CN114774423A - 一种大肠杆菌戊二胺响应启动子及应用 - Google Patents
一种大肠杆菌戊二胺响应启动子及应用 Download PDFInfo
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Abstract
本发明公开了一种大肠杆菌戊二胺响应启动子及应用。从头合成谷氨酸棒状杆菌中的丙酮酸羧化酶并在大肠杆菌中异源表达,PCR拷贝大肠杆菌中戊二胺响应启动子PgrcA及赖氨酸脱羧酶cadA,构建了PcadR‑PgrcA‑pyc‑trc‑cadA‑rrnB重组质粒。该质粒在赖氨酸高产的大肠杆菌中生长良好,基因表达稳定,葡萄糖被高效利用,能够响应戊二胺,发酵周期短,副产物少。从发酵结果可以看出,重组的大肠杆菌KAGPC的戊二胺产量较对照组有显著提高。
Description
技术领域
本发明属于菌株代谢改造领域,具体涉及一种大肠杆菌戊二胺响应启动子及应用。
背景技术
1,5-戊二胺(1,5-Diaminopentane) ,即尸胺(Cadaverine),又称为1,5-二氨基戊烷,是一种普遍存在于生物体内的生物胺。1,5-戊二胺在工业、农业和医药领域上有着广泛的应用。在工业上,1,5-戊二胺可与二元酸反应合成生物基高分子材料聚酰胺(俗称尼龙,PA);在农业上,可用于植物生长代谢的调节,果实质量和产量的提升;在医学上,可用于治疗痢疾的药物中。目前,聚酰胺在全球范围的需求量与日俱增。除了传统的化学合成1,5-戊二胺,生物法制备也日渐成熟,逐渐取代化学合成法,成为1,5-戊二胺合成的主流方法。其中,生物法主要分为生物催化法和细胞发酵法。催化法主要以L-赖氨酸为原料,通过赖氨酸脱羧酶脱羧生成1,5-戊二胺。但需要昂贵的辅酶且重复利用率低下。相较而言,发酵法具备条件温和,原料多样,生产成本低过程可控等优点。但1,5戊二胺对宿主菌具有毒性,导致细胞生产效率低下。
随着合成生物学的发展,大肠杆菌可作为模式工业菌株,进行大宗化学品的生产,其中就包括了1,5-戊二胺。但是1,5-戊二胺本身的物化性质,碱性,微毒,使大肠杆菌难以在高浓度的戊二胺中进行正常的生理活动。需要开发启动子满足生产需要。
发明内容
针对现有技术的不足,本发明提供了一种大肠杆菌戊二胺响应启动子及应用,通过挖掘大肠杆菌内源性戊二胺响应启动子并对关键基因进行调控,使戊二胺的产量得到进一步的提升达到14.1g/L。
为解决现有技术问题,本发明采取的技术方案:
一种大肠杆菌戊二胺响应启动子,所述启动子的核苷酸序列如SEQ ID No.7所示。
一种重组质粒PcadR-PgrcA-GFP,含有上述的大肠杆菌戊二胺响应启动子的核苷酸的质粒。
上述重组质粒PcadR-PgrcA-GFP构建的重组质粒PcadR-PgrcA-pyc-trc-cadA-rrnB构建方法,包括以下步骤:构建戊二胺响应启动子筛选质粒PcadR-GFP,所用引物中PET28a和Ptrc99a的引物带有NcoI和XhoI酶切位点,通过同源重组获得PcadR-GFP;筛选大肠杆菌内源性戊二胺响应启动子PgrcA,合成的PgrcA启动子上游引物带有BglII酶切位点,下游引物带有XbaI酶切位点,通过酶切链接获得PcadR-PgrcA-GFP;筛选出的响应启动子PgrcA用于调控谷氨酸棒状杆菌的基因丙酮酸羧化酶pyc,并将赖氨酸脱羧酶cadA构建在同一质粒上构建重组质粒PcadR-PgrcA-pyc-trc-cadA-rrnB;使用引物扩增丙酮酸羧化酶pyc和赖氨酸脱羧酶cadA,通过同源重组成功构建PcadR-PgrcA-pyc-trc-cadA-rrnB,将重组质粒导入克隆载体Trans1-T1,经过LB平板初筛后,选取平板上生长的单菌落进行PCR验证,再将阳性菌株送测。
一种重组菌株ka30-PcadR-PgrcA-pyc-trc-cadA-rrnB,表达上述的重组质粒PcadR-PgrcA-pyc-trc-cadA-rrnB的赖氨酸高产大肠杆菌ka30。
上述重组菌株ka30-PcadR-PgrcA-pyc-trc-cadA-rrnB在合成戊二胺上的应用。
有益效果:
与现有技术相比,本发明一种大肠杆菌戊二胺响应启动子及应用,通过挖掘大肠杆菌内源性戊二胺响应启动子并对关键基因进行调控,使戊二胺的产量得到进一步的提升达到14.1g/L。
附图说明
图1为本发明中HPLC检测戊二胺的生成,其中,(a)为戊二胺标准品;(b)为实施例戊二胺的产量与对照组的对比情况。
图2为重组蛋白表达情况。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明,但这并非是对本发明的限制,本领域技术人员根据本发明的基本思想,可以做出各种修改或改进,但是只要不脱离本发明的基本思想,均在本发明的范围之内。
实施例中未提及到的技术均为本领域常规技术,另外,使用的大肠杆菌Trans1-T1、pET28a, Ptrc99a等材料都是商业化产品,可直接购买。
实施例中所述的葡萄糖,戊二胺的浓度均指的是该体系中的终浓度。
戊二胺测定方法:Agilent YMC Carotenoid色谱柱,流动相为5%乙腈和0.5%三氟乙酸;流速0.8 mL·min-1;示差检测器。
实施例1 PcadR-GFP启动子筛选质粒的构建
以质粒PET28a和质粒Ptrc99a分别为模板进行PCR扩增,
所用PET28a为模板:
上游引物带有酶切位点NcoI,序列如SEQ ID NO.1:
GACGGAGCTCGAATTTTACTCTTTATCACCCAGCAGTACGGAT;
下游引物带有酶切位点XhoI,序列如SEQ ID NO.2:
AGGAGATATACCATGATGGCCACCCCACACAT;
所用Ptrc99a为模板
上游引物带有酶切位点NcoI,序列如SEQ ID NO.3所示:
CATGccatggaattcgagctcgg
下游引物带有酶切位点XhoI,序列如SEQ ID NO.4所示:
ccgCTCGAGaaaaggccatccgtcag。
反应条件为:95℃ 2 min ,95℃ 20 s,55℃ 20 s,72℃ 10 s,共30个循环;72℃5 min。得到的序列经1%琼脂糖凝胶电泳后回收相应片段。将回收的片段用Takara 公司的NcoI和XhoI酶切,酶切反应体系为:10×buffer 1 μL,NcoI 1 μL,XhoI 1 μL,两种回收片段各7 μL。酶切体系于37℃条件下反应1小时。两片段有相同的黏性末端,通过T4连接酶链接构成重组质粒,反应体系为:Takara公司的T4 Ligase 1 μL,10×T4 DNA Ligase Buffer1 μL,两种片段各4 μL。反应体系25℃反应1小时。将连接产物转化到大肠杆菌 Trans1-T1中,PCR筛选阳性菌株进行DNA测序验证,验证PcadR-GFP启动子筛选质粒的构建正确。
