CN114058635B - 一种用于靛玉红生产的大肠杆菌基因工程菌及其构建方法以及一种靛玉红的生产方法 - Google Patents
一种用于靛玉红生产的大肠杆菌基因工程菌及其构建方法以及一种靛玉红的生产方法 Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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Abstract
本发明提供一种用于靛玉红生产的大肠杆菌基因工程菌及其构建方法以及一种靛玉红的生产方法,构建方法包括:以大肠杆菌为出发菌,在其基因组上敲除pykA、pykF、trpR、ppc和yddG中的至少一种基因,并原位回补黄素单加氧酶fmo基因表达框,然后进一步以高拷贝质粒形式强化tnaA、fmoK223R/D317S中的至少一种,以及以中拷贝质粒形式强化色氨酸合成路径的ppsA、tktA、aroGfbr、aroL和plsB中的至少一种,即可获得一种大肠杆菌基因工程菌。本发明通过结合代谢改造和酶工程构建了一种靛玉红发酵工程菌,并将其用于制备靛玉红,在提高靛玉红的生产效率及生产产量的同时,降低了生产成本。
Description
技术领域
本发明涉及代谢工程及酶工程领域,具体地涉及一种用于靛玉红生产的大肠杆菌基因工程菌及其构建方法以及一种靛玉红的生产方法。
背景技术
靛玉红是一种天然吲哚生物碱产物,来源于植物的次级代谢,广泛存在于中药大青叶、青黛等。靛玉红的分子式为C16H10N2O2,室温下呈暗红色针状结晶,不溶于水,微溶于乙醇,溶于丙酮、乙酸乙酯、DMSO等有机溶剂。许多吲哚生物碱及其衍生物具有重要的药用价值,其中靛玉红也早已被开发成为药物,是传统中药“当归龙荟丸”的一种主要的生物活性成分。
由于靛玉红的天然植物来源中,存在靛玉红含量较低,提取和纯化过程较为困难,制药成本较高等问题。而化学合成法中所使用的底物成本昂贵,生产效率不够高。为了达到药品的市场需求,通过微生物的合成生物学方法生产靛玉红目前受到广泛的关注。
靛玉红在医药应用中可作为一种多靶点抑制剂,现代药理学研究中已经发现靛玉红针对人体代谢中多种疾病具有药理作用,故对于肿瘤或神经退行性疾病等综合性疾病来说,发病机制较为复杂,针对单一靶点的药物难以兼顾全面的治疗,则需要可以针对多靶点的物质,例如靛玉红,以开发成多重功效的药物。
靛玉红由中药青黛中分离得到,是抗白血病药物中的有效成分。靛玉红通过与ELANE基因结合,该基因与中性粒细胞数目相关,从而干预人骨髓中的中性粒细胞异常分化和增多,以达到治疗目的。目前靛玉红在临床上主要应用于治疗慢性粒细胞白血症,其临床治疗慢性粒细胞白血病的有效率达到87.3%以上。
同时靛玉红及其衍生物对神经退行性疾病的治疗,例如阿尔兹海默病等,以及对人脑皮层神经干细胞的神经保护作用等方面皆展现出良好的药用前景,具有非常好的研究价值和开发潜力。
因此通过基因工程和代谢工程的手段改造大肠杆菌菌株以提高靛玉红的产量,具有重要的意义和实际的应用价值。
发明内容
本发明的主要目的是提供一种用于靛玉红生产的大肠杆菌基因工程菌及其构建方法以及一种靛玉红的生产方法,从而解决现有技术中缺乏高效的靛玉红生产菌株的问题。
为了解决上述问题,本发明采用以下技术方案:
根据本发明的第一方面,提供一种用于靛玉红生产的大肠杆菌基因工程菌的构建方法,包括:以大肠杆菌为出发菌,在其基因组上敲除丙酮酸激酶IpykA、丙酮酸激酶IIpykF、色氨酸阻遏蛋白trpR、磷酸烯醇式丙酮酸激酶ppc和芳香族氨基酸转运蛋白yddG中的至少一种关键基因,并原位回补黄素单加氧酶fmo基因表达框,然后进一步以高拷贝质粒形式强化色氨酸酶tnaA、黄素单加氧酶突变体fmoK223R/D317S中的至少一种,以及以中拷贝质粒形式强化色氨酸合成路径的磷酸烯醇式丙酮酸合成酶ppsA、转酮醇酶ItktA、DAHP合成酶aroGfbr、莽草酸激酶IIaroL和细胞膜合成基因甘油-3-磷酸乙酰转移酶plsB中的至少一种,即可获得一种用于靛玉红生产的大肠杆菌基因工程菌;其中,所述原位回补黄素单加氧酶fmo基因表达框包括:将来自外源的黄素单加氧酶fmo基因构建于大肠杆菌质粒上;所述丙酮酸激酶IpykA、丙酮酸激酶IIpykF、色氨酸阻遏蛋白trpR、磷酸烯醇式丙酮酸激酶ppc、芳香族氨基酸转运蛋白yddG基因分别编码如SEQ ID NO.59-63所示的氨基酸序列的蛋白;所述色氨酸酶tnaA、磷酸烯醇式丙酮酸合成酶ppsA、转酮醇酶ItktA、DAHP合成酶aroGfbr、莽草酸激酶IIaroL和细胞膜合成基因甘油-3-磷酸乙酰转移酶plsB基因分别编码如SEQ IDNO.64-69所示的氨基酸序列的蛋白。
所述黄素单加氧酶fmo选自:野生型黄素单加氧酶fmo、突变体黄素单加氧酶fmoN291T、fmoK223R、fmoK223R/D317S中的任意一种。
所述野生型黄素单加氧酶fmo编码如SEQ ID NO.1所示氨基酸序列的蛋白;所述突变体黄素单加氧酶fmoN291T、fmoK223R、fmoK223R/D317S编码如SEQ ID NO.2-4所示氨基酸序列的蛋白。
根据本发明,对所述大肠杆菌基因组上关键基因的敲除包括单独敲除和组合敲除,所述单独敲除包括单独敲除pykF、pykA、trpR、ppc、yddG这样5种基因中的任意一种,所述组合敲除包括同时敲除pykF、pykA、trpR、ppc、yddG中的至少任意两种。
应当理解的是,这里的组合敲除包括同时敲除trpR+pykA,trpR+pykF,trpR+ppc,trpR+yddG,pykF+pykA,yddG+pykA,ppc+pykA,ppc+pykF,yddG+pykF,ppc+yddG,trpR+pykF+pykA,trpR+pykF+ppc,trpR+pykF+yddG,trpR+ppc+pykA,trpR+yddG+pykA,trpR+yddG+ppc,ppc+yddG+pykA,pykF+yddG+pykA,trpR+pykF+pykA,trpR+pykF+pykA,trpR+pykF+pykA+ppc,trpR+pykF+pykA+yddG,yddG+pykF+pykA+ppc,yddG+trpR+pykA+ppc,yddG+pykF+trpR+ppc,yddG+pykF+trpR+ppc+pykA这样26种组合。
根据本发明人的研究发现,无论是单独敲除以上任意一种基因并原位整合fmo基因,还是逐步敲除并依次原位整合fmo基因,都可以增加fmo基因的拷贝数,提高fmo表达水平,实现靛玉红产量的提升。因此,应当理解的是,以上限定的单独敲除和组合敲除均属于本发明的保护范围之内。
根据本发明,对所述关键基因的强化表达包括单独强化和组合强化,所述单独强化包括单独强化tnaA、fmoK223R/D317S、ppsA、tktA、aroGfbr、aroL和plsB这样7种质粒中的任意一种,所述组合强化包括同时强化tnaA、fmoK223R/D317S、ppsA、tktA、aroGfbr、aroL和plsB中的至少任意两种。
应当理解的是,这里的组合强化包括同时强化tnaA+fmoK223R/D317S、tnaA+tktA、tnaA+aroGfbr、tnaA+aroL、tnaA+plsB、fmoK223R/D317S+tktA、fmoK223R/D317S+aroL、fmoK223R/D317S+plsB、fmoK223R/D317S+aroGfbr、tktA+aroL、tktA+plsB、tktA+aroGfbr、aroGfbr+aroL、aroGfbr+plsB、tnaA+fmoK223R/D317S+plsB、tnaA+fmoK223R/D317S+tktA、tnaA+fmoK223R/D317S+aroGfbr、tnaA+fmoK223R/D317S+aroL、tnaA+tktA+aroGfbr、tnaA+tktA+aroL、tnaA+tktA+plsB、tnaA+aroGfbr+aroL、tnaA+aroGfbr+plsB、tnaA+aroL+plsB等等。应当理解是,这里仅作为示例而非穷举。
根据本发明人的研究发现,无论是单独强化以上任意一种基因,还是组合过表达强化,都可以实现提高前体或提高细胞膜的合成速率,实现靛玉红产量的提升。因此,应当理解的是,以上限定的单独强化和组合强化均属于本发明的保护范围之内。
根据本发明的一个优选方案,所述出发菌选自大肠杆菌W3110或者MG1655。
根据本发明的第二方面,提供一种根据上述构建方法构建而来的用于靛玉红生产的大肠杆菌基因工程菌。
根据本发明的第三方面,提供一种生产靛玉红的方法,发酵如上面所述的用于靛玉红生产的大肠杆菌基因工程菌,实现靛玉红的生产。
根据本发明的一个优选方案,提供一种用于靛玉红生产的大肠杆菌基因工程菌的构建方法,包括以下步骤:1)对黄素单加氧酶fmo进行定向进化,结合高通量筛选方法,得到酶活提高的突变体:fmoN291T,fmoK223R,fmoK223R/D317S;2)将pykF、pykA、trpR、ppc、yddG这五个关键基因敲除并原位整合fmo基因表达框;3)质粒形式过表达提高前体供应的基因:高拷贝质粒形式强化色氨酸酶tnaA、和黄素单加氧酶突变体fmoK223R/D317S以及中拷贝质粒形式强化色氨酸合成路径的磷酸烯醇式丙酮酸合成酶ppsA、转酮醇酶ItktA、DAHP合成酶aroGfbr、莽草酸激酶IIaroL和细胞膜合成基因甘油-3-磷酸乙酰转移酶plsB。
本领域技术人员应当理解的是,通过Crisper-Cas9系统对大肠杆菌基因组的pykF、pykA、trpR、ppc、yddG五个关键基因敲除,并原位回补fmo表达框。
采用Crisper-Cas9系统,参考于文献(Jiang Y,Chen B,Duan C,et al.MμLtigeneEditing in the Escherichia coli Genome via the CRISPR-Cas9 System[J].Applied&Environmental Microbiology,2015,81(7):2506.)。
本领域技术人员还应当理解的是,本发明中涉及的黄素单加氧酶可以是噬甲基菌Methylophaga sp.strain SK1来源,也可以是其他来源。
所述的fmo的序列见SEQ ID No.1。
所述的fmoN291T的序列见SEQ ID No.2。
所述的fmoK223R的序列见SEQ ID No.3。
所述的fmoK223R/D317S的序列见SEQ ID No.4。
所述的pykF的序列见SEQ ID No.59。
所述的pykA的序列见SEQ ID No.60。
所述的trpR的序列见SEQ ID No.61。
所述的ppc的序列见SEQ ID No.62。
所述的yddG的序列见SEQ ID No.63。
所述的tnaA的序列见SEQ ID No.64。
所述的ppsA的序列见SEQ ID No.65。
所述的tktA的序列见SEQ ID No.66。
所述的aroG的序列见SEQ ID No.70。
所述的aroGfbr的序列见SEQ ID No.67。
所述的aroL的序列见SEQ ID No.68。
所述的plsB的序列见SEQ ID No.69。
根据本发明的一个优选方案,本发明提供的一种靛玉红生产大肠杆菌整合菌株的构建方法,包括如下步骤:对应插入位点的gRNA质粒的构建;cas9表达质粒的引入;对应插入位点同源臂的扩增;对应基因(tnaA和fmoK223R/D317S)的高表达质粒的构建和对应基因(ppsA、tktA、aroGfbr、aroL和plsB)的中拷贝表达质粒的构建。
具体的,表达质粒选用常用的大肠杆菌蛋白表达质粒pET28a(addgene);高表达质粒pkk223-3(addgene);表达质粒选用常用的大肠杆菌中拷贝表达质粒pacyc-duet(addgene);Cas9的质粒为pCas9(addgene);gRNA质粒ptargetF(addgene)。
应当理解的是,将所用的质粒及片段转入大肠菌株中,采用电转化法。
为提高转化效率,先将对应质粒在大肠杆菌中表达,再将提取的质粒转化到电转感受态中,控制加入量(质粒为200ng,片段为800ng)。
根据本发明的一个优选方案,采用敲除基因pykF、pykA、trpR、ppc、yddG并原位整合五个拷贝fmo基因表达框,高拷贝质粒过表达tnaA和fmoK223R/D317S和中拷贝质粒过表达ppsA、tktA、aroGfbr、aroL和plsB,构建出一种靛玉红生产大肠杆菌重组菌株,其靛玉红的产量得到大幅提高,证明这些改造是有效的。
