大肠杆菌表达载体的构建与转化
将合成的基因片段经EcoRⅠ和HindⅢ双酶切后，连入相同酶切的载体pET-28a，得到重组质粒pET-clc。并将其通过热激转化大肠杆菌BL21(DE3)，在涂布在加有卡那霉素抗性人固体2YT平板上，37℃过夜培养后得到阳性克隆。对阳性克隆中的质粒进行酶切和DNA序列测定确定基因序列完整性和正确性。
实施例 3
阳性菌株对3-氯邻苯二酚、邻苯二酚4-氯邻苯二酚的降解作用
阳性菌株接菌于100毫升M9液体培养中(含1%甘油和50μg/ml卡那霉素)，37℃摇菌24小时(150rpm)，离心去上清，菌体用灭菌的蒸馏水洗一遍，之后用10毫升M9液体培养基重悬(含1%甘油、0.2%阿拉伯糖、50μg/ml卡那霉素和1mM IPTG)，向其中添加1mM的3-氯邻苯二酚，37℃摇菌处理，不同时间取菌液，通过HPLC检测培养基中残余的3-氯邻苯二酚含量（参见图2），并对终产物β-酮己二酸进行GC-MS检测（参见图3）。同时，检测阳性菌株对邻苯二酚和4-氯邻苯二酚的降解能力（参见图2）。
培养基中3-氯邻苯二酚、邻苯二酚和4-氯邻苯二酚的HPLC检测方法：
不同时间取500μL发酵液，12000rpm离心1分钟后取上清，过滤后经HPLC检测。
安捷伦1100高效液相色谱系统；C18柱（120Å，4.6×150mm，5μm）；流动相为10mM 磷酸溶液：乙腈=50：50，流速为1 ml/min；柱温为30℃；检测波长为203 nm；进样量为20 μL。
培养基中β-酮己二酸的GC-MS检测方法：
取10mL发酵液，使用液氮冻融的方法将细胞破壁，超声波提取后，离心取上清液，冻干后加入衍生化试剂BSTFA，60℃下衍生半小时，待GC-MS检测。
气相色谱-质谱联用仪（GC-MS/MS，7890B-7000C，美国Agilent公司）；HP-5 MS毛细管柱（30m×0.25mm×0.25μm，美国Agilent公司）；真空干燥箱（上海一恒科学仪器有限公司）；超声机（上海一恒科学仪器有限公司）、氮吹仪（上海安谱科技有限公司）、超纯水系统（美国Merck Millipore 公司）。
色谱条件：色谱柱：Agilent HP-5 MS毛细管柱（30 m×0.25 mm×0.25 µm）；载气He（99.999%），流速1.0mL/min；进样口温度290 ℃；升温程序：100 ℃以40 ℃/min升至160℃，再以10 ℃/min升至250 ℃，最后以20 ℃/min升至300 ℃；进样量1.0 μL，分流比50:1。
GC-MS质谱分析条件：电子轰击离子源（EI），电离能量70 eV；全扫描（scan）模式，扫描范围m/z：50 ~ 400；离子源温度230 ℃，四级杆温度150 ℃，接口温度300℃。
序列表
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<120> 3-氯邻苯二酚降解基因簇的优化重组与应用
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gtgtggcact caactcctga cggtctgtac tctggtatcc atgacaacat tcctgtggac 420
tactatcgtg gtaagctggt gactgactca caaggtaact accgtgttcg tactactatg 480
cctgttccat atcagattcc atacgaaggt cctactggtc gtcttcttgg tcatctgggt 540
tcacatactt ggcgtcctgc acacgtgcac ttcaaggttc gtaaggacgg tttcgaacca 600
ctgaccactc agtactactt cgaaggtggt aagtgggttg acgatgactg ctgtcatggt 660
gttactcctg acctgatcac tcctgagact atcgaagatg gtgttcgtgt gatgactctg 720
gacttcgtga tcgaacgtga acaagctgag cagcgtaagt ctgctactga gactgttgca 780
taa 783
<210> 2
<211> 1113
<212> DNA
<213> artifical synthesize
<400> 2
atgaagatcg aagcaatcga tgtgactctg gttgatgttc ctgcatcacg tcctatccag 60
atgtccttca ccactgtgca gaagcagtca tacgcaatcg ttcagatccg tgctggtggt 120
cttgtgggta tcggtgaagg ttcatctgtt ggtggtccaa cttggtcatc tgagtgcgct 180
gagaccatca aggtgatcat cgagacttac ctggcaccac tgctgatcgg taaggatgct 240
accaacctgc gtgagctgca acacctgatg gaacgtgctg ttactggtaa ctactctgct 300
aaggctgcaa tcgacgtggc actgcatgat ctgaaggcac gttcactgaa cctgccactg 360
tctgatctga tcggtggtgc aatccagcaa ggtatcccaa tcgcatggac tcttgcttct 420
ggtgacactc agcgtgacat cgcaatcgct gaggagatga tcgaacgtcg tcgtcacaac 480
