CN105112351B - 一种利用甘油发酵生产苯酚的工程菌株的构建方法及应用 - Google Patents
一种利用甘油发酵生产苯酚的工程菌株的构建方法及应用 Download PDFInfo
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- CN105112351B CN105112351B CN201510577825.4A CN201510577825A CN105112351B CN 105112351 B CN105112351 B CN 105112351B CN 201510577825 A CN201510577825 A CN 201510577825A CN 105112351 B CN105112351 B CN 105112351B
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Abstract
本发明涉及工程菌株构建技术领域,特别涉及一种利用甘油发酵生产苯酚的工程菌株的构建方法,在pTrc99a载体中分别插入序列表中序列2、3的碱基序列,得到的重组质粒,将重组质粒电转化ATCC31884菌株,获得利用甘油发酵生产苯酚的工程菌株。本发明构建的菌株高效表达酪氨酸苯酚裂解酶和苯丙氨酸4‑单加氧酶,所得的大肠杆菌工程菌株利用甘油生产苯酚的含量达到21.30‑57.66mg/L,从而实现了生物法发酵甘油生产苯酚,降低了苯酚的生产成本,提高了经济效益。
Description
技术领域
本发明涉及工程菌株构建技术领域,特别涉及一种利用甘油发酵生产苯酚的工程菌株的构建方法,还涉及得到的工程菌株的应用。
背景技术
苯酚是一种重要的基本有机化工原料,主要用于生产酚醛树脂、双酚A和己内酰胺、己二酸、苯胺等;苯酚衍生物如卤代酚、硝基酚、烷基酚可用于医药、染料、香料以及各种添加剂等的生产,因此苯酚市场具有良好的发展前景。
甘油又称丙三醇,分子式C3H5(OH)3,是一种粘稠液体,有甜味,能与水以任意比混溶,有强烈的吸湿性。甘油资源丰富、价格低廉,由于其C原子的高度还原性,在燃料和化学品生产中具有比普通糖如葡萄糖更高的收益,所以已经成为一个有吸引力的原料。
目前,世界上苯酚的生产方法主要是化学法合成:异丙苯法、甲苯-苯甲酸法、苯直接氧化法、苯硼酸法以及环己酮-环己醇法。其中异丙苯法是目前最主要的苯酚生产方法,生产能力占世界苯酚总生产能力的90%以上。其工艺步骤是:苯和丙烯反应合成异丙苯;异丙苯经氧气或空气氧化,生成过氧化氢异丙苯(CHP);CHP分解生成苯酚和丙酮。副产物丙酮具有毒性,所以化学法合成苯酚存在生产成本高、环境污染大、副产物多等一系列问题。
苯酚的生物合成由于自身毒性对微生物的伤害而受到了限制,因此关于生物法合成苯酚的研究较少。目前,国外已有关于以葡萄糖为原料生产苯酚的报道,根据相关文献报道(Byoungjin Kim,Hyegwon Park,Dokyun Na,Sang Yup Lee.Metabolic engineering ofEscherichia coli for the production of phenol from glucose.BiotechnolJournal,2014,9,621–629.),Byoungjin Kim利用葡萄糖生产苯酚的最高效价达到3.79g/L,但是没有利用甘油发酵生产苯酚的报道。
发明内容
为了解决以上现有技术中化学法生产苯酚存在的生产成本高、环境污染大、副产物多等问题,及生物法合成苯酚的研究较少的现状,本发明以甘油为原料,研究了一种利用甘油发酵生产苯酚的工程菌株的构建方法。
本发明还提供了所述得到的工程菌株的应用。
本发明是通过以下步骤得到的:
大肠杆菌利用甘油生产苯酚的途径如图1所示,其中涉及打通代谢条路的两个关键酶基因为tpl(编码酪氨酸苯酚裂解酶)和p4m(编码苯丙氨酸-4-单加氧酶),酪氨酸苯酚裂解酶催化酪氨酸生成苯酚,苯丙氨酸-4-单加氧酶则催化苯丙氨酸生成酪氨酸。涉及利用甘油生产苯酚的两个基因为glpX(编码果糖-1,6-二磷酸酶)和tktA(编码转酮醇酶)。果糖-1,6-二磷酸酶催化1,6-二磷酸果糖生成6-磷酸果糖,转酮醇酶催化6-磷酸果糖生成4-磷酸赤藓糖。
本发明的第一个目的是提供一种生产苯酚的大肠杆菌工程菌株的构建方法,该菌株共表达酪氨酸苯酚裂解酶(tpl基因编码)和苯丙氨酸-4-单加氧酶(p4m基因编码)。
所述酪氨酸苯酚裂解酶的氨基酸序列具体如序列表中的序列1所示,共由456个氨基酸组成;
所述酪氨酸苯酚裂解酶的编码基因是经过密码子优化的,其基因序列具体如序列表中的序列2所示,共由1373个核苷酸组成;
所述苯丙氨酸-4-单加氧酶的编码基因序列具体如序列表中的序列3所示,共由789个核苷酸组成。
一种生产苯酚的大肠杆菌工程菌株PH1的构建方法,其构建方法如下:
(1)选取酪氨酸苯酚裂解酶(tpl)的CDS全序列,经过设计和反复验证得到适合于在大肠杆菌中表达的优化型酪氨酸苯酚裂解酶基因序列,上述优化为在不改变相应酶的氨基酸序列的前提下,将野生型基因的密码子替换为大肠杆菌偏好(高频使用)的密码子优化序列,从而提高酪氨酸苯酚裂解酶在大肠杆菌培养环境中的表达水平。
(2)将tpl和p4m基因分别通过酶切位点Kpn I、Pst I和Pst I、Hind III克隆在pTrc99a中,得到重组质粒pTrc99a-tpl-p4m。将重组质粒pTrc99a-tpl-p4m电转化本实验室的高产苯丙氨酸菌株E.coli ATCC31884,获得重组菌命名为PH1即ATCC31884-pTrc99a-tpl-p4m。
本发明的第二个目的是提供一种高效利用甘油生产苯酚的大肠杆菌工程菌株的构建方法,该菌株在表达酪氨酸苯酚裂解酶(tpl基因编码)和苯丙氨酸-4-单加氧酶(p4m基因编码)的基础上,高表达果糖-1,6-二磷酸酶(glpX基因编码)和转酮醇酶(tktA基因编码),提高了工程菌株对甘油的利用效率。
一种高效利用甘油生产苯酚的大肠杆菌工程菌株PH2的构建方法如下:
(1)高产苯丙氨酸菌株E.coli ATCC31884基因组DNA的提取
取5mL E.coli ATCC31884菌株的菌液,12000rpm,离心1min,弃去上清,采用细菌基因组DNA提取试剂盒提取E.coli ATCC31884菌株基因组DNA。
(2)glpX和tktA基因的PCR扩增
以步骤(1)提取到的基因组DNA为模板,进行PCR扩增;将扩增产物用1%琼脂糖凝胶电泳检测,并采用胶回收试剂盒进行纯化,获得目的基因glpX和tktA。
(3)以pTrc99a质粒为模板,进行PCR扩增,得到PCR产物,该产物为trc启动子。
