CN114958890A - 构建稳定遗传的水杨酸生物合成的基因工程菌株的方法及其应用 - Google Patents
构建稳定遗传的水杨酸生物合成的基因工程菌株的方法及其应用 Download PDFInfo
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- CN114958890A CN114958890A CN202210505539.7A CN202210505539A CN114958890A CN 114958890 A CN114958890 A CN 114958890A CN 202210505539 A CN202210505539 A CN 202210505539A CN 114958890 A CN114958890 A CN 114958890A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
构建稳定遗传的水杨酸生物合成的基因工程菌株的方法及其应用涉及生物工程技术领域。构建基因工程菌株:应用CRISPR Cas9技术在宿主内敲除磷酸组氨酸搬运蛋白的基因、整合葡萄糖转运途径、解除途径酶的反馈抑制,然后在宿主内整合异分支酸丙酮酸裂解酶和异分支酸合酶的基因构建遗传稳定的产水杨酸工程菌。本发明优点(1)该基因工程菌碳收率为天然理论碳收率的50%,使工业可行性大大提高。(2)该基因工程菌的生产更节约成本,无需外加抗生素和诱导剂,只要有充足的碳源就可以在确保该菌株稳定遗传的同时实现水杨酸高效生物合成。本发明实现水杨酸的高产,可用作基于水杨酸为出发化合物的下游表达组件即插即用的生产平台大肠杆菌。
Description
技术领域
本发明涉及生物工程技术领域,具体涉及到水杨酸高产菌株的构建及其方法和应用。
背景技术
水杨酸是一种单羟基苯甲酸、一种酚酸和一种β-羟基酸。水杨醇是一种占有重要地位的精细化学品,主要用作大量合成水杨醛的原料,广泛作为中间体应用于合成具有抗炎功效的人用药品和农药,还可成为酚醛树脂等高分子材料的原料。最为人所知的是它在皮肤局部消费医疗(抗痤疮)的使用。水杨酸的工业合成基于Kolbe-Schmitt反应,在反应中使用大量的硫酸,产生了大量的酸污染和盐污染,故寻求生物合成的方法缓解该问题。生物合成法可以葡萄糖为碳源,构建质粒将途径基因转化到宿主菌中从头合成水杨酸,但是质粒的不稳定性遗传影响生产菌株的活性,且质粒系统有依赖抗生素的缺点,同时,引入质粒给宿主带来额外的代谢负担,影响产量及产率。
本发明通过改造大肠杆菌中心碳代谢,增强莽草酸途径,提高生产水杨酸途径的碳通量,并将全部途径基因整合到宿主菌的基因组,无需表达质粒就可以生产水杨酸,提高工程菌株生产稳定性,节约生产成本,生产水杨酸的产量为1.07g/L。向本发明所构建的基因工程菌中转入水杨醇生产质粒,可生产水杨醇的产量为1.60g/L。
发明内容
本发明提供了一种高产水杨酸的基因工程菌的构建方法。
本发明构建的基因工程菌,通过改造大肠杆菌中心碳代谢,改造莽草酸途径基因和整合生产水杨酸的途径基因,构建了一种高产水杨酸的基因工程菌株。实验结果表明,该工程菌株利用葡萄糖为简单碳源来生产水杨酸能达到1.07g/L的最终产量。
本发明提供了一种上述高产水杨酸的工程菌的构建方法,包括步骤:
构建基因工程菌株::应用CRISPR Cas9技术在宿主内敲除糖酵解途径基因pykA和pykF、敲除编码磷酸烯醇式丙酮酸-糖转移酶系统相关蛋白质的基因PtsH-I-crr、整合促葡萄糖转运酶(基因Glf编码)、葡萄糖激酶(基因Glk编码)、解除反馈抑制的3-脱氧-D- 阿拉伯庚糖酮酸-7-磷酸合酶(基因AroGfbr编码),然后在宿主内整合异分支酸丙酮酸裂解酶(基因pchB编码)、异分支酸合酶(基因entC编码)的基因构建遗传稳定的产水杨酸工程菌。
上述基因工程菌所述的整合位点,基因glfzm替换基因组上的ptsHI crr、基因glkEc、由10倍强度trc启动子启动的基因aroGfbr替换基因组上的假意义基因yeeP、基因entC-pchB 替换基因组上的假意义基因mbhA和yncI。