cgtttcaaga tcaagctggg tgtgcgttca cctgctgatg acctgcgtca catcgagaag 540
atcatcgaac gtgttggtga tcgtgctgct gtgcgtgttg acatcaacca ggcatgggat 600
gagaacactg catctgtgtg gattccacgt cttgaagcag caggtgtgga actggttgaa 660
caacctgttg cacgttccaa cttcgacgca cttcgtcgtc tgtctgctga caacggtgtt 720
gcaatcctgg ctgatgagtc actgtcatca ctggcatctg cattcgaact tgcacgtcat 780
cactgcgttg acgcattctc actgaagctg tgcaacatgg gtggtgtggc aaacactctg 840
aaggttgctg ctatcgctga agcatctggt atcgcatcct acggtggtac tatgttggac 900
tcatccatcg gtactgctgc tgcactgcat gtgtacgcaa ccttgccaac tatgccattc 960
ggttgtgaac tgcttggtcc ttgggtgctt gctgacactc tgactcagac tcaactggag 1020
atcaaggact tcgagatccg tctgccatct ggtcctggtc tgggtgttga catcgaccct 1080
gacaagctgc gtcacttcac tcgtgctggt taa 1113
<210> 3
<211> 711
<212> DNA
<213> artifical synthesize
<400> 3
atgttgactg aaggtatctc aattcagtca tacgatggtc ataccttcgg tgcacttgtt 60
ggttcaccag caaaggcacc agcacctgtg atcgtgatcg cacaagaaat cttcggtgtg 120
aacgcattca tgcgtgagac tgtgtcatgg ctggttgatc agggttacgc agctgtgtgt 180
cctgatctgt atgcacgtca ggcacctggt actgcacttg atccacagga tgaagcacaa 240
cgtgaacaag catacaagct gtggcaggca ttcgacatgg aagcaggtgt tggtgatctg 300
gaagcagcta tccgttatgc acgtcatcag ccatactcca acggtaaggt tggtctggtg 360
ggttactgcc ttggtggtgc acttgcattc ctggttgcag ctaagggtta cgttgatcgt 420
gctgttggtt actacggtgt tggtctggag aagcagctga acaaggtgcc tgaagtgaag 480
catccagcac tgttccacat gggtggtcaa gatcacttcg ttcctgcacc atcacgtcag 540
ctgatcactg aaggtttcgg tgctaaccca ctgctgcaag tgcactggta cgaagaagca 600
ggtcactcct tcgcacgtac ttcatcctct ggttacgtgg catctgctgc tgcactggct 660
aacgaacgta ctctggactt cctggcacca cttcagtcca agaagccata a 711
<210> 4
<211> 1059
<212> DNA
<213> artifical synthesize
<400> 4
atgaatttca tccatgacta tcgttcacca cgtgtgatct tcggtcctga ctcacttgca 60
cgtctgccac aggaactgga acgtcttggt atcgatcgtg cactggtgtt gactactcct 120
gaacaagcac cactgggtcg tcaagttgct gaacctgtga tcggtcatgt tgctgcattc 180
tacgatggtg ctactatgca tgttcctgca ctggtggctg aggaagcctg caagatcgca 240
cgtacttctg aggctaacgg tgtgatcgct atcggtggtg gttcaactat cggtctggct 300
aagatcgttg cactgcgtac tgagttgcct atcgttgctg tgcctactac ttacgctggt 360
tctgagatga cttcaatctt cggtatcact gaaggtggtg tgaagaagac tggtcgtgac 420
gctcgtgtca tgcctcgtgc tgtgatctac gaaccacgtc tgactctgga gctgccactg 480
tccatctctg tgacttctgc tatcaacgca atcgcacatg ctgttgaggg tctgtacgca 540
cctgatgcaa ctccactgct gaccatcatg gcacaggaag gtatcgctgc aactgttcgt 600
gctatctcac gtatgtatca gtcaccacgt gatcttcagg cacgtggtga tgcactgtat 660