本发明的有益效果在于:通过建立高通量筛选方法,得到黄素单加氧酶酶活提高的酶fmoN291T,fmoK223R,fmoK223R/D317S,使得产量提高了5.4倍,同时对大肠杆菌基因组进行改造,敲除基因pykF、pykA、trpR、ppc、yddG五个关键基因敲除并原位整合fmo基因表达框,提高了9.5倍。强化前体供应的基因色氨酸酶tnaA,强化色氨酸合成路径的磷酸烯醇式丙酮酸合成酶ppsA、转酮醇酶ItktA、DAHP合成酶aroGfbr、莽草酸激酶IIaroL和细胞膜合成基因甘油-3-磷酸乙酰转移酶plsB的大肠杆菌,提高了33%,通过上罐发酵可以达到860.4mg/L。
综上所述,根据本发明提供的一种用于靛玉红生产的大肠杆菌基因工程菌及其构建方法以及一种靛玉红的生产方法,实现了靛玉红高效生产菌株的改造与开发,大幅提高了靛玉红的生产效率。
附图说明
图1示出了fmo表达框质粒pET28a-fmo质粒图谱;
图2示出了fmo表达框质粒pkk-miniPtac-fmo质粒图谱;
图3示出了gRNA质粒ptarget-pykF质粒图谱;
图4示出了强化色氨酸酶tnaA、和黄素单加氧酶突变体fmoK223R/D317S质粒图谱;
图5示出了强化色氨酸合成路径的磷酸烯醇式丙酮酸合成酶ppsA、转酮醇酶ItktA、DAHP合成酶aroGfbr、莽草酸激酶IIaroL和细胞膜合成基因甘油-3-磷酸乙酰转移酶plsB质粒图谱;
图6示出了以W3110为出发菌株,分别单独敲除pykF、pykA、trpR、ppc、yddG这5个基因基因并在基因组上回补fmo基因的靛玉红发酵结果;
图7示出了以W3110为出发菌株,在基因组上连续敲除pykF、pykA、trpR、ppc、yddG这5个基因敲除并整合fmo表达框生产菌株的产量图;
图8示出了以构建的代谢菌株WFARPG为出发菌株,以质粒的形式过表达代谢路径上的关键基因ppsA、tktA、aroGfbr、aroL、plsB、fmo和tnaA的靛玉红产量图;
图9示出了以构建的代谢菌株WFARPG为出发菌株,组合强化高拷贝质粒和强化中拷贝质粒的生产菌株的产量图,其中W-pfAter代表以构建的代谢菌株WFARPG为出发菌株,携带高拷贝质粒过表达tnaA和fmoK223R/D317S的生产菌株,W-pfAter-ptGLB代表以构建的代谢菌株WFARPG为出发菌株,携带高拷贝质粒过表达tnaA和fmoK223R/D317S和中拷贝质粒过表达ppsA、tktA、aroGfbr、aroL和plsB的生产菌株;
图10示出了最终菌株上罐补料速率;
图11示出了最终菌株上罐的产量图。
具体实施方式
为更好的理解本发明的内容,下面结合具体实施例作进一步说明。应理解,以下实施例仅用于说明本发明而非用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法,通常按照常规条件,如《分子克隆:实验室手册》(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件进行。引物为上海捷瑞生物有限公司合成。
为更好的理解本发明的内容,以大肠杆菌中的一株W3110为出发菌株为具体实施例作进一步说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。
实施例1 高通量筛选方法的建立以及黄素单加氧酶定向进化
突变引物设计
从睿勉生物有限公司合成黄素单加氧酶基因(fmo,GenBank ID:AF494423.2),SEQID No.1。构建pET28a-fmo(其质粒图谱信息如图1所示)。以pET28a-fmo为模板,通过分子对接选定突变位点,设计饱和突变引物。
以位点291为例,设计引物为:
F:CTGCGTCTGGTCACCAATNNKCGTTTATGGCCGCTC;
R:GGTGACCAGACGCAGATCGT。
引物F的序列见SEQ ID No.71。
引物R的序列见SEQ ID No.72。
PCR反应体系组成为:15pmol上、下游引物,25μL Primerstar mix聚合酶(2×)、适量pET28a(+)-fmo模板DNA、和无菌超纯水,反应总体积为50μL。
PCR反应条件为:95℃变性3min后,按95℃10s,58℃10s,72℃8min的条件反应30个循环,最后在72℃延伸5min。
PCR结束后取样进行的琼脂糖凝胶电泳,凝胶浓度为1.2%。电泳缓冲液为1xTAE缓冲液。
1.2饱和文库的构建
将上述PCR反应得到的PCR产物用Dpn I在37.C酶解2小时,然后按以下步骤转化宿主构建饱和突变文库。取出保存于-80℃的大肠杆菌E.coli BL21(DE3)感受态细胞,冰浴解冻后加入10μL酶解PCR产物,轻轻摇匀后,冰浴放置30min。在42℃水浴90s后迅速冰浴冷却5min;然后立即加入1mL已预先温育至37℃的LB培养基(不含卡那霉素),再于37℃振荡培养1-1.5h。然后取菌液涂布LB筛选平板(含有30μg/mL卡那霉素,下同),每块平板涂布200μL菌液。涂布好的平板先正面向上在室温放置,直到液体被彻底吸收后再于37℃培养箱倒置培养16-24h,平板上长出的菌落就构成了所需的饱和突变文库。
1.3饱和突变文库筛选
母板:逐个将饱和突变文库中的克隆接种到每孔装有0.3mL LB液体培养基(含30pg/mL卡那霉素)的96孔板,37℃振荡培养24h作为母板。
子板:将母板转接到每孔装有0.5mL色氨酸液体培养基和半胱氨酸液体培养基(含30pg/mL卡那霉素和终浓度为0.5mmol/L IPTG)。37℃振荡培养18-24h。转接色氨酸培养基,主产物是靛蓝,而转接半胱氨酸培养基,培养基中只有靛玉红,靛蓝被半胱氨酸消耗完,将一个母板转接两个子板,就可以检测两种物质,用酶标仪进行扫描实现高通量筛选。
1.4检测方案
用DMSO萃取子板的产物然后用酶标仪扫描萃取液在540nm和620nm范围的光吸收曲线以测定产物的光谱特性,定性判定产物中靛玉红与靛蓝的多寡,进一步缩小筛选范围、挑选出产物中产量明显提高的克隆。然后用HPLC对这些克隆对应的催化产物萃取液进行定量分析,最终确定以靛玉红为主产物的目的突变株。
实施例2 fmo整合元件及pykF、pykA、trpR、ppc、yddG敲除元件的获取及过表达质粒的构建
2.1 fmo表达质粒的构建
从睿勉生物有限公司合成黄素单加氧酶基因(fmo,GenBank ID:AF494423.2),SEQID No.1。pkk-miniPtac-fmo(其质粒图谱信息如图2所示)的具体构建过程为以引物SEQ IDNo.41,42和pET281-fmo为模板,通过EcoRI和BglII双酶切质粒pkk223-3(addgene),将片段与质粒骨架进行无缝克隆(来源Vazyme,C113-01),转化到大肠杆菌,选取克隆测序,结果正确即为pkk-miniPtac-fmo。
miniPtac的序列通过引物SEQ ID No.41带入质粒pkk223-3。
所述的miniPtac的序列见SEQ ID No.73。
2.2各位点gRNA质粒的构建、
选取待敲除pykF、pykA、trpR、ppc、yddG基因序列中间大约500bp碱基序列。打开网站http://cctop.cos.uni-heidelberg.de/,将选取的500bp碱基序列输入到网站上进行查询,挑选出最高评分的sgRNA并标记(引物SEQ ID No.6-10)。通过引物pTarget-F将高评分的20bp的sgRNA引入,以pTarget质粒为模板,使用引物pTarget-F(引物SEQ ID No.5)和pTarget-R(引物SEQ ID No.6)进行PCR,再进行胶回收将线性DNA回收纯化。进行连接(T4连接体系)、转化。第二天挑取板上的单克隆进行稀释后,取1μL菌液作为模板进行PCR扩增验证,并将阳性克隆送测序公司进行测序。将测序正确的pTarget质粒菌扩增培养后,抽提质粒,放置于-4℃冰箱备用。位点的选择不仅仅局限于这些,包括其他报道的大肠杆菌常规的整合位点也可。
2.3对应位点上下游同源臂,及整合片段的构建
pykF上下游同源臂的获得,以W3110的基因组为模板,采用引物SEQ ID No.11-12扩增上游同源臂,引物SEQ ID No.15-16扩增下游同源臂。引物SEQ ID No.13-14以pkk-miniPtac-fmo为模板得到表达框FPTs。其他位点类似地使用对应引物pykA(引物SEQ IDNo.17,18,21,22),trpR(引物SEQ ID No.23,24,27,28),ppc(引物SEQ ID No.29,30,33,34)、yddG(引物SEQ ID No.35,36,39,40)来得到上下游同源臂,pykA(引物SEQ ID No.19-20),trpR(引物SEQ ID No.25-26),ppc(引物SEQ ID No.31-32),yddG(引物SEQ ID No.37-38)位点以pkk-miniPtac-fmo为模板扩增片段fmo基因表达框。
2.4各生产菌株的构建
通过电转化法(自制电转感受态W3110,电转转入pCas质粒),在W3110-pCas电转化感受态中(Jiang Y,Chen B,Duan C,et al.MμLtigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System[J].Applied&Environmental Microbiology,2015,81(7):2506.)中加入pykF的同源臂pykF-fmo-donor(具体片段的构建见实施例1.2,1.3得到的gRNA和扩增的元件),用含有卡那(Kan)和壮观霉素(Spe)抗生素的平板筛选,验证测序对的菌株得到的即W3110-ΔpykF::fmo,继续消除gRNA以便之后多拷贝的菌株的构建。
消除方法:通过将阳性克隆接入LB试管,加入IPTG,放置于30℃摇床进行过夜培养。划线(Kan平板)。挑菌稀释后取1-2μL菌液进行点板(Spe板)并标记编号,同时将剩余菌液接试管。将点好的Spe板放入30℃培养箱进行培养,试管放入37℃摇床培养。根据点板结果,将无菌落长出(pTarget质粒成功消除)的对应编号的试管中的菌进行后续实验和菌种保藏。
以W3110-ΔpykF::fmo菌株为底盘,接下来依次敲除pykA、trpR、ppc、yddG基因并整合fmo表达框,(具体片段的构建见实施例1.2,1.3得到的gRNA和扩增的元件)得到W3110-ΔpykF::fmo-ΔpykA::fmo-ΔtrpR::fmo-Δppc::fmo-ΔyddG::fmo。其中gRNA质粒ptarget-pykF质粒图谱信息如图3所示。
2.5过表达质粒的构建
使用引物SEQ ID No.43和引物SEQ ID No.44以大肠杆菌W3110基因组为模板,通过高保真酶扩增得到基因片段tnaA。通过KpnI和EcoRI双酶切质粒pKK223-3,将片段与质粒骨架进行无缝克隆,转化到大肠杆菌,选取克隆测序,结果正确即为pkk-tnaA。引物SEQ IDNo.45和引物SEQ ID No.46,以pkk-miniPtac-fmo为模板,通过高保真酶扩增得到基因片段FMO,通过BamHI单酶切质粒pkk-tnaA,将片段与质粒骨架进行无缝克隆,转化到大肠杆菌,选取克隆测序,结果正确即为pkk-tnaA-FMO(其质粒图谱信息如图4所示)。
引物SEQ ID No.45和SEQ ID No.46,引物SEQ ID No.47和SEQ ID No.48,引物SEQID No.49和SEQ ID No.50,引物SEQ ID No.51和SEQ ID No.52,引物SEQ ID No.53和SEQID No.54,引物SEQ ID No.55和SEQ ID No.56以大肠杆菌W3110基因组为模板,通过高保真酶扩增得到基因片段ppsA、tktA、aroG、aroL和plsB,其中引物SEQ ID No.57和SEQ IDNo.58可以得到pkk-aroGfbr,依次通过酶切连接转化构建得到pkk-ppsA-tktA-aroGfbr-aroL-plsB(其质粒图谱信息如图5所示)。
实施例3 大肠杆菌菌株发酵生产靛玉红
3.1大肠杆菌菌株的摇瓶及上罐发酵
从对应固体培养基上挑取过夜培育的单菌落在5mL LB试管中37℃活化过夜培养,1%接种量转接到摇瓶发酵培养基30mL TBT的250mL锥形瓶中初始OD600为0.4,加入抗生素(amp,50μg/mL)30℃培养48h,取样测定靛玉红的产量。
TBT培养基:Yeast extract 20g,蛋白胨8g,磷酸二氢钾2g,磷酸氢二钾4g,磷酸氢二钠7g,硫酸铵1g,色氨酸2g,半胱氨酸1g。