(4)以步骤(2)获得的glpX基因和步骤(3)获得的PCR产物为模板,通过OverlapPCR扩增得到PCR产物,该产物为trc启动子插入到glpX基因上游。
(5)将步骤(4)获得的PCR产物和步骤(2)获得的目的基因tktA分别通过酶切位点Kpn I、BamH I和BamH I、Xba I克隆在pTrc99a中,得到中间质粒pTrc99a-trcglpX-tktA。
(6)以重组质粒pTrc99a-tpl-p4m为模板,进行PCR扩增,得到PCR产物,该产物为tpl-p4m基因。
(7)将步骤(6)获得的PCR产物通过酶切位点BspH I、Sac I克隆在步骤(5)获得的中间质粒pTrc99a-trcglpX-tktA中,得到重组质粒pTrc99a-tpl-p4m-trcglpX-tktA。将重组质粒pTrc99a-tpl-p4m-trcglpX-tktA电转化本实验室的高产苯丙氨酸菌株E.coliATCC31884,获得重组菌命名为PH2即ATCC31884-pTrc99a-tpl-p4m-trcglpX-tktA(即tpl和p4m基因由一个trc启动子驱动,glpX和tktA基因由另外一个trc启动子驱动)。
为进一步增强苯酚合成能力,提高苯酚合成关键酶的表达量,在PH2菌株的基础上,对质粒上的p4m基因额外增加trc启动子驱动,构建过程如下:
(1)以pTrc99a质粒为模板,进行PCR扩增,得到PCR产物,该产物为trc启动子。
(2)以pTrc99a-tpl-p4m-trcglpX-tktA质粒为模板,进行PCR扩增,得到PCR产物。
(3)将步骤(1)获得的PCR产物通过酶切位点Xho I和Nco I克隆在步骤(2)获得的PCR产物中,得到重组质粒pTrc99a-tpl-trcp4m-trcglpX-tktA。将重组质粒pTrc99a-tpl-trcp4m-trcglpX-tktA电转化高产苯丙氨酸菌株E.coli ATCC31884,获得重组菌命名为PH3即ATCC31884-pTrc99a-tpl-trcp4m-trcglpX-tktA(即tpl和p4m基因分别由一个trc启动子驱动,glpX和tktA基因由另外一个trc启动子驱动)。
本发明的第三个目的是应用所述大肠杆菌工程菌株发酵生产苯酚。
所述大肠杆菌工程菌株优选:
PH1:ATCC31884-pTrc99a-tpl-p4m;
PH2:ATCC31884-pTrc99a-tpl-p4m-trcglpX-tktA;
PH3:ATCC31884-pTrc99a-tpl-trcp4m-trcglpX-tktA;
所述发酵生产苯酚涉及的培养基优选:
固体平板培养基(g/L):蛋白胨10,氯化钠10,酵母粉5,琼脂15,pH 7.0,必要时在培养基中添加Amp 100μg/mL;
发酵种子培养基(g/L):蛋白胨10,氯化钠10,酵母粉5,pH 7.0,装液量10mL/20mL,必要时在培养基中添加Amp 100μg/mL;
发酵培养基(g/L):甘油10,Na2HPO4·12H2017.11,KH2PO43,NaCl 0.5,NH4Cl 1,MgSO4·7H2O0.492,CaCl2·6H2O 0.022,装液量10mL/20mL,必要时在培养基中添加Amp 100μg/mL;
(1)种子培养:从固体培养基平板上挑取单菌落接种至试管中培养,装液量为10mL/20mL,在培养基中添加Amp 100μg/mL,37℃,200r/min培养12h,转接发酵培养基;
(2)发酵培养:将培养好的种子液按10%(v/v)的接种量,接种至装有10mL发酵培养基的20mL试管中进行培养,在培养基中添加Amp 100μg/mL,37℃,200r/min培养,当菌体生长至OD600为0.4-0.6时,添加0.4mM IPTG诱导表达,继续培养至48h。
目标产物苯酚的检测:高效液相色谱(HPLC)
苯酚标准溶液的配制:苯酚工作液:取苯酚标准品,用去离子水配制成1g/L的溶液。
待测样品溶液的制备:发酵结束后,将获得的发酵液12000rpm,2min离心后取上清,用0.22μm滤膜过滤,取到待上机的待测样品溶液。
色谱条件:
色谱分离柱:ZORBAX Eclipse XDB-C184.6×250mm,5μm
流动相:乙腈-水=40:60(v/v)
检测波长:210nm;流速:0.7mL/min;进样量:10μL;柱温:30℃。根据苯酚标准品溶液和待测样品溶液的HPLC图谱获得大肠杆菌工程菌株发酵液中苯酚的含量。
本发明的有益效果:
本发明在表达酪氨酸苯酚裂解酶(tpl基因编码)和苯丙氨酸4-单加氧酶(p4m基因编码)的基础上,高表达果糖-1,6-二磷酸酶(glpX基因编码)和转酮醇酶(tktA基因编码),取得了一定的技术效果:所得的大肠杆菌工程菌株PH3利用甘油生产苯酚的含量达到57.66mg/L,从而实现了生物法发酵甘油生产苯酚,降低了苯酚的生产成本,提高了经济效益。
附图说明
图1为基因tpl、p4m、glpX和tktA的PCR电泳验证图谱,其中泳道M为DNA分子量标准;
图2为苯酚标品和重组菌株PH1发酵液的HPLC图谱,其中,a为苯酚标品的HPLC图谱,b为重组菌株PH1发酵液的HPLC图谱;
图3为重组菌株PH1、PH2和PH3发酵液中苯酚含量图谱。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、用于甘油发酵生产苯酚的重组大肠杆菌的构建
一、重组表达载体pTrc99a-tpl-p4m的构建
1、tpl基因的选择与优化
选取酪氨酸苯酚裂解酶(tpl)的CDS全序列,经过设计和反复验证得到适合于在大肠杆菌中表达的优化型酪氨酸苯酚裂解酶基因序列,即将野生型酪氨酸苯酚裂解酶基因序列在不改变氨基酸序列的前提下转变成大肠杆菌偏好(高频使用)的密码子优化序列,从而提高酪氨酸苯酚裂解酶在大肠杆菌培养环境中的表达水平。
所述酪氨酸苯酚裂解酶的氨基酸序列具体如序列表中的序列1所示,共由456个氨基酸组成;
所述酪氨酸苯酚裂解酶的编码基因序列具体如序列表中的序列2所示,共由1373个核苷酸组成;
所述苯丙氨酸-4-单加氧酶的编码基因序列具体如序列表中的序列3所示,共由789个核苷酸组成。
2、表达载体pTrc99a-tpl-p4m的构建
以序列表中序列2所示DNA片段为模板,用引物1和2进行PCR扩增,得到PCR产物1,该产物为tpl基因(目的条带1373bp,图1-泳道1)。以序列表中序列3所示DNA片段为模板,用引物3和4进行PCR扩增,得到PCR产物2,该产物为p4m基因(目的条带817bp,图1-泳道2)。