ggtgcatggc tgtgtgcatc tgtgcttggt aacgtgtcta tggcactgca ccacaagctg 720
tgtcatactc tgggtggtac tctggacctg ccacatgcac agactcatac tgtggttctt 780
ccacatgcac tggcatacaa cgcacgtgct gtgcctgacg caatgcgtgt tctgcgtatc 840
gcactgggtc atgatgatcc accaactgca ctgtacgaac tggcacgtga caacggtgca 900
cctgtggcac tgcgtgatct gggtatgcgt gaggaggaca tcgagcatgt tggtgatctg 960
gcacttcagg atcgttaccc taacccacgt gaactggatc gtgatgcact gcttgcactg 1020
ctgcgtgatg catatcatgg tcgtccacca tctgcataa 1059
<210> 5
<211> 260
<212> PRT
<213> artifical synthesize
<400> 5
Met Asp Lys Arg Val Ala Glu Val Ala Gly Ala Ile Val Glu Ala Val
1 5 10 15
Arg Lys Ile Leu Leu Asp Lys Arg Val Thr Glu Ala Glu Tyr Arg Ala
20 25 30
Gly Val Asp Tyr Leu Thr Glu Val Ala Gln Thr Arg Glu Thr Ala Leu
35 40 45
Leu Leu Asp Val Phe Leu Asn Ser Thr Ile Ile Glu Gly Lys Ala Gln
50 55 60
Arg Ser Arg Thr Ser Ala Pro Ala Ile Gln Gly Pro Tyr Phe Leu Glu
65 70 75 80
Gly Ala Pro Val Val Glu Gly Val Leu Lys Thr Tyr Asp Thr Asp Asp
85 90 95
His Lys Pro Leu Ile Ile Arg Gly Thr Val Arg Ser Asp Thr Gly Glu
100 105 110
Leu Leu Ala Gly Ala Val Ile Asp Val Trp His Ser Thr Pro Asp Gly
115 120 125
Leu Tyr Ser Gly Ile His Asp Asn Ile Pro Val Asp Tyr Tyr Arg Gly
130 135 140
Lys Leu Val Thr Asp Ser Gln Gly Asn Tyr Arg Val Arg Thr Thr Met
145 150 155 160
Pro Val Pro Tyr Gln Ile Pro Tyr Glu Gly Pro Thr Gly Arg Leu Leu
165 170 175
Gly His Leu Gly Ser His Thr Trp Arg Pro Ala His Val His Phe Lys
180 185 190
Val Arg Lys Asp Gly Phe Glu Pro Leu Thr Thr Gln Tyr Tyr Phe Glu
195 200 205
Gly Gly Lys Trp Val Asp Asp Asp Cys Cys His Gly Val Thr Pro Asp
210 215 220
Leu Ile Thr Pro Glu Thr Ile Glu Asp Gly Val Arg Val Met Thr Leu
225 230 235 240
Asp Phe Val Ile Glu Arg Glu Gln Ala Glu Gln Arg Lys Ser Ala Thr
245 250 255
Glu Thr Val Ala
260
<210> 6
<211> 370
<212> PRT
<213> artifical synthesize
<400> 6
Met Lys Ile Glu Ala Ile Asp Val Thr Leu Val Asp Val Pro Ala Ser
1 5 10 15
Arg Pro Ile Gln Met Ser Phe Thr Thr Val Gln Lys Gln Ser Tyr Ala
20 25 30
Ile Val Gln Ile Arg Ala Gly Gly Leu Val Gly Ile Gly Glu Gly Ser
35 40 45
Ser Val Gly Gly Pro Thr Trp Ser Ser Glu Cys Ala Glu Thr Ile Lys
50 55 60
Val Ile Ile Glu Thr Tyr Leu Ala Pro Leu Leu Ile Gly Lys Asp Ala
65 70 75 80
Thr Asn Leu Arg Glu Leu Gln His Leu Met Glu Arg Ala Val Thr Gly
85 90 95
Asn Tyr Ser Ala Lys Ala Ala Ile Asp Val Ala Leu His Asp Leu Lys
100 105 110
Ala Arg Ser Leu Asn Leu Pro Leu Ser Asp Leu Ile Gly Gly Ala Ile
115 120 125
Gln Gln Gly Ile Pro Ile Ala Trp Thr Leu Ala Ser Gly Asp Thr Gln
130 135 140