从固体培养基上挑一个单菌落转接在5mL的试管中培养12-14h,将其转接至50mLLB的种子培养基(初始浓度为OD600=0.03)培养12-14h后,将其转接到2瓶100mL LB的250mL锥形瓶中。待其生长20h后,准备好发酵罐。首先使用饱和亚硫酸钠校准溶氧电极的0点,pH4和6.86的标准液校准pH电极。在罐中加入2.7L的罐中的培养基(不含葡萄糖),装好电极和补料瓶,115℃灭菌20min。待灭完菌之后,装载在操作台上连接在线监测装置,同时在超净台把另外灭的葡萄糖、硫酸镁加入到补料瓶中,晃动混匀。首先控制进气量为3vvm,搅拌速率为200r/min,在接种口点火接种(2瓶菌液200mL,还有单独灭的葡萄糖和硫酸镁溶液),此时进行溶氧电极的满点(100%)校准,待罐中的溶氧降到30%时,将进气量修改到3vvm,同时将浆叶的速率与溶氧进行自动关联区间为200r/min-800r/min之间,同时使用氨水来控制pH在7左右,之后设定补料速率(图10)即可,中间前期每2h取一次,后面可以改为4h取一次样。
5L发酵罐中的培养基(3L):葡萄糖15g,Yeast extract 60g,蛋白胨24g,磷酸二氢钾6g,磷酸氢二钾12g,磷酸氢二钠21g,硫酸铵3g,柠檬酸3g,无水MgSO4 3g,环糊精2.7g,色氨酸30g,半胱氨酸15g,消泡剂<1ml(其中葡萄糖、无水MgSO4单独灭菌115℃20min)
补料I:无水葡萄糖300g/500mL+酵母粉/硫酸氨/磷酸二氢钾/色氨酸/半胱氨酸(补料)(10/1/2/10/5g)/500mL(必须冷却后超净台混合)。
补料II:消泡剂(可不补,只是备用)。
补料III:氨水(瓶子空灭,后面加入氨水),用来调pH。
3.2发酵产物的HPLC分析
由发酵液中吸取1mL发酵液,12000rpm离心10min离心使菌体充分沉淀,由于靛玉红或靛蓝异源合成后积累在菌株的细胞膜上,离心后收集菌体即可将目的产物与培养基分离。向所收集的菌体中加入1mL DMSO,通过用移液枪吹打或者超声处理使菌体重悬,基于靛蓝或靛玉红的物化性质,超声菌体裂解后则目标产物可溶于DMSO中,12000rpm离心10min再次充分离心后收集上清,吸取500μL液体至液相小瓶进行通过高效液相色谱进行样品检测。
高效液相色谱检测条件:
使用美国Agilent公司的所生产的1260InfinityⅡ高效液相色谱仪进行样品的定性定量检测,选用C18柱进行目标产物的分离纯化及定量检测,以甲醇:水=8:2的比例的溶剂作为流动相,设置检测波长为289nm,流速0.8mL/min根据靛蓝或靛玉红标品的出峰时间及峰面积对待测样品进行定性检测及定量检测。
3.3 pykF、pykA、trpR、ppc、yddG五个关键基因敲除并整合fmo表达框的发酵结果
如图6中所示,WF代表以W3110为出发菌株,单独敲除pykF基因,得到菌株为W3110ΔpykF::fmo,发酵得到靛玉红的产量为3.4mg/L;WA代表以W3110为出发菌株,单独敲除pykA基因,得到菌株为W3110ΔpykA::fmo,发酵得到靛玉红的产量为4.4mg/L;WR代表以W3110为出发菌株,单独敲除trpR基因,得到菌株为W3110ΔtrpR::fmo,发酵得到靛玉红的产量为19.3mg/L;WP代表以W3110为出发菌株,单独敲除ppc基因,得到菌株为W3110Δppc::fmo,发酵得到靛玉红的产量为6.3mg/L;WG代表以W3110为出发菌株,单独敲除yddG基因,得到菌株为W3110ΔyddG::fmo,发酵得到靛玉红的产量为8.3mg/L。通过以上发酵结果说明单独敲除这5个基因中的任意一种并原位回补fmo基因都有利于靛玉红的积累。
如图7中所示,WF代表以W3110为出发菌株,单独敲除pykF基因,得到菌株为W3110ΔpykF::fmo,发酵得到靛玉红的产量为2.5mg/L;WFA代表以WF为出发菌株,敲除pykA基因,得到菌株为W3110ΔpykF::fmoΔpykA::fmo,发酵得到靛玉红的产量为12.33mg/L;WFAR代表以WFA为出发菌株,敲除trpR基因,得到菌株为W3110ΔpykF::fmoΔpykA::fmoΔtrpR::fmo,发酵得到靛玉红的产量为44.38mg/L;WFARP代表以WFAR为出发菌株,敲除ppc基因,得到菌株为W3110ΔpykF::fmoΔpykA::fmoΔtrpR::fmoΔppc::fmo,发酵得到靛玉红的产量为69.14mg/L;WFARPG代表以WFARP为出发菌株,敲除yddG基因,得到菌株为W3110ΔpykF::fmoΔpykA::fmoΔtrpR::fmoΔppc::fmoΔyddG::fmo,发酵得到靛玉红的产量为112.68mg/L。以上发酵结果表明,基因组上连续敲除关键基因,并回补外源基因fmo,有利于产物的积累,第一步整合使原本不生产靛玉红的大肠杆菌开始积累靛玉红,而后得到的代谢菌株WFARPG,相较于第一步整合结果,产量提高了45倍。
通过以上实验证明,以W3110为出发菌株进行pykF、pykA、trpR、ppc、yddG五个关键基因敲除并整合fmo表达框,不断增加fmo的拷贝数的策略是有效的,每整合一步,产量随拷贝数的增加也是逐级递增的,其产量达到100.2mg/L,是单敲除pykF并整合fmo表达框的9.3倍。
3.4强化过表达质粒的菌株的发酵结果
如图8所示,以构建的代谢菌株WFARPG为出发菌株,以质粒的形式过表达代谢路径上的关键基因tnaA、fmo、ppsA、tktA、aroGfbr、aroL和plsB的菌株的靛玉红产量各不相同。W代表代谢菌株WFARPG。将关键基因都构建在高拷贝质粒pkk-223-3-miniptac,比较各个基因单独过表达,对于靛玉红产量的影响。当在发酵培养基中培养(发酵操作如实施例3.1所示)时,过表达tnaA、fmo、ppsA、tktA、aroGfbr、aroL、plsB,使靛玉红的产量增加到1.2倍,达到123.4mg/L(图8)。由结果所示,过表达这些基因对于产量提升都有明显的效果,后期采用组合过表达的形式过表达这些基因。从图中可见,细胞膜工程有利于增加细菌的耐受性。其中,通过强化plsB,有效增加细胞膜的耐受性,发酵培养的具体操作见实施例3.1所示,达到160.2mg/L,表明plsB的强化特别有利于靛玉红的合成。
3.5细胞膜改造的生产菌株的发酵结果
图9示出了以构建的代谢菌株WFARPG为出发菌株,组合强化高拷贝质粒和强化中拷贝质粒的生产菌株的靛玉红产量图。其中W-pfAter代表以构建的代谢菌株WFARPG为出发菌株,携带高拷贝质粒过表达tnaA和fmoK223R/D317S的生产菌株。其中W-pfAter-PTGLB代表以构建的代谢菌株WFARPG为出发菌株,携带高拷贝质粒过表达tnaA和fmoK223R/D317S和中拷贝质粒过表达ppsA、tktA、aroGfbr、aroL和plsB的生产菌株。
3.6上罐发酵
上罐发酵的具体操作见实施例3.1所示,最终菌株上罐葡萄糖补料速率如图10所示。
通过上罐发酵产量可以达到860.4mg/L(图11)。
以上所述的,仅为本发明的较佳实施例,并非用以限定本发明的范围,本发明的上述实施例还可以做出各种变化。即凡是依据本发明申请的权利要求书及说明书内容所作的简单、等效变化与修饰,皆落入本发明专利的权利要求保护范围。本发明未详尽描述的均为常规技术内容。
序列表
<110> 华东理工大学
<120> 一种用于靛玉红生产的大肠杆菌基因工程菌及其构建方法以及一种靛玉红的生产方法
<160> 73
<170> PatentIn version 3.5
<210> 1
<211> 1686
<212> DNA
<213> Methylophaga sulfidovorans
<400> 1
cattttcacc gtttgatatc cattatcgtt taaccagggc agatggcact gaagtcagcg 60
tccaggataa aggccggggg ctgtactcga aaagcggtat ggtacttggt gttgaaggca 120
ttttgttact cactaacaag tgataacaac ctgattagat ttattttttt caggctaatc 180
tacccccttt tttacccttt tgggataccc acacaagagg atttctccca caaaaaaagc 240
gagctatttt ggttattaag cgctgtcgat tttcgcagca tgattgacta aataacactg 300
tatggagaga cccctatggc aactcgtatt gcgatacttg gtgcaggccc aagtggtatg 360
gcacaactca gagcattcca atccgcccag gaaaaaggtg ctgagatccc tgaactcgtt 420
tgttttgaaa aacaagctga ttggggcggc cagtggaatt acacatggcg cactggttta 480
gatgaaaatg gcgaacctgt tcatagcagt atgtatcgct atctgtggtc aaacggcccg 540
aaagaatgtc ttgaatttgc tgattacacg tttgacgaac actttggtaa gcccatcgcc 600
tcttatccac cccgtgaagt cttatgggac tatattaaag gccgtgttga aaaagccggc 660
gtcagaaaat atatccgttt taataccgct gttcgtcatg ttgaattcaa cgaagacagc 720
caaactttta ccgttaccgt gcaggaccat actactgaca caatttactc tgaagagttt 780
gactatgttg tctgttgtac cggtcacttc tcaacacctt acgtgcctga atttgaaggc 840
tttgaaaaat ttggtggccg cattctgcat gcccatgact tccgtgacgc attagaattt 900
aaagacaaaa ctgtattact ggtcggcagc agttactcag ctgaagatat cggctcacaa 960
tgttataaat acggcgcgaa aaaactgatc agctgctacc gtaccgcacc gatgggttat 1020
aaatggcctg aaaactggga tgaaagaccc aacctggttc gtgttgatac tgaaaacgct 1080
tattttgccg atggttcatc agaaaaagtc gatgcgatta ttctgtgtac cggttatatc 1140
catcacttcc ccttcctcaa tgacgatctg cgtctggtca ccaataaccg tttatggccg 1200
ctcaaccttt ataaaggcgt ggtgtgggaa gataatccaa aattcttcta cattggcatg 1260
caggatcaat ggtacagctt caatatgttt gatgcccaag cctggtatgc ccgtgatgtg 1320
attatgggtc gactgccatt gccatcaaaa gaagagatga aagccgacag catggcctgg 1380
cgtgaaaaag aactgacgct ggttacggct gaagaaatgt acacctacca gggtgactac 1440
attcagaatc tgattgatat gactgactat ccgtcatttg atattccggc aaccaacaaa 1500
actttcctgg aatggaaaca tcacaaaaaa gaaaacatca tgactttccg tgaccactca 1560
taccgttcac tgatgactgg cacgatggca ccgaaacatc acacaccatg gatagatgca 1620
ctggatgatt ctctggaagc ctatctctct gataagagcg aaattcctgt ggctaaagaa 1680
gcttaa 1686
<210> 2
<211> 456
<212> PRT
<213> Methylophaga sulfidovorans
<400> 2
Met Ala Thr Arg Ile Ala Ile Leu Gly Ala Gly Pro Ser Gly Met Ala
1 5 10 15
Gln Leu Arg Ala Phe Gln Ser Ala Gln Glu Lys Gly Ala Glu Ile Pro
20 25 30
Glu Leu Val Cys Phe Glu Lys Gln Ala Asp Trp Gly Gly Gln Trp Asn
35 40 45
Tyr Thr Trp Arg Thr Gly Leu Asp Glu Asn Gly Glu Pro Val His Ser
50 55 60