在PcadR-GFP启动子筛选质粒上插入GFP荧光蛋白作为报告基因用于启动子筛选。GFP基因可合成,作为模板进行PCR扩增。其序列如下:
SEQ ID NO.5
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
对合成的GFP基因为模板进行PCR
所用上游引物序列如SEQ ID NO.6:
ATGGTGAGCAAGGGCGAG
所用下游引物为序列如SEQ ID NO.7:
TACTTGTACAGCTCGTCCATGCC
反应条件为:95℃ 2 min ,95℃ 20 s,55℃ 20 s,72℃ 10 s,共30个循环;72℃5 min。得到的序列经1%琼脂糖凝胶电泳后回收相应片段。同时对PcadR-GFP分别用Takara公司的限制性内切酶SalI和NcoI酶切,酶切反应体系为:10×buffer 1 μL,NcoI1 μL,SalI1 μL,回收片段7 μL。将回收的GFP基因和PcadR-GFP用T4连接酶连接,反应体系为:Takara公司的T4 Ligase 1 μL,10×T4 DNA Ligase Buffer 1 μL,两种片段各4 μL。反应体系25℃反应1小时。将连接产物转化到大肠杆菌 Trans1-T1中,PCR筛选阳性菌株PcadR-GF进行DNA测序验证
实施例2 PcadR-PgrcA-GFP质粒的构建
通过转录组分析大肠杆菌对戊二胺做出响应的基因,转录分析方法和结果可见:Wang X , Guo X , Wang J , et al. Ameliorating end-product inhibition toimprove cadaverine production in engineered Escherichia coli and itsapplication in the synthesis of bio-based diisocyanates[J]. Synthetic andSystems Biotechnology, 2021, 6( 4):243-253.,得到确认启动子,
戊二胺下调启动子PgrcA的核苷酸序列如SEQ ID No.8所示:
TTGCGACCATACTTGATGTGTGGTTTTTATTGATTTAAATCAAAGATTCAAGGGTGTTTGAGGAGTATATATACACTCAAGCAACAATGGTTTTACCAATTGGCCGCGACAGGCTGAACAAATCAAATAATTTTGCCGGGGAGGCATCAC
以大肠杆菌Trans1-T1的基因组为模板,以启动子上下游序列为引物。
所用上游引物带有酶切位点XbaI,序列如SEQ ID NO.9所示:
AGATCTTTGCGACCATACTTGATGT。
下游引物带有酶切位点BglII,序列如SEQ ID NO.10所示:
TCTAGAGTGATGCCTCCCCGGC。
反应条件为:95℃ 2 min ,95℃ 20 s,55℃ 20 s,72℃ 10 s,共30个循环;72℃5 min。得到的序列经1%琼脂糖凝胶电泳后回收相应片段。将回收的片段及上述构建的重组质粒PcadR-GFP分别用Takara 公司的限制性内切酶XbaI和BglII酶切,酶切反应体系为:10×buffer 1 μL,XbaI1 μL,BglII 1 μL,回收片段7 μL。将回收的启动子和PcadR-GFP启动子筛选质粒用T4连接酶连接,反应体系为:Takara公司的T4 Ligase 1 μL,10×T4 DNALigase Buffer 1 μL,两种片段各4 μL。反应体系25℃反应1小时。将连接产物转化到大肠杆菌 Trans1-T1中,PCR筛选阳性菌株进行DNA测序验证,验证重组质粒PcadR-PgrcA-GFP构建正确。
实施例3 响应启动子PgrcA的表征
将实施例2的阳性菌株接种含不同戊二胺浓度的LB液体培养基于96孔板,LB液体培养基组成为:蛋白胨10 g/L、酵母粉5 g/L、氯化钠5 g/L,其中含戊二胺的浓度分别为0g/L、10 g/L、20 g/L、30 g/L、40 g/L,于37 ℃、200 rpm 条件下振荡培养过夜。12小时后测量菌体的生物量(OD600)和荧光值(GFP),并计算GFP/OD600,筛选出对戊二胺响应较好的启动子PgrcA。
实施例4 Trans1-PcadR-PgrcA-pyc表达菌株的构建
以具有丙酮酸羧化酶(pyc)的谷氨酸棒状杆菌(该菌可购于中国普通微生物菌种保藏管理中心,编号ATCC13032)的基因组(如SEQ NO.12所示)为模板,通过常规PCR扩增丙酮酸羧化酶的核苷酸的序列。
所用上游引物带有同源臂,序列如SEQ ID NO.11所示:
CATCGCCACC GTGTCGATTCACACATCTTCAACG
下游引物带有同源臂,序列如SEQ ID NO.12所示:
GCAGGTCGACTTAGGAAACGACGACGATCAAGTC
反应条件为:95℃ 2 min ,95℃ 15 s,55℃ 20 s,72℃ 210 s,共30个循环;72℃5 min。得到的序列经1%琼脂糖凝胶电泳后回收相应片段。
将PcadR-PgrcA-GFP质粒中GFP基因删除,所需PCR引物如下
所用上游引物带有同源臂,序列如为SEQ ID NO.13所示:
GAGTCGACACGGTGGCGATGGATCCG
下游引物带有同源臂,序列如为SEQ ID NO.14所示:
CGTTTCCTAAAGTTGGCTGCTGCCAC
反应条件为: 95℃ 2 min ,95℃ 15 s,55℃ 20 s,72℃ 210 s,共30个循环;72℃ 5 min。得到的序列经1%琼脂糖凝胶电泳后回收相应片段。将上述PCR扩增得到的两个片段通过同源臂进行同源重组获得重组质粒,重组反应体系为:使用Vazyme公司的5 X CE IIbuffer 4 μL,Exnase II 2 μL,两个基本片段各7 μL。连接于37℃下反应1小时。将连接产物转化大肠杆菌Trans1-T1。PCR筛选阳性菌株Trans1-PcadR-PgrcA-pyc并进行DNA测序,验证重组质粒PcadR-PgrcA-pyc构建正确。