Arg Asp Ile Ala Ile Ala Glu Glu Met Ile Glu Arg Arg Arg His Asn
145 150 155 160
Arg Phe Lys Ile Lys Leu Gly Val Arg Ser Pro Ala Asp Asp Leu Arg
165 170 175
His Ile Glu Lys Ile Ile Glu Arg Val Gly Asp Arg Ala Ala Val Arg
180 185 190
Val Asp Ile Asn Gln Ala Trp Asp Glu Asn Thr Ala Ser Val Trp Ile
195 200 205
Pro Arg Leu Glu Ala Ala Gly Val Glu Leu Val Glu Gln Pro Val Ala
210 215 220
Arg Ser Asn Phe Asp Ala Leu Arg Arg Leu Ser Ala Asp Asn Gly Val
225 230 235 240
Ala Ile Leu Ala Asp Glu Ser Leu Ser Ser Leu Ala Ser Ala Phe Glu
245 250 255
Leu Ala Arg His His Cys Val Asp Ala Phe Ser Leu Lys Leu Cys Asn
260 265 270
Met Gly Gly Val Ala Asn Thr Leu Lys Val Ala Ala Ile Ala Glu Ala
275 280 285
Ser Gly Ile Ala Ser Tyr Gly Gly Thr Met Leu Asp Ser Ser Ile Gly
290 295 300
Thr Ala Ala Ala Leu His Val Tyr Ala Thr Leu Pro Thr Met Pro Phe
305 310 315 320
Gly Cys Glu Leu Leu Gly Pro Trp Val Leu Ala Asp Thr Leu Thr Gln
325 330 335
Thr Gln Leu Glu Ile Lys Asp Phe Glu Ile Arg Leu Pro Ser Gly Pro
340 345 350
Gly Leu Gly Val Asp Ile Asp Pro Asp Lys Leu Arg His Phe Thr Arg
355 360 365
Ala Gly
370
<210> 7
<211> 236
<212> PRT
<213> artifical synthesize
<400> 7
Met Leu Thr Glu Gly Ile Ser Ile Gln Ser Tyr Asp Gly His Thr Phe
1 5 10 15
Gly Ala Leu Val Gly Ser Pro Ala Lys Ala Pro Ala Pro Val Ile Val
20 25 30
Ile Ala Gln Glu Ile Phe Gly Val Asn Ala Phe Met Arg Glu Thr Val
35 40 45
Ser Trp Leu Val Asp Gln Gly Tyr Ala Ala Val Cys Pro Asp Leu Tyr
50 55 60
Ala Arg Gln Ala Pro Gly Thr Ala Leu Asp Pro Gln Asp Glu Ala Gln
65 70 75 80
Arg Glu Gln Ala Tyr Lys Leu Trp Gln Ala Phe Asp Met Glu Ala Gly
85 90 95
Val Gly Asp Leu Glu Ala Ala Ile Arg Tyr Ala Arg His Gln Pro Tyr
100 105 110
Ser Asn Gly Lys Val Gly Leu Val Gly Tyr Cys Leu Gly Gly Ala Leu
115 120 125
Ala Phe Leu Val Ala Ala Lys Gly Tyr Val Asp Arg Ala Val Gly Tyr
130 135 140
Tyr Gly Val Gly Leu Glu Lys Gln Leu Asn Lys Val Pro Glu Val Lys
145 150 155 160
His Pro Ala Leu Phe His Met Gly Gly Gln Asp His Phe Val Pro Ala
165 170 175
Pro Ser Arg Gln Leu Ile Thr Glu Gly Phe Gly Ala Asn Pro Leu Leu
180 185 190
Gln Val His Trp Tyr Glu Glu Ala Gly His Ser Phe Ala Arg Thr Ser
195 200 205
Ser Ser Gly Tyr Val Ala Ser Ala Ala Ala Leu Ala Asn Glu Arg Thr
210 215 220
Leu Asp Phe Leu Ala Pro Leu Gln Ser Lys Lys Pro
225 230 235
<210> 8
<211> 352
<212> PRT
<213> artifical synthesize
<400> 8
Met Asn Phe Ile His Asp Tyr Arg Ser Pro Arg Val Ile Phe Gly Pro
1 5 10 15
Asp Ser Leu Ala Arg Leu Pro Gln Glu Leu Glu Arg Leu Gly Ile Asp
20 25 30