Ser Met Tyr Arg Tyr Leu Trp Ser Asn Gly Pro Lys Glu Cys Leu Glu
65 70 75 80
Phe Ala Asp Tyr Thr Phe Asp Glu His Phe Gly Lys Pro Ile Ala Ser
85 90 95
Tyr Pro Pro Arg Glu Val Leu Trp Asp Tyr Ile Lys Gly Arg Val Glu
100 105 110
Lys Ala Gly Val Arg Lys Tyr Ile Arg Phe Asn Thr Ala Val Arg His
115 120 125
Val Glu Phe Asn Glu Asp Ser Gln Thr Phe Thr Val Thr Val Gln Asp
130 135 140
His Thr Thr Asp Thr Ile Tyr Ser Glu Glu Phe Asp Tyr Val Val Cys
145 150 155 160
Cys Thr Gly His Phe Ser Thr Pro Tyr Val Pro Glu Phe Glu Gly Phe
165 170 175
Glu Lys Phe Gly Gly Arg Ile Leu His Ala His Asp Phe Arg Asp Ala
180 185 190
Leu Glu Phe Lys Asp Lys Thr Val Leu Leu Val Gly Ser Ser Tyr Ser
195 200 205
Ala Glu Asp Ile Gly Ser Gln Cys Tyr Lys Tyr Gly Ala Lys Arg Leu
210 215 220
Ile Ser Cys Tyr Arg Thr Ala Pro Met Gly Tyr Lys Trp Pro Glu Asn
225 230 235 240
Trp Asp Glu Arg Pro Asn Leu Val Arg Val Asp Thr Glu Asn Ala Tyr
245 250 255
Phe Ala Asp Gly Ser Ser Glu Lys Val Asp Ala Ile Ile Leu Cys Thr
260 265 270
Gly Tyr Ile His His Phe Pro Phe Leu Asn Asp Asp Leu Arg Leu Val
275 280 285
Thr Asn Asn Arg Leu Trp Pro Leu Asn Leu Tyr Lys Gly Val Val Trp
290 295 300
Glu Asp Asn Pro Lys Phe Phe Tyr Ile Gly Met Gln Asp Gln Trp Tyr
305 310 315 320
Ser Phe Asn Met Phe Asp Ala Gln Ala Trp Tyr Ala Arg Asp Val Ile
325 330 335
Met Gly Arg Leu Pro Leu Pro Ser Lys Glu Glu Met Lys Ala Asp Ser
340 345 350
Met Ala Trp Arg Glu Lys Glu Leu Thr Leu Val Thr Ala Glu Glu Met
355 360 365
Tyr Thr Tyr Gln Gly Asp Tyr Ile Gln Asn Leu Ile Asp Met Thr Asp
370 375 380
Tyr Pro Ser Phe Asp Ile Pro Ala Thr Asn Lys Thr Phe Leu Glu Trp
385 390 395 400
Lys His His Lys Lys Glu Asn Ile Met Thr Phe Arg Asp His Ser Tyr
405 410 415
Arg Ser Leu Met Thr Gly Thr Met Ala Pro Lys His His Thr Pro Trp
420 425 430
Ile Asp Ala Leu Asp Asp Ser Leu Glu Ala Tyr Leu Ser Asp Lys Ser
435 440 445
Glu Ile Pro Val Ala Lys Glu Ala
450 455
<210> 3
<211> 456
<212> PRT
<213> Methylophaga sulfidovorans
<400> 3
Met Ala Thr Arg Ile Ala Ile Leu Gly Ala Gly Pro Ser Gly Met Ala
1 5 10 15
Gln Leu Arg Ala Phe Gln Ser Ala Gln Glu Lys Gly Ala Glu Ile Pro
20 25 30
Glu Leu Val Cys Phe Glu Lys Gln Ala Asp Trp Gly Gly Gln Trp Asn
35 40 45
Tyr Thr Trp Arg Thr Gly Leu Asp Glu Asn Gly Glu Pro Val His Ser
50 55 60
Ser Met Tyr Arg Tyr Leu Trp Ser Asn Gly Pro Lys Glu Cys Leu Glu
65 70 75 80
Phe Ala Asp Tyr Thr Phe Asp Glu His Phe Gly Lys Pro Ile Ala Ser
85 90 95
Tyr Pro Pro Arg Glu Val Leu Trp Asp Tyr Ile Lys Gly Arg Val Glu
100 105 110
Lys Ala Gly Val Arg Lys Tyr Ile Arg Phe Asn Thr Ala Val Arg His
115 120 125
Val Glu Phe Asn Glu Asp Ser Gln Thr Phe Thr Val Thr Val Gln Asp
130 135 140
His Thr Thr Asp Thr Ile Tyr Ser Glu Glu Phe Asp Tyr Val Val Cys
145 150 155 160
Cys Thr Gly His Phe Ser Thr Pro Tyr Val Pro Glu Phe Glu Gly Phe
165 170 175
Glu Lys Phe Gly Gly Arg Ile Leu His Ala His Asp Phe Arg Asp Ala
180 185 190
Leu Glu Phe Lys Asp Lys Thr Val Leu Leu Val Gly Ser Ser Tyr Ser
195 200 205
Ala Glu Asp Ile Gly Ser Gln Cys Tyr Lys Tyr Gly Ala Lys Lys Leu
210 215 220
Ile Ser Cys Tyr Arg Thr Ala Pro Met Gly Tyr Lys Trp Pro Glu Asn
225 230 235 240
Trp Asp Glu Arg Pro Asn Leu Val Arg Val Asp Thr Glu Asn Ala Tyr
245 250 255
Phe Ala Asp Gly Ser Ser Glu Lys Val Asp Ala Ile Ile Leu Cys Thr
260 265 270
Gly Tyr Ile His His Phe Pro Phe Leu Asn Asp Asp Leu Arg Leu Val
275 280 285
Thr Asn Thr Arg Leu Trp Pro Leu Asn Leu Tyr Lys Gly Val Val Trp
290 295 300
Glu Asp Asn Pro Lys Phe Phe Tyr Ile Gly Met Gln Asp Gln Trp Tyr
305 310 315 320
Ser Phe Asn Met Phe Asp Ala Gln Ala Trp Tyr Ala Arg Asp Val Ile
325 330 335
Met Gly Arg Leu Pro Leu Pro Ser Lys Glu Glu Met Lys Ala Asp Ser
340 345 350
Met Ala Trp Arg Glu Lys Glu Leu Thr Leu Val Thr Ala Glu Glu Met
355 360 365
Tyr Thr Tyr Gln Gly Asp Tyr Ile Gln Asn Leu Ile Asp Met Thr Asp
370 375 380
Tyr Pro Ser Phe Asp Ile Pro Ala Thr Asn Lys Thr Phe Leu Glu Trp
385 390 395 400
Lys His His Lys Lys Glu Asn Ile Met Thr Phe Arg Asp His Ser Tyr
405 410 415
Arg Ser Leu Met Thr Gly Thr Met Ala Pro Lys His His Thr Pro Trp
420 425 430
Ile Asp Ala Leu Asp Asp Ser Leu Glu Ala Tyr Leu Ser Asp Lys Ser
435 440 445
Glu Ile Pro Val Ala Lys Glu Ala
450 455
<210> 4
<211> 456
<212> PRT
<213> Methylophaga sulfidovorans
<400> 4
Met Ala Thr Arg Ile Ala Ile Leu Gly Ala Gly Pro Ser Gly Met Ala
1 5 10 15
Gln Leu Arg Ala Phe Gln Ser Ala Gln Glu Lys Gly Ala Glu Ile Pro
20 25 30
Glu Leu Val Cys Phe Glu Lys Gln Ala Asp Trp Gly Gly Gln Trp Asn
35 40 45
Tyr Thr Trp Arg Thr Gly Leu Asp Glu Asn Gly Glu Pro Val His Ser
50 55 60
Ser Met Tyr Arg Tyr Leu Trp Ser Asn Gly Pro Lys Glu Cys Leu Glu
65 70 75 80
Phe Ala Asp Tyr Thr Phe Asp Glu His Phe Gly Lys Pro Ile Ala Ser
85 90 95
Tyr Pro Pro Arg Glu Val Leu Trp Asp Tyr Ile Lys Gly Arg Val Glu
100 105 110
Lys Ala Gly Val Arg Lys Tyr Ile Arg Phe Asn Thr Ala Val Arg His
115 120 125
Val Glu Phe Asn Glu Asp Ser Gln Thr Phe Thr Val Thr Val Gln Asp
130 135 140
His Thr Thr Asp Thr Ile Tyr Ser Glu Glu Phe Asp Tyr Val Val Cys
145 150 155 160
Cys Thr Gly His Phe Ser Thr Pro Tyr Val Pro Glu Phe Glu Gly Phe
165 170 175
Glu Lys Phe Gly Gly Arg Ile Leu His Ala His Asp Phe Arg Asp Ala
180 185 190
Leu Glu Phe Lys Asp Lys Thr Val Leu Leu Val Gly Ser Ser Tyr Ser
195 200 205
Ala Glu Asp Ile Gly Ser Gln Cys Tyr Lys Tyr Gly Ala Lys Arg Leu
210 215 220
Ile Ser Cys Tyr Arg Thr Ala Pro Met Gly Tyr Lys Trp Pro Glu Asn
225 230 235 240
Trp Asp Glu Arg Pro Asn Leu Val Arg Val Asp Thr Glu Asn Ala Tyr
245 250 255
Phe Ala Asp Gly Ser Ser Glu Lys Val Asp Ala Ile Ile Leu Cys Thr
260 265 270
Gly Tyr Ile His His Phe Pro Phe Leu Asn Asp Asp Leu Arg Leu Val