引物1:
5'-ATAGGTACCATGAATTACCCGGCCGAACCTT-3'(下划线部分为限制性内切酶
Kpn I的识别序列)
引物2:
5'-GCGCTGCAGTTAAATAAAATCAAAACGGGCGG-3'(下划线部分为限制性内切酶Pst I的识别序列)
引物3:5'-ATACTGCAGCGCACAACAGAGGAAGAGGAGGTAACATATGAAACAGACGCAATACGTGGCACGC-3'(下划线部分为限制性内切酶Pst I的识别序列,倾斜部分为RBS)
引物4:
5'-ATAAAGCTTTCAGGCAGCGACCTTGGGTGGAAACTTCGGTGCATGCAGACCCAGCTGCAT-3'(下划线部分为限制性内切酶Hind III的识别序列)
用限制性内切酶Kpn I和Pst I双酶切PCR产物1,胶回收后与经过同样双酶切的pTrc99a载体骨架大片段相连,得到中间质粒pTrc99a-tpl。将构建的中间质粒送样测序,测序结果与预期结果一致,证明中间质粒pTrc99a-tpl构建正确。
再用限制性内切酶Pst I和Hind III双酶切PCR产物2,胶回收后与经过Pst I和Hind III双酶切的中间质粒pTrc99a-tpl载体骨架大片段相连,得到重组质粒并将重组质粒送样测序。将经测序表明在pTrc99a载体的多克隆位点Kpn I和Pst I之间插入序列表中序列2所示DNA片段,同时在多克隆位点Pst I和Hind III之间插入序列表中序列3所示DNA片段后得到的重组质粒命名为pTrc99a-tpl-p4m。
将重组质粒pTrc99a-tpl-p4m电转化本实验室的高产苯丙氨酸菌株E.coliATCC31884,获得重组菌PH1即ATCC31884-pTrc99a-tpl-p4m。
二、重组表达载体pTrc99a-tpl-p4m-trcglpX-tktA的构建
以pTrc99a质粒为模板,用引物5和引物6进行PCR扩增,得到PCR产物3,该产物为trc启动子。
以ATCC31884基因组为模板,用引物7和引物8进行PCR扩增,得到PCR产物4,该产物为glpX基因(目的条带1011bp,图1-泳道3,碱基序列见序列表4);再以PCR产物3和PCR产物4为模板,用引物5和引物8为模板,进行Overlap PCR扩增得到PCR产物5,该产物为trc启动子插入到glpX基因上游。
引物5:
5'-GCGGGTACCATATTCTGAAATGAGCTGTTGACAATTAATCATCCGG-3'(下划线部分为限制性内切酶Kpn I的识别序列)
引物6:
5'-TCGATGGCAAGTTCTCGTCTCATGGTCTGTTTCCTGTGTGAAATT-3'
引物7:
5'-AATTTCACACAGGAAACAGACCATGAGACGAGAACTTGCCATCGA-3'
引物8:
5'-TATATATATAGGATCCTCAGAGGATGTGCACCTGCATTTCCGGGT-3'(下划线部分为限制性内切酶BamH I的识别序列)
用限制性内切酶Kpn I和BamH I双酶切PCR产物5,胶回收后与经过Kpn I和BamH I双酶切的质粒pTrc99a载体骨架大片段相连,得到中间质粒。将构建的中间质粒送样测序,测序结果与预期结果一致,证明中间质粒pTrc99a-trcglpX构建正确。
以ATCC31884基因组为模板,用引物9和引物10进行PCR扩增,得到PCR产物6,该产物为tktA基因(目的条带1997bp,图1-泳道4,碱基序列见序列表5)。
引物9:
5'-ATAGGATCCTG3'(下划线部分为限制性内切酶BamH I的识别序列,方框部分为RBS)
引物10:
5'-GCCTCTAGATTACAGCAGTTCTTTTGCTTTCGCAACAACGTTATCAACAGTGAAGCCGAACTCTTCAAACAGCAGC-3'(下划线部分为限制性内切酶Xba I的识别序列)
用限制性内切酶BamH I和Xba I双酶切PCR产物6,胶回收后与经过BamH I和Xba I双酶切的中间质粒pTrc99a-trcglpX载体骨架大片段相连,得到中间质粒pTrc99a-trcglpX-tktA。将构建的中间质粒送样测序,测序结果与预期结果一致,证明中间质粒pTrc99a-trcglpX-tktA构建正确。
以重组质粒pTrc99a-tpl-p4m为模板,用引物11和引物12进行PCR扩增,得到PCR产物7,该产物为tpl-p4m基因。
引物11:
5'-GCCGTCATGAATTACCCGGCCGAACCTT-3'(下划线部分为限制性内切酶BspH I的识别序列)
引物12:
5'-ATAGAGCTCTCAGGCAGCGACCTTGGGT-3'(下划线部分为限制性内切酶Sac I的识别序列)
用限制性内切酶BspH I(与Nco I为同尾酶)和Sac I双酶切PCR产物7,胶回收后与经过Nco I和Xba I双酶切的中间质粒pTrc99a-trcglpX-tktA载体骨架大片段相连,得到重组质粒。将重组质粒送样测序,测序结果与预期结果一致,证明重组质粒构建正确,并命名为pTrc99a-tpl-p4m-trcglpX-tktA。
将重组质粒pTrc99a-tpl-p4m-trcglpX-tktA电转化本实验室的高产苯丙氨酸菌株E.coli ATCC31884,获得重组菌PH2即ATCC31884-pTrc99a-tpl-p4m-trcglpX-tktA。
三、重组表达载体pTrc99a-tpl-trcp4m-trcglpX-tktA的构建
以pTrc99a质粒为模板,用引物13和引物14进行PCR扩增,得到PCR产物8,该产物为trc启动子。
以pTrc99a-tpl-p4m-trcglpX-tktA质粒为模板,用引物15和引物16进行PCR扩增,得到PCR产物9。
引物13:
5'-GCGCTCGAGATATTCTGAAATGAGCTGTTGACAATTAATCATCCG-3'(下划线部分为限制性内切酶Xho I的识别序列)
引物14:
5'-TGTACCATGGTCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAA-3'(下划线部分为限制性内切酶Nco I的识别序列)
引物15:
5'-AATATCCATGGACATGAAACAGACGCAATACGTGGCACGC-3'(下划线部分为限制性内切酶Nco I的识别序列)
引物16:
5'-AGCGCTCGAGTTAAATAAAATCAAAACGGGCGGTGAAGAAACG-3'(下划线部分为限制性内切酶Xho I的识别序列)