谷氨酸棒状杆菌来源的合成丙酮酸羧化酶基因pyc的核苷酸序列
SEQ ID No.15
gtgtcgactcacacatcttcaacgcttccagcattcaaaaagatcttggtagcaaaccgcggcgaaatcgcggtccgtgctttccgtgcagcactcgaaaccggtgcagccacggtagctatttacccccgtgaagatcggggatcattccaccgctcttttgcttctgaagctgtccgcattggtaccgaaggctcaccagtcaaggcgtacctggacatcgatgaaattatcggtgcagctaaaaaagttaaagcagatgccatttacccgggatacggcttcctgtctgaaaatgcccagcttgcccgcgagtgtgcggaaaacggcattacttttattggcccaaccccagaggttcttgatctcaccggtgataagtctcgcgcggtaaccgccgcgaagaaggctggtctgccagttttggcggaatccaccccgagcaaaaacatcgatgagatcgttaaaagcgctgaaggccagacttaccccatctttgtgaaggcagttgccggtggtggcggacgcggtatgcgttttgttgcttcacctgatgagcttcgcaaattagcaacagaagcatctcgtgaagctgaagcggctttcggcgatggcgcggtatatgtcgaacgtgctgtgattaaccctcagcatattgaagtgcagatccttggcgatcacactggagaagttgtacacctttatgaacgtgactgctcactgcagcgtcgtcaccaaaaagttgtcgaaattgcgccagcacagcatttggatccagaactgcgtgatcgcatttgtgcggatgcagtaaagttctgccgctccattggttaccagggcgcgggaaccgtggaattcttggtcgatgaaaagggcaaccacgtcttcatcgaaatgaacccacgtatccaggttgagcacaccgtgactgaagaagtcaccgaggtggacctggtgaaggcgcagatgcgcttggctgctggtgcaaccttgaaggaattgggtctgacccaagataagatcaagacccacggtgcagcactgcagtgccgcatcaccacggaagatccaaacaacggcttccgcccagataccggaactatcaccgcgtaccgctcaccaggcggagctggcgttcgtcttgacggtgcagctcagctcggtggcgaaatcaccgcacactttgactccatgctggtgaaaatgacctgccgtggttccgactttgaaactgctgttgctcgtgcacagcgcgcgttggctgagttcaccgtgtctggtgttgcaaccaacattggtttcttgcgtgcgttgctgcgggaagaggacttcacttccaagcgcatcgccaccggattcattgccgatcacccgcacctccttcaggctccacctgctgatgatgagcagggacgcatcctggattacttggcagatgtcaccgtgaacaagcctcatggtgtgcgtccaaaggatgttgcagctcctatcgataagctgcctaacatcaaggatctgccactgccacgcggttcccgtgaccgcctgaagcagcttggcccagccgcgtttgctcgtgatctccgtgagcaggacgcactggcagttactgataccaccttccgcgatgcacaccagtctttgcttgcgacccgagtccgctcattcgcactgaagcctgcggcagaggccgtcgcaaagctgactcctgagcttttgtccgtggaggcctggggcggcgcgacctacgatgtggcgatgcgtttcctctttgaggatccgtgggacaggctcgacgagctgcgcgaggcgatgccgaatgtaaacattcagatgctgcttcgcggccgcaacaccgtgggatacaccccgtacccagactccgtctgccgcgcgtttgttaaggaagctgccagctccggcgtggacatcttccgcatcttcgacgcgcttaacgacgtctcccagatgcgtccagcaatcgacgcagtcctggagaccaacaccgcggtagccgaggtggctatggcttattctggtgatctctctgatccaaatgaaaagctctacaccctggattactacctaaagatggcagaggagatcgtcaagtctggcgctcacatcttggccattaaggatatggctggtctgcttcgcccagctgcggtaaccaagctggtcaccgcactgcgccgtgaattcgatctgccagtgcacgtgcacacccacgacactgcgggtggccagctggcaacctactttgctgcagctcaagctggtgcagatgctgttgacggtgcttccgcaccactgtctggcaccacctcccagccatccctgtctgccattgttgctgcattcgcgcacacccgtcgcgataccggtttgagcctcgaggctgtttctgacctcgagccgtactgggaagcagtgcgcggactgtacctgccatttgagtctggaaccccaggcccaaccggtcgcgtctaccgccacgaaatcccaggcggacagttgtccaacctgcgtgcacaggccaccgcactgggccttgcggatcgtttcgaactcatcgaagacaactacgcagccgttaatgagatgctgggacgcccaaccaaggtcaccccatcctccaaggttgttggcgacctcgcactccacctcgttggtgcgggtgtggatccagcagactttgctgccgatccacaaaagtacgacatcccagactctgtcatcgcgttcctgcgcggcgagcttggtaaccctccaggtggctggccagagccactgcgcacccgcgcactggaaggccgctccgaaggcaaggcacctctgacggaagttcctgaggaagagcaggcgcacctcgacgctgatgattccaaggaacgtcgcaatagcctcaaccgcctgctgttcccgaagccaaccgaagagttcctcgagcaccgtcgccgcttcggcaacacctctgcgctggatgatcgtgaattcttctacggcctggtcgaaggccgcgagactttgatccgcctgccagatgtgcgcaccccactgcttgttcgcctggatgcgatctctgagccagacgataagggtatgcgcaatgttgtggccaacgtcaacggccagatccgcccaatgcgtgtgcgtgaccgctccgttgagtctgtcaccgcaaccgcagaaaaggcagattcctccaacaagggccatgttgctgcaccattcgctggtgttgtcaccgtgactgttgctgaaggtgatgaggtcaaggctggagatgcagtcgcaatcatcgaggctatgaagatggaagcaacaatcactgcttctgttgacggcaaaatcgatcgcgttgtggttcctgctgcaacgaaggtggaaggtggcgacttgatcgtcgtcgtttcctaa
实施例5 构建BL21(DE3)- PcadR-grcA-pyc-trc-cadA重组菌株
从菌株KA30中获取赖氨酸脱羧酶cadA,该菌株获取为:CN113817762A。以该基因为模板,通过常规PCR扩增赖氨酸脱羧酶(cadA)
所用上游引物带有同源臂,序列如SEQ ID NO.16所示:
GCTGCTAACATTGCGACCATACTTGATGTGTG
下游引物带有同源臂,序列如SEQ ID NO.17所示:
GCAGCCAACTTTAGGAAACGACGACGATCAAGTC
反应条件为:95℃ 2 min ,95℃ 15 s,55℃ 20 s,72℃ 180 s,共30个循环;72℃5 min。得到的序列经1%琼脂糖凝胶电泳后回收相应片段。将载体质粒PcadR-PgrcA-pyc通过PCR进行线性化,所用PCR引物如下:
所用上游引物带有同源臂,序列如SEQ ID NO.18所示:
ATGGTCGCAATGTTAGCAGCCGGATCTCA
所用下游引物带有同源臂,序列SEQ ID NO.19所示:
CGTTTCCTAAAGTTGGCTGCTGCCAC
反应条件为:95℃ 2 min ,95℃ 15 s,55℃ 20 s,72℃ 360 s,共30个循环;72℃5 min。得到的序列经1%琼脂糖凝胶电泳后回收相应片段。将目的片段和载体片段通过同源重组构成新的质粒。所用的同源重组体系如下:使用Vazyme公司的5 X CE II buffer 4 μL,Exnase II 2 μL,基因片段7 μL,载体7 μL。连接于37℃下反应1小时。将连接产物转化到Trans1大肠杆菌中,PCR筛选阳性菌株Trans1-PcadR-PgrcA-pyc-trc-cadA-rrnB并进行DNA测序,验证重组质粒Trans1-PcadR-PgrcA-pyc-trc-cadA-rrnB构建正确。将重组菌株在LB/Kan 5 mL培养基中过夜培养,使用天根质粒小提试剂盒提取质粒转化到BL21(DE3)中,构建BL21(DE3)-PcadR-PgrcA-pyc-trc-cadA重组菌株。
合成赖氨酸脱羧酶基因CadA的核酸序列,序列如SEQ ID No.20所示:
ATGAACGTTATTGCAATATTGAATCACATGGGGGTTTATTTTAAAGAAGAACCCATCCGTGAACTTCATCGCGCGCTTGAACGTCTGAACTTCCAGATTGTTTACCCGAACGACCGTGACGACTTATTAAAACTGATCGAAAACAATGCGCGTCTGTGCGGCGTTATTTTTGACTGGGATAAATATAATCTCGAGCTGTGCGAAGAAATTAGCAAAATGAACGAGAACCTGCCGTTGTACGCGTTCGCTAATACGTATTCCACTCTCGATGTAAGCCTGAATGACCTGCGTTTACAGATTAGCTTCTTTGAATATGCGCTGGGTGCTGCTGAAGATATTGCTAATAAGATCAAGCAGACCACTGACGAATATATCAACACTATTCTGCCTCCGCTGACTAAAGCACTGTTTAAATATGTTCGTGAAGGTAAATATACTTTCTGTACTCCTGGTCACATGGGCGGTACTGCATTCCAGAAAAGCCCGGTAGGTAGCCTGTTCTATGATTTCTTTGGTCCGAATACCATGAAATCTGATATTTCCATTTCAGTATCTGAACTGGGTTCTCTGCTGGATCACAGTGGTCCACACAAAGAAGCAGAACAGTATATCGCTCGCGTCTTTAACGCAGACCGCAGCTACATGGTGACCAACGGTACTTCCACTGCGAACAAAATTGTTGGTATGTACTCTGCTCCGGCAGGCAGCACCATTCTGATTGACCGTAACTGCCACAAATCGCTGACCCACCTGATGATGATGAGCGATGTTACGCCAATCTATTTCCGCCCGACCCGTAACGCTTACGGTATTCTTGGTGGTATCCCACAGAGTGAATTCCAGCACGCTACCATTGCTAAGCGCGTGAAAGAAACACCAAACGCAACCTGGCCGGTACATGCTGTAATTACCAACTCTACCTATGATGGTCTGCTGTACAACACCGACTTCATCAAGAAAACACTGGATGTGAAATCCATCCACTTTGACTCCGCGTGGGTGCCTTACACCAACTTCTCACCGATTTACGAAGGTAAATGCGGTATGAGCGGTGGCCGTGTAGAAGGGAAAGTGATTTACGAAACCCAGTCCACTCACAAACTGCTGGCGGCGTTCTCTCAGGCTTCCATGATCCACGTTAAAGGTGACGTAAACGAAGAAACCTTTAACGAAGCCTACATGATGCACACCACCACTTCTCCGCACTACGGTATCGTGGCGTCCACTGAAACCGCTGCGGCGATGATGAAGGGTAATGCTGGTAAGCGTCTGATCAACGGTTCCATTGAACGTGCGATCAAATTCCGTAAAGAGATCAAACGTCTGAGAACGGAATCTGATGGCTGGTTCTTTGATGTTTGGCAGCCGGATCATATCGATACGACTGAATGCTGGCCGCTGCGTTCTGACAGCACCTGGCACGGCTTCAAAAACATCGATAACGAGCACATGTATCTTGACCCGATCAAAGTCACCCTGCTGACTCCGGGGATGGAAAAAGACGGCACCATGAGCGACTTTGGTATTCCGGCCAGCATCGTGGCGAAATACCTCGACGAACATGGCATCGTTGTTGAGAAAACCGGTCCGTATAACCTGCTGTTCCTGTTCAGCATCGGTATCGATAAGACCAAAGCACTGAGCCTGCTGCGTGCTCTGACTGACTTCAAACGTGCGTTCGACCTGAACCTGCGTGTGAAAAACATGCTGCCGTCTCTGTATCGTGAAGATCCTGAATTCTATGAAAACATGCGTATTCAGGAACTGGCTCAAAATATCCACAAACTGATTGTTCACCACAATCTGCCGGATCTGATGTATCGCGCATTTGAAGTGCTGCCGACGATGGTAATGACTCCGTATGCTGCGTTCCAGAAAGAGCTGCACGGTATGACCGAAGAAGTTTACCTCGACGAAATGGTAGGTCGTATTAACGCCAATATGATCCTTCCGTATCCGCCGGGAGTTCCTCTGGTAATGCCGGGTGAAATGATCACCGAAGAAAGCCGTCCGGTTCTGGAGTTCCTGCAGATGCTGTGTGAAATCGGCGCTCACTATCCGGGCTTTGAAACCGATATTCACGGTGCATACCGTCAGGCTGATGGCCGCTATACCGTTAAGGTATT
实施例6 丙酮酸羧化酶和赖氨酸脱羧酶的表达
将得到的BL21(DE3)-PcadR-PgrcA-pyc-trc-cadA重组菌株在500mL锥形瓶装液量100mL的LB培养基中37℃,200rpm培养,当OD600达到0.4-0.6时,添加1‰IPTG诱导,诱导温度为18℃,培养24小时后收菌超声破碎,将破碎后的菌液离心上清和沉淀分离,对上清和沉淀跑蛋白胶验证(图2)。
实施例7 重组质粒PcadR-PgrcA-pyc-trc-cadA导入赖氨酸高产的大肠杆菌菌株ka30,该菌株获得来源:CN113817762A
使用天根质粒小提试剂盒提取上述实施例5中的重组菌株的质粒,将提取出来的重组质粒导入赖氨酸高产的大肠杆菌的感受态中进行发酵验证ka30-PcadR-PgrcA-pyc-trc-cadA。
大肠杆菌感受态制备的方法:
在LB平板上,挑取单菌落,然后接入摇管(LB培养基),温度37℃,200rpm,培养10-12h。按以1%的接种量转接到500 mL摇瓶LB的液体培养基中,装液量为50ml,温度37 ℃,200rmp进行培养。待菌液光密度值OD 600生长至0.4-0.5,将摇瓶放到冰上冰浴20 min(停止生长活动)后,在已预冷的离心机中,4 ℃下,4000 rpm,离心10 min,去除上清后收集菌体。用10 mL 0.1mol/L预冷(4℃)的CaCl2(包含20%甘油)溶液重悬菌体,放置于冰上静置15min,4 ℃下,4000 rpm,离心10 min(该步骤重复两次)。去除上清,最后添加1 mL提前放置冰箱预冷的含15%甘油的0.1 mol·L-1CaCl2溶液悬浮细胞,冰上放置5 min后,即制成了感受态细胞悬液,分装成80μL/管的小份-80℃保存。
实施例8 重组大肠杆菌ka30-PcadR-PgrcA-pyc-trc-cadA一步法发酵产戊二胺的发酵验证
将重组大肠杆菌在50ml离心管中接入5ml种子培养基(种子培养基的配方表1所示),并富集培养至OD600为0.8,将富集的种子培养基接入30ml的发酵培养基(发酵培养基的配方如表2所示)中,接入初糖20g/L,当OD600为0.6时,加入诱导剂IPTG诱导赖氨酸脱羧酶的表达,分别在发酵24h和48h取样,测残糖含量和戊二胺产量,直到残糖耗完发酵结束。
表1 种子液的液体培养基的配方
表2 发酵液的液体培养基的配方
本发明通过对大肠杆菌内源性戊二胺响应启动子的挖掘,获得戊二胺响应启动子PgrcA并用于关键基因的表达调控,使大肠杆菌发酵产戊二胺的产量得到进一步的提升达到14.1g/L,具有良好的市场前景。
序列表
<110> 南京工业大学
<120> 一种大肠杆菌戊二胺响应启动子及应用
<160> 20
<170> SIPOSequenceListing 1.0
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ccgctcgaga aaaggccatc cgtcag 26
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<213> 人工序列(Artificial Sequence)
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atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 6
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
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atggtgagca agggcgag 18
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<213> 人工序列(Artificial Sequence)
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tacttgtaca gctcgtccat gcc 23