275 280 285
Thr Asn Asn Arg Leu Trp Pro Leu Asn Leu Tyr Lys Gly Val Val Trp
290 295 300
Glu Asp Asn Pro Lys Phe Phe Tyr Ile Gly Met Gln Ser Gln Trp Tyr
305 310 315 320
Ser Phe Asn Met Phe Asp Ala Gln Ala Trp Tyr Ala Arg Asp Val Ile
325 330 335
Met Gly Arg Leu Pro Leu Pro Ser Lys Glu Glu Met Lys Ala Asp Ser
340 345 350
Met Ala Trp Arg Glu Lys Glu Leu Thr Leu Val Thr Ala Glu Glu Met
355 360 365
Tyr Thr Tyr Gln Gly Asp Tyr Ile Gln Asn Leu Ile Asp Met Thr Asp
370 375 380
Tyr Pro Ser Phe Asp Ile Pro Ala Thr Asn Lys Thr Phe Leu Glu Trp
385 390 395 400
Lys His His Lys Lys Glu Asn Ile Met Thr Phe Arg Asp His Ser Tyr
405 410 415
Arg Ser Leu Met Thr Gly Thr Met Ala Pro Lys His His Thr Pro Trp
420 425 430
Ile Asp Ala Leu Asp Asp Ser Leu Glu Ala Tyr Leu Ser Asp Lys Ser
435 440 445
Glu Ile Pro Val Ala Lys Glu Ala
450 455
<210> 5
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 5
actagtatta tacctaggac tgag 24
<210> 6
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 6
gacggcatca tggttgcgcg gttttagagc tagaaatagc aagtt 45
<210> 7
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 7
tgcgcgtcag ctaaaccgag gttttagagc tagaaatagc aagtt 45
<210> 8
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 8
gccagatgag cgcgaagcgt gttttagagc tagaaatagc aagtt 45
<210> 9
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 9
gtccgcttct gccgcgcaac gttttagagc tagaaatagc aagtt 45
<210> 10
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 10
tgggtgttag gcggtgacaa gttttagagc tagaaatagc aagtt 45
<210> 11
<211> 25
<212> DNA
<213> Escherichia coli
<400> 11
tcacactgac aacttcggca ccaga 25
<210> 12
<211> 40
<212> DNA
<213> Escherichia coli
<400> 12
gtcaacagct cggatccgcg gacagtctta gtctttaagt 40
<210> 13
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 13
acttaaagac taagactgtc cgcggatccg agctgttgac 40
<210> 14
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 14
attaattcac aaaagcaata acgcaaaaag gccatccgtc 40
<210> 15
<211> 40
<212> DNA
<213> Escherichia coli
<400> 15
gacggatggc ctttttgcgt tattgctttt gtgaattaat 40
<210> 16
<211> 23
<212> DNA
<213> Escherichia coli
<400> 16
cagagtagaa cccaggatta ccg 23
<210> 17
<211> 26
<212> DNA
<213> Escherichia coli
<400> 17
agcaacgctg gcaattaccc tcgacg 26
<210> 18
<211> 40
<212> DNA
<213> Escherichia coli
<400> 18
tgattaattg tcaacagctc gtaatactcc gttgactgaa 40
<210> 19
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 19
ttcagtcaac ggagtattac gagctgttga caattaatca 40
<210> 20
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 20
tgtaggccgg atgtggcgtt agagtttgta gaaacgcaaa 40
<210> 21
<211> 40
<212> DNA
<213> Escherichia coli
<400> 21
tttgcgtttc tacaaactct aacgccacat ccggcctaca 40
<210> 22
<211> 30
<212> DNA
<213> Escherichia coli
<400> 22
ttgcccgcga ttggtcgttt gatgaaagtg 30
<210> 23
<211> 22
<212> DNA
<213> Escherichia coli
<400> 23
cagcgaccca tacggtgaag at 22
<210> 24
<211> 40
<212> DNA
<213> Escherichia coli
<400> 24
gtcaacagct cggatccgcg aatatgtcgc cattgttagc 40
<210> 25
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 25
gctaacaatg gcgacatatt cgcggatccg agctgttgac 40
<210> 26
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 26
gtcttatcag gcctacaaaa acgcaaaaag gccatccgtc 40
<210> 27
<211> 40
<212> DNA
<213> Escherichia coli
<400> 27
gacggatggc ctttttgcgt ttttgtaggc ctgataagac 40
<210> 28
<211> 23
<212> DNA
<213> Escherichia coli
<400> 28
ccaatgcccg ccgtttactt cca 23
<210> 29
<211> 22
<212> DNA
<213> Escherichia coli
<400> 29
tccgaccgac agtgactcaa ac 22
<210> 30
<211> 40
<212> DNA
<213> Escherichia coli
<400> 30
gtcaacagct cggatccgcg attaccccag acaccccatc 40
<210> 31
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 31
gatggggtgt ctggggtaat cgcggatccg agctgttgac 40
<210> 32
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 32
gggtttgcag aagaggaaga acgcaaaaag gccatccgtc 40
<210> 33
<211> 40
<212> DNA
<213> Escherichia coli
<400> 33
gacggatggc ctttttgcgt tcttcctctt ctgcaaaccc 40
<210> 34
<211> 23
<212> DNA
<213> Escherichia coli
<400> 34
caaccacagc gaactgaata aca 23
<210> 35
<211> 22
<212> DNA
<213> Escherichia coli
<400> 35
aaagccgaga cgggcataag tt 22
<210> 36
<211> 40
<212> DNA
<213> Escherichia coli
<400> 36
gtcaacagct cggatccgcg aataactgcc gggtctacgg 40
<210> 37
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 37
ccgtagaccc ggcagttatt cgcggatccg agctgttgac 40
<210> 38
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 38
agagagacaa aacagcgagc acgcaaaaag gccatccgtc 40
<210> 39
<211> 40
<212> DNA
<213> Escherichia coli
<400> 39
gacggatggc ctttttgcgt gctcgctgtt ttgtctctct 40
<210> 40
<211> 23
<212> DNA
<213> Escherichia coli
<400> 40
gccatcaccc aggctataca tca 23
<210> 41
<211> 91
<212> DNA
<213> Artificial Sequence
<400> 41
cgcggatccg agctgttgac aattaatcat cggctcgtat aatgtgtgga attgtaagaa 60
ggagatatac ccattttcac cgtttgatat c 91
<210> 42
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 42
tgattaattg tcaacagctc agagtttgta gaaacgcaaa 40
<210> 43
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 43
gaaggagata taccggtacc atggaaaact ttaaacatct ccctg 45
<210> 44
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 44
aagcttggct gcaggaattc ttaaacttct ttaagttttg cggtg 45
<210> 45
<211> 32
<212> DNA
<213> Artificial Sequence
<400> 45
aaggagatat acccattttc accgtttgat at 32
<210> 46
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 46
tggctgcagg tcgacttaag cttctttagc cacaggaa 38
<210> 47
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 47
catcaccaca gccaggatcc atgtccaaca atggctcgtc 40
<210> 48
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 48