Arg Ala Leu Val Leu Thr Thr Pro Glu Gln Ala Pro Leu Gly Arg Gln
35 40 45
Val Ala Glu Pro Val Ile Gly His Val Ala Ala Phe Tyr Asp Gly Ala
50 55 60
Thr Met His Val Pro Ala Leu Val Ala Glu Glu Ala Cys Lys Ile Ala
65 70 75 80
Arg Thr Ser Glu Ala Asn Gly Val Ile Ala Ile Gly Gly Gly Ser Thr
85 90 95
Ile Gly Leu Ala Lys Ile Val Ala Leu Arg Thr Glu Leu Pro Ile Val
100 105 110
Ala Val Pro Thr Thr Tyr Ala Gly Ser Glu Met Thr Ser Ile Phe Gly
115 120 125
Ile Thr Glu Gly Gly Val Lys Lys Thr Gly Arg Asp Ala Arg Val Met
130 135 140
Pro Arg Ala Val Ile Tyr Glu Pro Arg Leu Thr Leu Glu Leu Pro Leu
145 150 155 160
Ser Ile Ser Val Thr Ser Ala Ile Asn Ala Ile Ala His Ala Val Glu
165 170 175
Gly Leu Tyr Ala Pro Asp Ala Thr Pro Leu Leu Thr Ile Met Ala Gln
180 185 190
Glu Gly Ile Ala Ala Thr Val Arg Ala Ile Ser Arg Met Tyr Gln Ser
195 200 205
Pro Arg Asp Leu Gln Ala Arg Gly Asp Ala Leu Tyr Gly Ala Trp Leu
210 215 220
Cys Ala Ser Val Leu Gly Asn Val Ser Met Ala Leu His His Lys Leu
225 230 235 240
Cys His Thr Leu Gly Gly Thr Leu Asp Leu Pro His Ala Gln Thr His
245 250 255
Thr Val Val Leu Pro His Ala Leu Ala Tyr Asn Ala Arg Ala Val Pro
260 265 270
Asp Ala Met Arg Val Leu Arg Ile Ala Leu Gly His Asp Asp Pro Pro
275 280 285
Thr Ala Leu Tyr Glu Leu Ala Arg Asp Asn Gly Ala Pro Val Ala Leu
290 295 300
Arg Asp Leu Gly Met Arg Glu Glu Asp Ile Glu His Val Gly Asp Leu
305 310 315 320
Ala Leu Gln Asp Arg Tyr Pro Asn Pro Arg Glu Leu Asp Arg Asp Ala
325 330 335
Leu Leu Ala Leu Leu Arg Asp Ala Tyr His Gly Arg Pro Pro Ser Ala
340 345 350

Claims (4)

1.人工优化后的3-氯邻苯二酚降解基因簇包含四个相关基因，其特征在于，这四个基因的核苷酸序列分别如SEQ ID No 1、SEQ ID No 2、SEQ ID No 3和SEQ ID No 4所示。
2.根据权利要求1所述的四个基因的核苷酸序列，其特征在于，其编码的蛋白质的氨基酸序列如SEQ ID No 5、SEQ ID No 6、SEQ ID No 7和SEQ ID No 8所示。
3.根据权利要求1所述的四个基因的核苷酸序列构建大肠杆菌表达载体pET-clc，其特征在于，每段基因序列均与T7启动子和终止子相连组成一个表达单元，将四个表达单元串连插入表达载体。
4.根据权利要求3所述的大肠杆菌表达载体，其特征在于，该载体转化大肠杆菌后，阳性菌株能够有效去除3-氯邻苯二酚、邻苯二酚和4-氯邻苯二酚。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108486141A (zh) * 2018-03-13 2018-09-04 南京乐夫朗生物科技有限公司 一种邻苯二酚类化合物生物转化制备法
CN109517833A (zh) * 2018-11-17 2019-03-26 上海市农业科学院 邻苯二酚降解相关四个基因的优化重组与应用

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Publication number Priority date Publication date Assignee Title
CN108486141A (zh) * 2018-03-13 2018-09-04 南京乐夫朗生物科技有限公司 一种邻苯二酚类化合物生物转化制备法
CN109517833A (zh) * 2018-11-17 2019-03-26 上海市农业科学院 邻苯二酚降解相关四个基因的优化重组与应用

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Title
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