用限制性内切酶Xho I和Nco I分别双酶切PCR产物8和PCR产物9,胶回收后与经过Nco I和Xho I双酶切的中间质粒pTrc99a-tpl-p4m-trcglpX-tktA载体骨架大片段相连,得到重组质粒,并将重组质粒送样测序。将经测序表明在pTrc99a-tpl-p4m-trcglpX-tktA载体的多克隆位点Xho I和Nco I之间插入酶切后的PCR产物8后得到的重组质粒命名为pTrc99a-tpl-trcp4m-trcglpX-tktA。
将重组质粒pTrc99a-tpl-trcp4m-trcglpX-tktA电转化本实验室的高产苯丙氨酸菌株E.coli ATCC31884,获得重组菌PH3即ATCC31884-pTrc99a-tpl-trcp4m-trcglpX-tktA。
实施例2、以甘油为底物发酵生产苯酚
一、利用甘油生产苯酚的发酵过程
所述大肠杆菌工程菌株优选:
PH1:ATCC31884-pTrc99a-tpl-p4m;
PH2:ATCC31884-pTrc99a-tpl-p4m-trcglpX-tktA;
PH3:ATCC31884-pTrc99a-tpl-trcp4m-trcglpX-tktA;
所述发酵生产苯酚涉及的培养基优选:
固体平板培养基(g/L):蛋白胨10,氯化钠10,酵母粉5,琼脂15,pH 7.0,必要时在培养基中添加Amp 100μg/mL;
发酵种子培养基(g/L):蛋白胨10,氯化钠10,酵母粉5,pH 7.0,装液量10mL/20mL,必要时在培养基中添加Amp 100μg/mL;
发酵培养基(g/L):甘油10,Na2HPO4·12H2017.11,KH2PO43,NaCl 0.5,NH4Cl 1,MgSO4·7H2O0.492,CaCl2·6H2O 0.022,装液量10mL/20mL,必要时在培养基中添加Amp 100μg/mL;
(1)种子培养:从固体培养基平板上挑取单菌落接种至试管中培养,装液量为10mL/20mL,在培养基中添加Amp 100μg/mL,37℃,200r/min培养12h,转接发酵培养基;
(2)发酵培养:将培养好的种子液按10%(v/v)的接种量,接种至装有10mL发酵培养基的20mL试管中进行培养,在培养基中添加Amp 100μg/mL,37℃,200r/min培养,当菌体生长至OD600为0.4-0.6时,添加0.4mM IPTG诱导表达,继续培养至48h。
二、目标产物苯酚含量的测定:高效液相色谱(HPLC)检测
苯酚标准溶液的配制:苯酚工作液:取苯酚标准品,用去离子水配制成1g/L的溶液。
待测样品溶液的制备:发酵结束后,将获得的发酵液12000rpm,2min离心后取上清,用0.22μm滤膜过滤,取到待上机的待测样品溶液。
色谱条件:
色谱分离柱:ZORBAX Eclipse XDB-C184.6×250mm,5μm
流动相:乙腈-水=40:60(v/v)
检测波长:210nm;流速:0.7mL/min;进样量:10μL;柱温:30℃。根据苯酚标准品溶液和待测样品溶液的HPLC图谱获得大肠杆菌工程菌株发酵液中苯酚的含量。
结果显示,苯酚标准品的HPLC图谱如图2中a所示,从图中可以看出,苯酚标准品的出峰时间为7.014min。重组菌株PH1发酵液的HPLC图谱如图2中b所示,在与苯酚标准品保留时间一致的位置也出现了目标峰。根据以上结果,判定重组菌株PH1的发酵液中含有苯酚,即利用甘油生产苯酚的途径已打通。
利用甘油生产苯酚产量结果如图3所示:
重组菌PH1的发酵液中苯酚产量为21.30mg/L;
重组菌PH2的发酵液中苯酚产量为27.68mg/L;
重组菌PH3的发酵液中苯酚产量为57.66mg/L。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受实施例的限制,其它任何未背离本发明的精神实质与原理下所做的改变、修饰、组合、替代、简化均应为等效替换方式,都包含在本发明的保护范围之内。
<110> 曲阜师范大学
<120>一种利用甘油发酵生产苯酚的工程菌株的构建方法及应用
<160> 21
<210> 1
<211> 455
<212> PCR
<213>大肠杆菌种(E.coli)
<400> 1
Met Asn Tyr Pro Ala Glu Pro Phe Arg Ile Lys Ser Val Glu Thr Val
1 5 10 15
Ser Met Ile Ser Arg Asp Glu Arg Val Lys Lys Met Gln Glu Ala Gly
20 25 30
Tyr Asn Thr Phe Leu Leu Asn Ser Lys Asp Ile Tyr Ile Asp Leu Leu
35 40 45
Thr Asp Ser Gly Thr Asn Ala Met Ser Asp Lys Gln Trp Ala Gly Met
50 55 60
Met Ile Gly Asp Glu Ala Tyr Ala Gly Ser Glu Asn Phe Tyr His Leu
65 70 75 80
Glu Lys Thr Val Lys Glu Leu Phe Gly Phe Lys His Ile Val Pro Thr
85 90 95
His Gln Gly Arg Gly Ala Glu Asn Leu Leu Ser Gln Leu Ala Ile Lys
100 105 110
Pro Gly Gln Tyr Val Ala Gly Asn Met Tyr Phe Thr Thr Thr Arg Phe
115 120 125
His Gln Glu Lys Asn Gly Ala Thr Phe Val Asp Ile Val Arg Asp Glu
130 135 140
Ala His Asp Ala Ser Leu Asn Leu Pro Phe Lys gly Asp Ile Asp Leu
145 150 155 160
Asn Lys Leu Ala Thr Leu Ile Lys Glu Lys Gly Ala Glu Asn Ile Ala
165 170 175
Tyr Ile Cys Leu Ala Val Thr Val Asn Leu Ala Gly Gly Gln Pro Val
180 185 190
Ser Met Ala Asn Met Arg Ala Val His Glu Met Ala Ser Thr Tyr Gly
195 200 205
Ile Lys Ile Phe Tyr Asp Ala Thr Arg Cys Val Glu Asn Ala Tyr Phe