caagcttggc tgcaggaatt atttcttcag ttcagccagg c 41
<210> 49
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 49
gaagaaataa ttcctgcagc tgttgacaat taatcatcgg 40
<210> 50
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 50
gcgcgccgag ctcgaattct tacagcagtt cttttgct 38
<210> 51
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 51
tgtaagaagg agatatacca tgaattatca gaacgacg 38
<210> 52
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 52
agccgatgat taattgtcaa ttacccgcga cgcgctttta 40
<210> 53
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 53
taaaagcgcg tcgcgggtaa ttgacaatta atcatcggct 40
<210> 54
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 54
tttctttacc agactcgagt caacaattga tcgtctgt 38
<210> 55
<211> 39
<212> DNA
<213> Artificial Sequence
<400> 55
cgtctactag cgcagcttaa ttgacaatta atcatcggc 39
<210> 56
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 56
tggcagcagc ctaggttaac agccaagctt ggctgcag 38
<210> 57
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 57
gcaggtgagt ttctcaatat gatcacccca 30
<210> 58
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 58
atattgagaa actcacctgc cgctggcaga 30
<210> 59
<211> 1413
<212> DNA
<213> Escherichia coli
<400> 59
atgaaaaaga ccaaaattgt ttgcaccatc ggaccgaaaa ccgaatctga agagatgtta 60
gctaaaatgc tggacgctgg catgaacgtt atgcgtctga acttctctca tggtgactat 120
gcagaacacg gtcagcgcat tcagaatctg cgcaacgtga tgagcaaaac tggtaaaacc 180
gccgctatcc tgcttgatac caaaggtccg gaaatccgca ccatgaaact ggaaggcggt 240
aacgacgttt ctctgaaagc tggtcagacc tttactttca ccactgataa atctgttatc 300
ggcaacagcg aaatggttgc ggtaacgtat gaaggtttca ctactgacct gtctgttggc 360
aacaccgtac tggttgacga tggtctgatc ggtatggaag ttaccgccat tgaaggtaac 420
aaagttatct gtaaagtgct gaacaacggt gacctgggcg aaaacaaagg tgtgaacctg 480
cctggcgttt ccattgctct gccagcactg gctgaaaaag acaaacagga cctgatcttt 540
ggttgcgaac aaggcgtaga ctttgttgct gcttccttta ttcgtaagcg ttctgacgtt 600
atcgaaatcc gtgagcacct gaaagcgcac ggcggcgaaa acatccacat catctccaaa 660
atcgaaaacc aggaaggcct caacaacttc gacgaaatcc tcgaagcctc tgacggcatc 720
atggttgcgc gtggcgacct gggtgtagaa atcccggtag aagaagttat cttcgcccag 780
aagatgatga tcgaaaaatg tatccgtgca cgtaaagtcg ttatcactgc gacccagatg 840
ctggattcca tgatcaaaaa cccacgcccg actcgcgcag aagccggtga cgttgcaaac 900
gccatcctcg acggtactga cgcagtgatg ctgtctggtg aatccgcaaa aggtaaatac 960
ccgctggaag cggtttctat catggcgacc atctgcgaac gtaccgaccg cgtgatgaac 1020
agccgtctcg agttcaacaa tgacaaccgt aaactgcgca ttaccgaagc ggtatgccgt 1080
ggtgccgttg aaactgctga aaaactggat gctccgctga tcgtggttgc tactcagggc 1140
ggtaaatctg ctcgcgcagt acgtaaatac ttcccggatg ccaccatcct ggcactgacc 1200
accaacgaaa aaacggctca tcagttggta ctgagcaaag gcgttgtgcc gcagcttgtt 1260
aaagagatca cttctactga tgatttctac cgtctgggta aagaactggc tctgcagagc 1320
ggtctggcac acaaaggtga cgttgtagtt atggtttctg gtgcactggt accgagcggc 1380
actactaaca ccgcatctgt tcacgtcctg taa 1413
<210> 60
<211> 1443
<212> DNA
<213> Escherichia coli
<400> 60
atgtccagaa ggcttcgcag aacaaaaatc gttaccacgt taggcccagc aacagatcgc 60
gataataatc ttgaaaaagt tatcgcggcg ggtgccaacg ttgtacgtat gaacttttct 120
cacggctcgc ctgaagatca caaaatgcgc gcggataaag ttcgtgagat tgccgcaaaa 180
ctggggcgtc atgtggctat tctgggtgac ctccaggggc ccaaaatccg tgtatccacc 240
tttaaagaag gcaaagtttt cctcaatatt ggggataaat tcctgctcga cgccaacctg 300
ggtaaaggtg aaggcgacaa agaaaaagtc ggtatcgact acaaaggcct gcctgctgac 360
gtcgtgcctg gtgacatcct gctgctggac gatggtcgcg tccagttaaa agtactggaa 420
gttcagggca tgaaagtgtt caccgaagtc accgtcggtg gtcccctctc caacaataaa 480
ggtatcaaca aacttggcgg cggtttgtcg gctgaagcgc tgaccgaaaa agacaaagca 540
gacattaaga ctgcggcgtt gattggcgta gattacctgg ctgtctcctt cccacgctgt 600
ggcgaagatc tgaactatgc ccgtcgcctg gcacgcgatg caggatgtga tgcgaaaatt 660
gttgccaagg ttgaacgtgc ggaagccgtt tgcagccagg atgcaatgga tgacatcatc 720
ctcgcctctg acgtggtaat ggttgcacgt ggcgacctcg gtgtggaaat tggcgacccg 780
gaactggtcg gcattcagaa agcgttgatc cgtcgtgcgc gtcagctaaa ccgagcggta 840
atcacggcga cccagatgat ggagtcaatg attactaacc cgatgccgac gcgtgcagaa 900
gtcatggacg tagcaaacgc cgttctggat ggtactgacg ctgtgatgct gtctgcagaa 960
actgccgctg ggcagtatcc gtcagaaacc gttgcagcca tggcgcgcgt ttgcctgggt 1020
gcggaaaaaa tcccgagcat caacgtttct aaacaccgtc tggacgttca gttcgacaat 1080
gtggaagaag ctattgccat gtcagcaatg tacgcagcta accacctgaa aggcgttacg 1140
gcgatcatca ccatgaccga atcgggtcgt accgcgctga tgacctcccg tatcagctct 1200
ggtctgccaa ttttcgccat gtcgcgccat gaacgtacgc tgaacctgac tgctctctat 1260
cgtggcgtta cgccggtgca ctttgatagc gctaatgacg gcgtagcagc tgccagcgaa 1320
gcggttaatc tgctgcgcga taaaggttac ttgatgtctg gtgacctggt gattgtcacc 1380
cagggcgacg tgatgagtac cgtgggttct actaatacca cgcgtatttt aacggtagag 1440
taa 1443
<210> 61
<211> 327
<212> DNA
<213> Escherichia coli
<400> 61
atggcccaac aatcacccta ttcagcagcg atggcagaac agcgtcacca ggagtggtta 60
cgttttgtcg acctgcttaa gaatgcctac caaaacgatc tccatttacc gttgttaaac 120
ctgatgctga cgccagatga gcgcgaagcg ttggggactc gcgtgcgtat tgtcgaagag 180
ctgttgcgcg gcgaaatgag ccagcgtgag ttaaaaaatg aactcggcgc aggcatcgcg 240
acgattacgc gtggatctaa cagcctgaaa gccgcgcccg tcgagctgcg ccagtggctg 300
gaagaggtgt tgctgaaaag cgattga 327
<210> 62
<211> 2652
<212> DNA
<213> Escherichia coli
<400> 62
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> 63
<211> 882
<212> DNA
<213> Escherichia coli
<400> 63
atgacacgac aaaaagcaac gctcataggg ctgatagcga tcgtcctgtg gagcacgatg 60
gtaggattga ttcgcggtgt cagtgagggg ctcggcccgg tcggcggcgc agctgctatc 120
tattcattaa gcgggctgct gttaatcttc acggttggat ttccgcgtat tcggcaaatc 180
ccgaaaggct atttactcgc cgggagtctg ttattcgtca gctatgaaat ctgtctggcg 240