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ttgcgaccat acttgatgtg tggtttttat tgatttaaat caaagattca agggtgtttg 60
aggagtatat atacactcaa gcaacaatgg ttttaccaat tggccgcgac aggctgaaca 120
aatcaaataa ttttgccggg gaggcatcac 150
<210> 9
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agatctttgc gaccatactt gatgt 25
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tctagagtga tgcctccccg gc 22
<210> 11
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
catcgccacc gtgtcgattc acacatcttc aacg 34
<210> 12
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gcaggtcgac ttaggaaacg acgacgatca agtc 34
<210> 13
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gagtcgacac ggtggcgatg gatccg 26
<210> 14
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cgtttcctaa agttggctgc tgccac 26
<210> 15
<211> 3423
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gtgtcgactc acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 60
ggcgaaatcg cggtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 120
atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 180
attggtaccg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 240
gctaaaaaag ttaaagcaga tgccatttac ccgggatacg gcttcctgtc tgaaaatgcc 300
cagcttgccc gcgagtgtgc ggaaaacggc attactttta ttggcccaac cccagaggtt 360
cttgatctca ccggtgataa gtctcgcgcg gtaaccgccg cgaagaaggc tggtctgcca 420
gttttggcgg aatccacccc gagcaaaaac atcgatgaga tcgttaaaag cgctgaaggc 480
cagacttacc ccatctttgt gaaggcagtt gccggtggtg gcggacgcgg tatgcgtttt 540
gttgcttcac ctgatgagct tcgcaaatta gcaacagaag catctcgtga agctgaagcg 600
gctttcggcg atggcgcggt atatgtcgaa cgtgctgtga ttaaccctca gcatattgaa 660
gtgcagatcc ttggcgatca cactggagaa gttgtacacc tttatgaacg tgactgctca 720
ctgcagcgtc gtcaccaaaa agttgtcgaa attgcgccag cacagcattt ggatccagaa 780
ctgcgtgatc gcatttgtgc ggatgcagta aagttctgcc gctccattgg ttaccagggc 840
gcgggaaccg tggaattctt ggtcgatgaa aagggcaacc acgtcttcat cgaaatgaac 900
ccacgtatcc aggttgagca caccgtgact gaagaagtca ccgaggtgga cctggtgaag 960
gcgcagatgc gcttggctgc tggtgcaacc ttgaaggaat tgggtctgac ccaagataag 1020
atcaagaccc acggtgcagc actgcagtgc cgcatcacca cggaagatcc aaacaacggc 1080
ttccgcccag ataccggaac tatcaccgcg taccgctcac caggcggagc tggcgttcgt 1140
cttgacggtg cagctcagct cggtggcgaa atcaccgcac actttgactc catgctggtg 1200
aaaatgacct gccgtggttc cgactttgaa actgctgttg ctcgtgcaca gcgcgcgttg 1260
gctgagttca ccgtgtctgg tgttgcaacc aacattggtt tcttgcgtgc gttgctgcgg 1320
gaagaggact tcacttccaa gcgcatcgcc accggattca ttgccgatca cccgcacctc 1380
cttcaggctc cacctgctga tgatgagcag ggacgcatcc tggattactt ggcagatgtc 1440
accgtgaaca agcctcatgg tgtgcgtcca aaggatgttg cagctcctat cgataagctg 1500
cctaacatca aggatctgcc actgccacgc ggttcccgtg accgcctgaa gcagcttggc 1560
ccagccgcgt ttgctcgtga tctccgtgag caggacgcac tggcagttac tgataccacc 1620