210 215 220
Ile Lys Glu Gln Glu Ala Gly Tyr Glu Asn Val Ser Ile Lys Asp Ile
225 230 235 240
Val His Glu Met Phe Ser Tyr Ala Asp Gly Cys Thr Met Ser Gly Lys
245 250 255
Lys Asp Cys Leu Val Asn Ile Gly Gly Phe Leu Cys Met Asn Asp Glu
260 265 270
Glu Met Phe Ser Ala Ala Lys Glu Leu Val Val Val Tyr Glu Gly Met
275 280 285
Pro Ser Tyr Gly Gly Leu Ala Gly Arg Asp Met Glu Ala Met Ala Ile
290 295 300
Gly Leu Arg Glu Ala Met Gln Tyr Glu Tyr Ile Glu His Arg Val Lys
305 310 315 320
Gln Val Arg Tyr Leu Gly Asp Lys Leu Arg Glu Ala Gly Val Pro Ile
325 330 335
Val Glu Pro Thr Gly Gly His Ala Val Phe Leu Asp Ala Arg Arg Phe
340 345 350
Cys Pro His Leu Thr Gln Asp Gln Phe Pro Ala Gln Ser Leu Ala Ala
355 360 365
Ser Ile Tyr Met Glu Thr Gly Val Arg Ser Met Glu Arg Gly Ile Val
370 375 380
Ser Ala Gly Arg Ser Lys Glu Thr Gly Glu Asn His Arg Pro Lys Leu
385 390 395 400
Glu Thr Val Arg Leu Thr Ile Pro Arg Arg Val Tyr Thr Tyr Ala His
405 410 415
Met Asp Val Val Ala Asp Gly Ile Ile Lys Leu Tyr Gln His Lys Glu
420 425 430
Asp Ile Arg Gly Leu Thr Phe Val Tyr Glu Pro Lys Gln Leu Arg Phe
435 440 445
Phe Thr Ala Arg Phe Asp Phe Ile
450 455
<210> 2
<211> 1373
<212> DNA
<213>大肠杆菌种(E.coli)
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atgaattacc cggccgaacc tttccgcatc aagagtgtgg agaccgtgag tatgatcagc 60
cgcgacgaac gtgttaagaa gatgcaagag gccggctaca acacctttct gctgaacagc 120
aaagatatct atatcgacct gctgacagat agcggcacca acgccatgag cgataaacag 180
tgggccggca tgatgattgg tgatgaagca tacgccggca gcgagaactt ttaccacctg 240
gagaagaccg tgaaagagct gttcggcttt aagcacatcg tgccgacaca tcagggccgc 300
ggtgccgaaa acctgctgag ccagctggcc attaaaccgg gtcagtacgt tgccggtaac 360
atgtacttca ccacaacccg ctttcaccag gagaaaaatg gcgccacctt tgtggacatc 420
gtgcgtgatg aggcccatga tgccagcctg aatctgccgt tcaaaggcga catcgacctg 480
aacaaactgg ccaccctgat taaagaaaaa ggcgccgaga acatcgcata tatttgcctg 540
gccgtgaccg ttaatctggc aggtggtcaa cctgtgagca tggccaatat gcgtgccgtg 600
catgagatgg ccagcaccta tggcatcaag atcttttacg acgccacccg ttgcgtggaa 660
aacgcctact tcatcaaaga acaagaagct ggctatgaaa acgtgagcat taaagatatc 720
gttcacgaga tgttcagcta cgccgatggt tgcaccatga gcggcaagaa agattgcctg 780
gtgaacattg gcggcttcct gtgcatgaac gatgaggaaa tgtttagcgc cgccaaagag 840
ctggtggtgg tgtatgaagg catgccgagc tatggcggtt tagccggccg tgacatggag 900
gcaatggcca tcggtctgcg cgaagccatg cagtatgaat atattgagca tcgtgtgaaa 960
caggtgcgct atctgggtga taagctgcgt gaggccggtg tgccgattgt tgaaccgacc 1020
ggtggtcacg cagtttttct ggatgcccgc cgcttctgcc cgcatctgac ccaggatcag 1080
ttcccggcac agagtctggc cgccagcatt tacatggaaa ccggcgtgcg cagtatggaa 1140
cgtggtatcg ttagtgcagg ccgtagcaaa gagaccggcg agaaccaccg tccgaaactg 1200
gagaccgttc gcctgaccat tcctcgtcgc gtttacacct atgcccacat ggatgtggtg 1260
gccgatggca tcattaaact gtaccagcat aaggaagata ttcgcggcct gaccttcgtg 1320