ctttccttag ggtatgcggc gacccatcat caggcgattg aagtgggtat ggtgaactat 300
ctgtggccca gcctgacaat tctctttgcc attctgttta atggtcagaa aaccaactgg 360
ttgattgtac ctggattatt attagccctc gtcggcgtct gttgggtgtt aggcggtgac 420
aatgggttac attatgatga aatcatcaat aatatcacca ccagcccatt gagttatttc 480
ctggcgttca ttggtgcgtt tatctgggca gcctattgca cagtaacgaa taaatacgca 540
cgcggattta atggaattac cgtttttgtc ctgctaacgg gagcaagtct gtgggtttac 600
tattttctta cgccacaacc agaaatgata tttagcacgc ccgtcatgat taaactcatc 660
tctgcggcat ttaccttagg atttgcttat gctgcatgga atgtcggtat attgcatggc 720
aatgtcacca ttatggcggt aggttcgtat tttacgcctg tactttcctc agcgcttgca 780
gccgtgctgc tcagcgcccc gctgtcgttc tcgttctggc aaggcgcgct gatggtctgc 840
ggcggttccc tgctctgctg gctggcgaca cgtcgtggtt aa 882
<210> 64
<211> 1416
<212> DNA
<213> Escherichia coli
<400> 64
atggaaaact ttaaacatct ccctgaaccg ttccgcattc gtgttattga gccagtaaaa 60
cgtaccactc gcgcttatcg tgaagaggca attattaaat ccggtatgaa cccgttcctg 120
ctggatagcg aagatgtttt tatcgattta ctgaccgaca gcggcaccgg ggcggtgacg 180
cagagcatgc aggctgcgat gatgcgcggc gacgaagcct acagcggcag tcgtagctac 240
tatgcgttag ccgagtcagt gaaaaatatc tttggttatc aatacaccat tccgactcac 300
cagggccgtg gcgcagagca aatctatatt ccggtactga ttaaaaaacg cgagcaggaa 360
aaaggcctgg atcgcagcaa aatggtggcg ttctctaact atttctttga taccacgcag 420
ggccatagcc agatcaacgg ctgtaccgtg cgtaacgtct atatcaaaga agccttcgat 480
acgggcgtgc gttacgactt taaaggcaac tttgaccttg agggattaga acgcggtatt 540
gaagaagttg gtccgaataa cgtgccgtat atcgttgcaa ccatcaccag taactctgca 600
ggtggtcagc cggtttcact ggcaaactta aaagcgatgt acagcatcgc gaagaaatac 660
gatattccgg tggtaatgga ctccgcgcgc tttgctgaaa acgcctattt catcaagcag 720
cgtgaagcag aatacaaaga ctggaccatc gagcagatca cccgcgaaac ctacaaatat 780
gccgatatgc tggcgatgtc cgccaagaaa gatgcgatgg tgccgatggg cggcctgctg 840
tgcatgaaag acgacagctt ctttgatgtg tacaccgagt gcagaaccct ttgcgtggtg 900
caggaaggct tcccgacata tggcggcctg gaaggcggcg cgatggagcg tctggcggta 960
ggtctgtatg acggcatgaa tctcgactgg ctggcttatc gtatcgcgca ggtacagtat 1020
ctggtcgatg gtctggaaga gattggcgtt gtctgccagc aggcgggcgg tcacgcggca 1080
ttcgttgatg ccggtaaact gttgccgcat atcccggcag accagttccc ggcacaggcg 1140
ctggcctgcg agctgtataa agtcgccggt atccgtgcgg tagaaattgg ctctttcctg 1200
ttaggccgcg atccgaaaac cggtaaacaa ctgccatgcc cggctgaact gctgcgttta 1260
accattccgc gcgcaacata tactcaaaca catatggact tcattattga agcctttaaa 1320
catgtgaaag agaacgcggc gaatattaaa ggattaacct ttacgtacga accgaaagta 1380
ttgcgtcact tcaccgcaaa acttaaagaa gtttaa 1416
<210> 65
<211> 2379
<212> DNA
<213> Escherichia coli
<400> 65
atgtccaaca atggctcgtc accgctggtg ctttggtata accaactcgg catgaatgat 60
gtagacaggg ttgggggcaa aaatgcctcc ctgggtgaaa tgattactaa tctttccgga 120
atgggtgttt ccgttccgaa tggtttcgcc acaaccgccg acgcgtttaa ccagtttctg 180
gaccaaagcg gcgtaaacca gcgcatttat gaactgctgg ataaaacgga tattgacgat 240
gttactcagc ttgcgaaagc gggcgcgcaa atccgccagt ggattatcga cactcccttc 300
cagcctgagc tggaaaacgc catccgcgaa gcctatgcac agctttccgc cgatgacgaa 360
aacgcctctt ttgcggtgcg ctcctccgcc accgcagaag atatgccgga cgcttctttt 420
gccggtcagc aggaaacctt cctcaacgtt cagggttttg acgccgttct cgtggcagtg 480
aaacatgtat ttgcttctct gtttaacgat cgcgccatct cttatcgtgt gcaccagggt 540
tacgatcacc gtggtgtggc gctctccgcc ggtgttcaac ggatggtgcg ctctgacctc 600
gcatcatctg gcgtgatgtt ctccattgat accgaatccg gctttgacca ggtggtgttt 660
atcacttccg catggggcct tggtgagatg gtcgtgcagg gtgcggttaa cccggatgag 720
ttttacgtgc ataaaccgac actggcggcg aatcgcccgg ctatcgtgcg ccgcaccatg 780
gggtcgaaaa aaatccgcat ggtttacgcg ccgacccagg agcacggcaa gcaggttaaa 840
atcgaagacg taccgcagga acagcgtgac atcttctcgc tgaccaacga agaagtgcag 900
gaactggcaa aacaggccgt acaaattgag aaacactacg gtcgcccgat ggatattgag 960
tgggcgaaag atggccacac cggtaaactg ttcattgtgc aggcgcgtcc ggaaaccgtg 1020
cgctcacgcg gtcaggtcat ggagcgttat acgctgcatt cacagggtaa gattatcgcc 1080
gaaggccgtg ctatcggtca tcgcatcggt gcgggtccgg tgaaagtcat ccatgacatc 1140
agcgaaatga accgcatcga acctggcgac gtgctggtta ctgacatgac cgacccggac 1200
tgggaaccga tcatgaagaa agcatctgcc atcgtcacca accgtggcgg tcgtacctgt 1260
cacgcggcga tcatcgctcg tgaactgggc attccggcgg tagtgggctg tggagatgca 1320
acagaacgga tgaaagacgg tgagaacgtc actgtttctt gtgccgaagg tgataccggt 1380
tacgtctatg cggagttgct ggaatttagc gtgaaaagct ccagcgtaga aacgatgccg 1440
gatctgccgt tgaaagtgat gatgaacgtc ggtaacccgg accgtgcttt cgacttcgcc 1500
tgcctaccga acgaaggcgt gggccttgcg cgtctggaat ttatcatcaa ccgtatgatt 1560
ggcgtccacc cacgcgcact gcttgagttt gacgatcagg aaccgcagtt gcaaaacgaa 1620
atccgcgaga tgatgaaagg ttttgattct ccgcgtgaat tttacgttgg tcgtctgact 1680
gaagggatcg cgacgctggg tgccgcgttt tatccgaagc gcgtcattgt ccgtctctct 1740
gattttaaat cgaacgaata tgccaacctg gtcggtggtg agcgttacga gccagatgaa 1800
gagaacccga tgctcggctt ccgtggcgcg ggccgctatg tttccgacag cttccgcgac 1860
tgtttcgcgc tggagtgtga agcagtgaaa cgtgtgcgca acgacatggg actgaccaac 1920
gttgagatca tgatcccgtt cgtgcgtacc gtagatcagg cgaaagcggt ggttgaagaa 1980
ctggcgcgtc aggggctgaa acgtggcgag aacgggctga aaatcatcat gatgtgtgaa 2040
atcccgtcca acgccttgct ggccgagcag ttcctcgaat atttcgacgg cttctcaatt 2100
ggctcaaacg atatgacgca gctggcgctc ggtctggacc gtgactccgg cgtggtgtct 2160
gaattgttcg atgagcgcaa cgatgcggtg aaagcactgc tgtcgatggc tatccgtgcc 2220
gcgaagaaac agggcaaata tgtcgggatt tgcggtcagg gtccgtccga ccacgaagac 2280
tttgccgcat ggttgatgga agaggggatc gatagcctgt ctctgaaccc ggacaccgtg 2340
gtgcaaacct ggttaagcct ggctgaactg aagaaataa 2379
<210> 66
<211> 1992
<212> DNA
<213> Escherichia coli
<400> 66
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 aa 1992
<210> 67