ttccgcgatg cacaccagtc tttgcttgcg acccgagtcc gctcattcgc actgaagcct 1680
gcggcagagg ccgtcgcaaa gctgactcct gagcttttgt ccgtggaggc ctggggcggc 1740
gcgacctacg atgtggcgat gcgtttcctc tttgaggatc cgtgggacag gctcgacgag 1800
ctgcgcgagg cgatgccgaa tgtaaacatt cagatgctgc ttcgcggccg caacaccgtg 1860
ggatacaccc cgtacccaga ctccgtctgc cgcgcgtttg ttaaggaagc tgccagctcc 1920
ggcgtggaca tcttccgcat cttcgacgcg cttaacgacg tctcccagat gcgtccagca 1980
atcgacgcag tcctggagac caacaccgcg gtagccgagg tggctatggc ttattctggt 2040
gatctctctg atccaaatga aaagctctac accctggatt actacctaaa gatggcagag 2100
gagatcgtca agtctggcgc tcacatcttg gccattaagg atatggctgg tctgcttcgc 2160
ccagctgcgg taaccaagct ggtcaccgca ctgcgccgtg aattcgatct gccagtgcac 2220
gtgcacaccc acgacactgc gggtggccag ctggcaacct actttgctgc agctcaagct 2280
ggtgcagatg ctgttgacgg tgcttccgca ccactgtctg gcaccacctc ccagccatcc 2340
ctgtctgcca ttgttgctgc attcgcgcac acccgtcgcg ataccggttt gagcctcgag 2400
gctgtttctg acctcgagcc gtactgggaa gcagtgcgcg gactgtacct gccatttgag 2460
tctggaaccc caggcccaac cggtcgcgtc taccgccacg aaatcccagg cggacagttg 2520
tccaacctgc gtgcacaggc caccgcactg ggccttgcgg atcgtttcga actcatcgaa 2580
gacaactacg cagccgttaa tgagatgctg ggacgcccaa ccaaggtcac cccatcctcc 2640
aaggttgttg gcgacctcgc actccacctc gttggtgcgg gtgtggatcc agcagacttt 2700
gctgccgatc cacaaaagta cgacatccca gactctgtca tcgcgttcct gcgcggcgag 2760
cttggtaacc ctccaggtgg ctggccagag ccactgcgca cccgcgcact ggaaggccgc 2820
tccgaaggca aggcacctct gacggaagtt cctgaggaag agcaggcgca cctcgacgct 2880
gatgattcca aggaacgtcg caatagcctc aaccgcctgc tgttcccgaa gccaaccgaa 2940
gagttcctcg agcaccgtcg ccgcttcggc aacacctctg cgctggatga tcgtgaattc 3000
ttctacggcc tggtcgaagg ccgcgagact ttgatccgcc tgccagatgt gcgcacccca 3060
ctgcttgttc gcctggatgc gatctctgag ccagacgata agggtatgcg caatgttgtg 3120
gccaacgtca acggccagat ccgcccaatg cgtgtgcgtg accgctccgt tgagtctgtc 3180
accgcaaccg cagaaaaggc agattcctcc aacaagggcc atgttgctgc accattcgct 3240
ggtgttgtca ccgtgactgt tgctgaaggt gatgaggtca aggctggaga tgcagtcgca 3300
atcatcgagg ctatgaagat ggaagcaaca atcactgctt ctgttgacgg caaaatcgat 3360
cgcgttgtgg ttcctgctgc aacgaaggtg gaaggtggcg acttgatcgt cgtcgtttcc 3420
taa 3423
<210> 16
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gctgctaaca ttgcgaccat acttgatgtg tg 32
<210> 17
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gcagccaact ttaggaaacg acgacgatca agtc 34
<210> 18
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
atggtcgcaa tgttagcagc cggatctca 29
<210> 19
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cgtttcctaa agttggctgc tgccac 26
<210> 20
<211> 2126
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60
gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120
gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180
aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240
gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300
agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360
actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420
cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480
agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540
tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600
gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660
tccactgcga acaaaattgt tggtatgtac tctgctccgg caggcagcac cattctgatt 720
gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780
tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840
cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900
gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960
acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020
ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080
gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140
aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200
ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa gggtaatgct 1260
ggtaagcgtc tgatcaacgg ttccattgaa cgtgcgatca aattccgtaa agagatcaaa 1320
cgtctgagaa cggaatctga tggctggttc tttgatgttt ggcagccgga tcatatcgat 1380
acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440
aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500
gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560
catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620
atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgacttcaa acgtgcgttc 1680
gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740
tatgaaaaca tgcgtattca ggaactggct caaaatatcc acaaactgat tgttcaccac 1800
aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860
tatgctgcgt tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920
gtaggtcgta ttaacgccaa tatgatcctt ccgtatccgc cgggagttcc tctggtaatg 1980
ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040
gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100
gatggccgct ataccgttaa ggtatt 2126
Claims (5)
1.一种大肠杆菌戊二胺响应启动子,其特征在于,所述启动子的核苷酸序列如SEQ IDNo.7所示。
2.一种重组质粒PcadR-PgrcA-GFP,其特征在于,含有编码权利要求1所述的大肠杆菌戊二胺响应启动子的核苷酸的质粒。
3.一种基于权利要求2所述重组质粒PcadR-PgrcA-GFP构建的重组质粒PcadR-PgrcA-pyc-trc-cadA-rrnB构建方法,其特征在于,包括以下步骤:构建戊二胺响应启动子筛选质粒PcadR-GFP,所用引物中PET28a和Ptrc99a的引物带有NcoI 和XhoI酶切位点,通过同源重组获得PcadR-GFP;筛选大肠杆菌内源性戊二胺响应启动子PgrcA,合成的PgrcA启动子上游引物带有BglII 酶切位点,下游引物带有XbaI酶切位点,通过酶切链接获得PcadR-PgrcA-GFP;筛选出的响应启动子PgrcA用于调控谷氨酸棒状杆菌的基因丙酮酸羧化酶pyc,并将赖氨酸脱羧酶cadA构建在同一质粒上构建重组质粒PcadR-PgrcA-pyc-trc-cadA-rrnB;使用引物扩增丙酮酸羧化酶pyc和赖氨酸脱羧酶cadA,通过同源重组成功构建PcadR-PgrcA-pyc-trc-cadA-rrnB,将重组质粒导入克隆载体Trans1-T1,经过LB平板初筛后,选取平板上生长的单菌落进行PCR验证,再将阳性菌株送测。
4.一种重组菌株ka30-PcadR-PgrcA-pyc-trc-cadA-rrnB,其特征在于,表达权利要求3所述的重组质粒PcadR-PgrcA-pyc-trc-cadA-rrnB的赖氨酸高产大肠杆菌ka30。
5.基于权利要求4的重组菌株ka30-PcadR-PgrcA-pyc-trc-cadA-rrnB在合成戊二胺上的应用。
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