tacgaaccga aacaactgcg tttcttcacc gcccgttttg attttattta aaa 1373
<210> 3
<211> 789
<212> DNA
<213>大肠杆菌种(E.coli)
<400> 3
atgaaacaga cgcaatacgt ggcacgcgag cccgatgcgc atggttttat cgattacccg 60
cagcaagagc atgcggtgtg gaacaccctg atcacccgcc agctgaaagt gatcgaaggc 120
cgtgcgtgcc aggaatacct ggacggcatc gaccagctga aattgccgca tgaccgcatt 180
ccgcaactgg gcgagatcaa caaggtgctg ggtgccacca ccggctggca ggttgcccgg 240
gttccggcgc tgatcccctt ccagaccttc ttcgaattgc tggccagcaa gcgctttccg 300
gtcgccacct tcatccgcac cccggaagag ctggactacc tgcaagagcc ggatatcttc 360
cacgagatct tcggccactg cccgctgctg accaatccct ggttcgccga attcacccac 420
acctacggca agctcggcct ggccgcgacc aaggaacaac gtgtgtacct ggcacgcttg 480
tactggatga ccatcgagtt tggcctgatg gaaaccgcgc aaggccgcaa aatctatggt 540
ggtggcatcc tctcgtcgcc gaaagagacc gtctacagtc tgtctgacga gcctgagcac 600
caggccttcg acccgatcga ggccatgcgt acaccctacc gcatcgacat tctgcaaccg 660
gtgtatttcg tactgccgaa catgaagcgc ctgttcgacc tggcccacga ggacatcatg 720
ggcatggtcc ataaagccat gcagctgggt ctgcatgcac cgaagtttcc acccaaggtc 780
gctgcctga 789
<210> 4
<211> 1011
<212> DNA
<213>大肠杆菌种(E.coli)
<400> 4
atgagacgag aacttgccat cgaattttcc cgcgtcaccg aatcagcggc gctggctggc 60
tacaaatggt taggacgcgg cgataaaaac accgcggacg gcgcggcggt aaacgccatg 120
cgtattatgc tcaaccaggt caacattgac ggcaccatcg tcattggtga aggtgaaatc 180
gacgaagcac cgatgctcta cattggtgaa aaagtcggta ctggtcgcgg cgacgcggta 240
gatattgctg ttgatccgat tgaaggcacg cgcatgacgg cgatgggcca ggctaacgcg 300
ctggcggtgc tggcagtagg cgataaaggc tgcttcctca atgcgccgga tatgtatatg 360
gagaagctga ttgtcgggcc gggagccaaa ggcaccattg atctgaacct gccgctggcg 420
gataacctgc gcaatgtagc ggcggcgctc ggcaaaccgt tgagcgaact gacggtaacg 480
attctggcta aaccacgcca cgatgccgtt atcgctgaaa tgcagcaact cggcgtacgc 540
gtatttgcta ttccggacgg cgacgttgcg gcctcaattc tcacctgtat gccagacagc 600
gaagttgacg tgctgtacgg tattggtggc gcgccggaag gcgtagtttc tgcggcggtg 660
atccgcgcat tagatggcga catgaacggt cgtctgctgg cgcgtcatga cgtcaaaggc 720
gacaacgaag agaatcgtcg cattggcgag caggagctgg cacgctgcaa agcgatgggc 780
atcgaagccg gtaaagtatt gcgcctgggc gatatggcgc gcagcgataa cgtcatcttc 840
tctgccaccg gtattaccaa aggcgatctg ctggaaggca ttagccgcaa aggcaatatc 900
gcgactaccg aaacgctgct gatccgcggc aagtcacgca ccattcgccg cattcagtcc 960
atccactatc tggatcgcaa agacccggaa atgcaggtgc acatcctctg a 1011
<210> 5
<211> 1997
<212> DNA
<213>大肠杆菌种(E.coli)
<400> 5
atgtcctcac gtaaagagct tgccaatgct attcgtgcgc tgagcatgga cgcagtacag 60
aaagccaaat ccggtcaccc gggtgcccct atgggtatgg ctgacattgc cgaagtcctg 120
tggcgtgatt tcctgaaaca caacccgcag aatccgtcct gggctgaccg tgaccgcttc 180
gtgctgtcca acggccacgg ctccatgctg atctacagcc tgctgcacct caccggttac 240
gatctgccga tggaagaact gaaaaacttc cgtcagctgc actctaaaac tccgggtcac 300
ccggaagtgg gttacaccgc tggtgtggaa accaccaccg gtccgctggg tcagggtatt 360
gccaacgcag tcggtatggc gattgcagaa aaaacgctgg cggcgcagtt taaccgtccg 420