<211> 1053
<212> DNA
<213> Escherichia coli
<400> 67
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> 68
<211> 525
<212> DNA
<213> Escherichia coli
<400> 68
atgacacaac ctctttttct gatcgggcct cggggctgtg gtaaaacaac ggtcggaatg 60
gcccttgccg attcgcttaa ccgtcggttt gtcgataccg atcagtggtt gcaatcacag 120
ctcaatatga cggtcgcgga gatcgtcgaa agggaagagt gggcgggatt tcgcgccaga 180
gaaacggcgg cgctggaagc ggtaactgcg ccatccaccg ttatcgctac aggcggcggc 240
attattctga cggaatttaa tcgtcacttc atgcaaaata acgggatcgt ggtttatttg 300
tgtgcgccag tatcagtcct ggttaaccga ctgcaagctg caccggaaga agatttacgg 360
ccaaccttaa cgggaaaacc gctgagcgaa gaagttcagg aagtgctgga agaacgcgat 420
gcgctatatc gcgaagttgc gcatattatc atcgacgcaa caaacgaacc cagccaggtg 480
atttctgaaa ttcgcagcgc cctggcacag acgatcaatt gttga 525
<210> 69
<211> 2424
<212> DNA
<213> Escherichia coli
<400> 69
atgtccggct ggccacgaat ttactacaaa ttactgaatt taccattaag catcctggta 60
aaaagcaagt ctattccggc agatcctgcc ccggaactgg ggctggatac ctctcgtcca 120
attatgtacg ttttaccgta caactcgaaa gcagatttgc tgacgttgcg cgcccagtgt 180
ctggcacatg acttgcctga cccgttagag ccgctggaaa tcgacggcac gctactgccg 240
cgctatgtgt tcattcacgg cgggccgcgt gtgttcacct attacacgcc gaaagaagag 300
tctattaagc tgttccacga ctatctcgat ttgcaccgta gcaacccaaa tctggatgtg 360
cagatggtgc cagtgtcggt gatgtttggt cgcgcgccgg ggcgtgaaaa aggcgaagtg 420
aacccgccgc tgcgtatgct taacggcgta cagaaatttt tcgctgtact gtggctcggt 480
cgcgacagtt ttgtgcgttt ctcgccgtca gtttcgctgc gccgtatggc ggatgaacac 540
ggcacggata aaactatcgc tcagaaactg gcgcgcgtgg cgcgtatgca ctttgcccgt 600
caacgtctgg ctgccgtagg cccacgtttg cctgctcgtc aggatctgtt taataagctg 660
ctcgcctccc gcgccattgc caaagcggta gaagatgaag cgcgcagcaa aaaaatctcc 720
catgaaaaag cgcagcagaa cgcgattgca ctgatggaag agattgcggc gaatttctct 780
tacgagatga ttcgcctgac tgaccgtatt ctgggcttca cctggaaccg actttaccag 840
ggcatcaacg tccataacgc tgagcgcgtt cgccagctgg cccacgacgg tcatgagctg 900
gtatatgtgc cttgccaccg cagtcacatg gactacctgc tgctttctta cgtgctgtat 960
caccaggggc tggtgccgcc gcatatcgcc gccgggatca acctgaattt ctggcctgcc 1020
gggccgattt tccgccgtct gggggcgttc tttattcgcc gtacgtttaa aggcaataaa 1080
ctttattcca ccgttttccg ggagtatctc ggcgaactgt tcagccgtgg ttattccgtc 1140
gagtacttcg tggaaggcgg tcgttcccgt acggggcgtt tgctggatcc gaaaactggt 1200
acgctgtcga tgaccattca ggcgatgctg cgtggcggca cgcgtccgat tacgctgatt 1260
ccgatctata tcggttatga gcacgtcatg gaagtgggta cttacgccaa agaactgcgc 1320
ggcgcgacga aagagaaaga gagcctgccg cagatgctgc gcggtttaag caagctgcgt 1380
aatctcggtc agggttacgt caacttcggt gaaccaatgc cgttgatgac ctaccttaac 1440
cagcatgtac ctgactggcg tgaatctatc gatcccatcg aagcggtgcg tccggcatgg 1500
ttaacgccga cggtcaataa tattgctgcc gatctgatgg tacgcattaa caacgcaggc 1560
gcggcaaacg ccatgaacct gtgctgtact gcgctactgg catcacgtca gcgctcactc 1620
acccgcgagc agttaaccga gcaactcaac tgctacctgg atctgatgcg caacgtgccc 1680
tactccacgg actctaccgt tccttcagcc agcgccagcg agcttatcga tcacgcgctg 1740
caaatgaaca agtttgaagt cgagaaagac acaatcggcg acatcatcat tctgccgcgc 1800
gagcaagcgg tgctgatgac ctactatcgc aacaacattg cgcatatgtt ggtgctgcct 1860
tcgctgatgg cggcaatcgt cacccagcat cgccacatct cccgcgacgt attgatggag 1920
cacgtcaatg tgctttaccc aatgctgaaa gcggagctgt tcctgcgctg ggatcgcgac 1980
gagttgccgg acgttattga tgcgctggca aatgagatgc aacgtcaggg gctgattacc 2040
ctgcaagatg atgagttgca tatcaacccg gcgcattctc gcacgctaca gctgctggcc 2100
gcaggcgcgc gcgaaacgct gcaacgttat gccatcacct tctggttgtt gagtgccaac 2160
ccgtcgatca accgcggtac gctggagaaa gagagccgca ccgtcgcgca acgtctctcc 2220
gtgctgcacg gcatcaacgc gccggagttc ttcgacaagg cggtgttcag ttctctggtg 2280
ctgacactgc gtgatgaagg gtatatcagc gatagcggcg atgccgaacc ggcagaaacg 2340
atgaaggttt atcagttgct ggcggagttg attacatcag acgtgcgttt gacgattgag 2400
agtgcgacgc agggcgaagg gtaa 2424
<210> 70
<211> 1053
<212> DNA
<213> Escherichia coli
<400> 70
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 ttctcgatat 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> 71
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 71
ctgcgtctgg tcaccaatnn kcgtttatgg ccgctc 36
<210> 72
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 72
ggtcaccaga cgcagatcgt 20
<210> 73
<211> 56
<212> DNA
<213> Artificial Sequence
<400> 73
ttgacaatta atcatcggct cgtataatgt gtggaattgt aagaaggaga tatacc 56
Claims (4)
1.一种用于靛玉红生产的大肠杆菌基因工程菌的构建方法,其特征在于,包括:以W3110大肠杆菌为出发菌,在其基因组上连续敲除丙酮酸激酶IpykA、丙酮酸激酶IIpykF、色氨酸阻遏蛋白trpR、磷酸烯醇式丙酮酸激酶ppc和芳香族氨基酸转运蛋白yddG这五种基因,并分别原位回补黄素单加氧酶fmo基因表达框,然后进一步以高拷贝质粒pkk-223-3-miniptac为表达质粒,以高拷贝质粒形式强化色氨酸酶tnaA、黄素单加氧酶突变体fmo K223R /D317S这两种基因,以及以中拷贝质粒形式强化色氨酸合成路径的磷酸烯醇式丙酮酸合成酶ppsA、转酮醇酶ItktA、DAHP合成酶aroG fbr 、莽草酸激酶IIaroL和细胞膜合成基因甘油-3-磷酸乙酰转移酶plsB这五种基因,即可获得一种用于靛玉红生产的大肠杆菌基因工程菌;其中,
所述丙酮酸激酶IpykA、丙酮酸激酶IIpykF、色氨酸阻遏蛋白trpR、磷酸烯醇式丙酮酸激酶ppc、芳香族氨基酸转运蛋白yddG基因分别编码如SEQ ID NO.59-63所示的氨基酸序列的蛋白;
所述色氨酸酶tnaA、磷酸烯醇式丙酮酸合成酶ppsA、转酮醇酶ItktA、DAHP合成酶aroG fbr 、莽草酸激酶IIaroL和细胞膜合成基因甘油-3-磷酸乙酰转移酶plsB基因分别编码如SEQ ID NO.64-69所示的氨基酸序列的蛋白;所述黄素单加氧酶fmo选自:野生型黄素单加氧酶fmo、突变体黄素单加氧酶fmo N291T、突变体黄素单加氧酶fmo K223R、突变体黄素单加氧酶fmo K223R/D317S中的任意一种;
所述野生型黄素单加氧酶fmo编码如SEQ ID NO. 1所示氨基酸序列的蛋白;所述突变体黄素单加氧酶fmo N291T、 fmo K223R、 fmo K223R/D317S分别编码如SEQ ID NO. 2-4所示氨基酸序列的蛋白。
2.根据权利要求1所述的构建方法,其特征在于,对大肠杆菌基因组上进行丙酮酸激酶IpykA、丙酮酸激酶IIpykF、色氨酸阻遏蛋白trpR、磷酸烯醇式丙酮酸激酶ppc和芳香族氨基酸转运蛋白yddG基因的敲除以及原位回补黄素单加氧酶fmo基因表达框通过Crisper-Cas9系统实现。
3.一种根据权利要求1~2中任意一项所述的构建方法构建而来的用于靛玉红生产的大肠杆菌基因工程菌。
4.一种生产靛玉红的方法,其特征在于,发酵根据权利要求3所述的用于靛玉红生产的大肠杆菌基因工程菌,实现靛玉红的生产。
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Developing synthetic microbes to produce indirubin-derivatives;Sandipty Kayastha et al.;Biocatalysis andAgriculturalBiotechnology;全文 * |
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