ggccacgaca ttgtcgacca ctacacctac gccttcatgg gcgacggctg catgatggaa 480
ggcatctccc acgaagtttg ctctctggcg ggtacgctga agctgggtaa actgattgca 540
ttctacgatg acaacggtat ttctatcgat ggtcacgttg aaggctggtt caccgacgac 600
accgcaatgc gtttcgaagc ttacggctgg cacgttattc gcgacatcga cggtcatgac 660
gcggcatcta tcaaacgcgc agtagaagaa gcgcgcgcag tgactgacaa accttccctg 720
ctgatgtgca aaaccatcat cggtttcggt tccccgaaca aagccggtac ccacgactcc 780
cacggtgcgc cgctgggcga cgctgaaatt gccctgaccc gcgaacaact gggctggaaa 840
tatgcgccgt tcgaaatccc gtctgaaatc tatgctcagt gggatgcgaa agaagcaggc 900
caggcgaaag aatccgcatg gaacgagaaa ttcgctgctt acgcgaaagc ttatccgcag 960
gaagccgctg aatttacccg ccgtatgaaa ggcgaaatgc cgtctgactt cgacgctaaa 1020
gcgaaagagt tcatcgctaa actgcaggct aatccggcga aaatcgccag ccgtaaagcg 1080
tctcagaatg ctatcgaagc gttcggtccg ctgttgccgg aattcctcgg cggttctgct 1140
gacctggcgc cgtctaacct gaccctgtgg tctggttcta aagcaatcaa cgaagatgct 1200
gcgggtaact acatccacta cggtgttcgc gagttcggta tgaccgcgat tgctaacggt 1260
atctccctgc acggtggctt cctgccgtac acctccacct tcctgatgtt cgtggaatac 1320
gcacgtaacg ccgtacgtat ggctgcgctg atgaaacagc gtcaggtgat ggtttacacc 1380
cacgactcca tcggtctggg cgaagacggc ccgactcacc agccggttga gcaggtcgct 1440
tctctgcgcg taaccccgaa catgtctaca tggcgtccgt gtgaccaggt tgaatccgcg 1500
gtcgcgtgga aatacggtgt tgagcgtcag gacggcccga ccgcactgat cctctcccgt 1560
cagaacctgg cgcagcagga acgaactgaa gagcaactgg caaacatcgc gcgcggtggt 1620
tatgtgctga aagactgcgc cggtcagccg gaactgattt tcatcgctac cggttcagaa 1680
gttgaactgg ctgttgctgc ctacgaaaaa ctgactgccg aaggcgtgaa agcgcgcgtg 1740
gtgtccatgc cgtctaccga cgcatttgac aagcaggatg ctgcttaccg tgaatccgta 1800
ctgccgaaag cggttactgc acgcgttgct gtagaagcgg gtattgctga ctactggtac 1860
aagtatgttg gcctgaacgg tgctatcgtc ggtatgacca ccttcggtga atctgctccg 1920
gcagagctgc tgtttgaaga gttcggcttc actgttgata acgttgttgc gaaagcaaaa 1980
gaactgctgt aatgtaa 1997
<210> 6
<211> 31
<212> DNA
<213>人工合成
<400> 6
ATAGGTACCA TGAATTACCC GGCCGAACCT T 31
<210> 7
<211> 32
<212> DNA
<213>人工合成
<400>7
GCGCTGCAGT TAAATAAAAT CAAAACGGGC GG 32
<210> 8
<211> 64
<212> DNA
<213>人工合成
<400>8
ATACTGCAGC GCACAACAGA GGAAGAGGAG GTAACATATG AAACAGACGC AATACGTGGC 60
ACGC 64
<210> 9
<211> 60
<212> DNA
<213>人工合成
<400>9
ATAAAGCTTT CAGGCAGCGA CCTTGGGTGG AAACTTCGGT GCATGCAGAC CCAGCTGCAT 60
<210>10
<211> 46
<212> DNA
<213>人工合成
<400>10
GCGGGTACCA TATTCTGAAA TGAGCTGTTG ACAATTAATC ATCCGG 46
<210>11
<211> 45
<212> DNA
<213>人工合成
<400>11
TCGATGGCAA GTTCTCGTCT CATGGTCTGT TTCCTGTGTG AAATT 45
<210> 12
<211> 45
<212> DNA
<213>人工合成
<400>12
AATTTCACAC AGGAAACAGA CCATGAGACG AGAACTTGCC ATCGA 45
<210> 13
<211> 45
<212> DNA
<213>人工合成
<400>13
TATATATATA GGATCCTCAG AGGATGTGCA CCTGCATTTC CGGGT 45
<210> 14
<211> 76
<212> DNA
<213>人工合成
<400>14
ATAGGATCCT GTGGAATTGT GAGCGGATAA CAATTTCACA CAGGAAACAG ACCATGTCCT 60
CACGTAAAGA GCTTGC 76
<210> 15
<211> 76
<212> DNA
<213>人工合成
<400>15
GCCTCTAGAT TACAGCAGTT CTTTTGCTTT CGCAACAACG TTATCAACAG TGAAGCCGAA 60
CTCTTCAAAC AGCAGC 76
<210> 16
<211> 28
<212> DNA
<213>人工合成
<400>16
GCCGTCATGA ATTACCCGGC CGAACCTT 28
<210>17
<211> 28
<212> DNA
<213>人工合成
<400>17
ATAGAGCTCT CAGGCAGCGA CCTTGGGT 28
<210> 18
<211> 45
<212> DNA
<213>人工合成
<400>18
GCGCTCGAGA TATTCTGAAA TGAGCTGTTG ACAATTAATC ATCCG 45
<210> 19
<211> 45
<212> DNA
<213>人工合成
<400>19
TGTACCATGG TCTGTTTCCT GTGTGAAATT GTTATCCGCT CACAA 45
<210> 20
<211> 40
<212> DNA
<213>人工合成
<400>20
AATATCCATG GACATGAAAC AGACGCAATA CGTGGCACGC 40
<210> 21
<211> 43
<212> DNA
<213>人工合成
<400>21
AGCGCTCGAG TTAAATAAAA TCAAAACGGG CGGTGAAGAA ACG 43
Claims (5)
1.一种工程菌株在利用甘油发酵生产苯酚中的应用,其特征在于对所述工程菌株进行种子液培养,然后接种到含有甘油的发酵培养基中,进行发酵培养;
所述工程菌株是通过以下步骤构建得到的:
(2)在pTrc99a载体的多克隆位点Kpn I和Pst I之间插入序列表中序列2的碱基序列,同时在多克隆位点Pst I和Hind III之间插入序列表中序列3的碱基序列后得到的重组质粒命名为pTrc99a-tpl-p4m;
(2)将从质粒pTrc99a扩增的trc启动子插入到序列表中序列4的glpX基因上游,并一起插入到质粒pTrc99a的多克隆位点 Kpn I和BamH I之间,得到中间质粒pTrc99a-trcglpX,将序列表中序列5的tktA基因接入中间质粒pTrc99a-trcglpX的多克隆位点BamH I和Xba I之间,得到中间质粒pTrc99a-trcglpX-tktA,以重组质粒pTrc99a-tpl-p4m为模板,扩增得到tpl-p4m基因,通过BspH I和Sac I酶切位点接入中间质粒pTrc99a-trcglpX-tktA,得到重组质粒pTrc99a-tpl-p4m-trcglpX-tktA;
(3)从质粒pTrc99a扩增trc启动子,在pTrc99a-tpl-p4m-trcglpX-tktA载体的多克隆位点Xho I和Nco I之间插入酶切后的trc启动子后得到重组质粒pTrc99a-tpl-trcp4m-trcglpX-tktA,电转化ATCC31884,获得利用甘油发酵生产苯酚的工程菌株;
发酵培养基中含有甘油 10 g/L,Na2HPO4·12H20 17.11 g/L,KH2PO4 3 g/L,NaCl 0.5g/L,NH4Cl 1 g/L,MgSO4·7H2O 0.492 g/L,CaCl2·6H2O 0.022 g/L,装液量10mL/20mL。
2.根据权利要求1所述的应用,其特征在于种子液培养为从固体培养基平板上挑取单菌落接种至试管中培养,装液量为10mL/20mL,在培养基中添加Amp 100μg/mL,37℃,200r/min培养12h,转接发酵培养基。
3.根据权利要求2所述的应用,其特征在于种子液培养基中含有蛋白胨 10 g/L,氯化钠 10 g/L,酵母粉 5 g/L,pH 7.0。
4.根据权利要求2所述的应用,其特征在于固体平板培养基中含有蛋白胨 10 g/L,氯化钠 10 g/L,酵母粉 5 g/L,琼脂 15 g/L,pH 7.0。
5.根据权利要求1所述的应用,其特征在于发酵培养操作为:
将培养好的种子液按10%(v/v)的接种量,接种至装有10mL发酵培养基的20mL试管中进行培养,在培养基中添加Amp 100μg/mL,37℃,200r/min培养,当菌体生长至OD600为0.4-0.6时,添加0.4mM IPTG诱导表达,继续培养至48h。
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Non-Patent Citations (5)
Title |
---|
Construction of a novel phenol synthetic pathway in Escherichia coli through 4-hydroxybenzoate decarboxylation;Miao LT et al;《Appl Microbiol Biotechnol》;20150312;第99卷(第12期);5163-5173 |
Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose;Wierckx N et al;《Applied and Environmental Microbiology》;20051231;第72卷(第12期);8221-8227 |
Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: the role of extra copies of glpK, glpX, and tktA genes;Gottlieb K et al;《Microbial Cell Factories》;20140711;第13卷(第1期);1-16 |
Metabolic engineering of Escherichia for the production of phenol from glucose;Kim B et al;《Biotechnol J》;20131011;第9卷(第5期);621-629 |
Production of tyrosine through phenylalanine hydroxylation bypasses the intrinsic feedback inhibition in Escherichia coli;Huang J et al;《J Ind Microbiol Biotechnol》;20150203;第42卷(第4期);655-659 |
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