本发明还提供一种高产水杨酸的基因工程菌的应用,其中,将上述工程菌划线于无抗生素的平板上,37℃过夜培养,挑取阳性单克隆转化子,得到水杨酸的基因工程菌株,将其接种到发酵培养基中,30℃进行发酵从头合成生产水杨酸。上述的发酵培养基包括20g/L 葡萄糖,5g/L酵母粉,1g/L MOPS,5g/L NaHPO4,1g/L NaCl,3g/L KH2PO4,1g/LNH4Cl, 250mg/L MgSO4,15mg/L CaCl2,溶剂为水。
本发明提供的基因工程菌是通过改造大肠杆菌中心碳代谢,改造莽草酸途径基因,然后在上述工程菌中共表达异分支酸丙酮酸裂解酶(PchB)的基因pchB和异分支酸合酶(EntC) 的基因entC,从头合成水杨酸,最终水杨酸产量可达到1.07g/L。
本发明还提供一种高产水杨醇的基因工程菌的应用,其中,将上述工程菌划线于无抗生素的平板上,37℃过夜培养,挑取阳性单克隆转化子,得到水杨酸的基因工程菌株,电化学转入水杨醇生产质粒pET1-Car-Sfp,其携带来源于海洋分枝杆菌的羧酸还原酶(Car) 的基因car、来源于枯草芽孢杆菌的磷酰转移酶(Sfp)基因sfp,将其接种到发酵培养基中, 30℃进行发酵从头合成生产水杨醇。
基于上述,所述的发酵培养基包括20g/L葡萄糖,5g/L酵母粉,1g/L MOPS,5g/LNaHPO4,1g/L NaCl,3g/L KH2PO4,1g/L NH4Cl,250mg/L MgSO4,15mg/L CaCl2,溶剂为水。
本发明提供的基因工程菌是通过改造大肠杆菌中心碳代谢,改造莽草酸途径基因,然后在上述工程菌中共表达异分支酸丙酮酸裂解酶(PchB)和异分支酸合酶(EntC)的基因,从头合成水杨酸,最终水杨酸产量可达到1.07g/L。通过引入水杨醇生产质粒,在上述工程菌中共表达羧酸还原酶(Car)和磷酰转移酶(Sfp)的基因,最终水杨醇产量可达到1.60g/L。
本发明提供的上述基因工程菌与原始的需要导入质粒才能生产水杨酸重组工程菌相比,其优点在于(1)该基因工程菌碳收率为天然理论碳收率的50%,使工业可行性大大提高。(2)该基因工程菌的生产更节约成本,这是因为通过对野生型大肠杆菌的基因改造构建了一株天然生产水杨酸的菌株,即无需外加抗生素和诱导剂,只要有充足的碳源就可以在确保该菌株稳定遗传的同时实现水杨酸高效生物合成。
本发明提供的上述基因工程菌实现水杨酸的高产,可用作基于水杨酸为出发化合物的下游表达组件即插即用的生产平台大肠杆菌。
具体实施方式
下面将对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。
上述基因工程菌,所述的目的菌为大肠杆菌。大肠杆菌菌株BW25113为常用大肠杆菌菌株,可市售获得。
以下实施实例对本发明做进一步说明
实施例1构建重组质粒
重组质粒pET1-Car-Sfp,主要是将基因car-sfp替换pET载体kpnI和XbaI酶切位点间的DNA片段得到的重组载体,最终构建为pET1-Car-Sfp。
重组质粒pCS-PchB-EntC,主要是将基因pchB-entC替换pCS载体kpnI和BamHI酶切位点间的DNA片段得到的重组载体,最终构建为pCS-EP。
重组质粒pCS-Glf-Glk,主要是将基因glf-glk替换pCS载体kpnI和BamHI酶切位点间的DNA片段得到的重组载体,最终构建为pCS-Glf-Glk。
重组质粒pCS-aroGfbr,主要是将基因aroGfbr替换pCS载体kpnI和BamHI酶切位点间的DNA片段得到的重组载体,最终构建为pCS-aroGfbr。
实施例2构建基因工程大肠杆菌
应用crispr cas9技术将基因glfzm替换基因组上的ptsHI crr、glkEc替换基因组上的假意义基因yghX,由10倍trc启动子启动的基因aroGfbr替换基因组上的假意义基因yeeP、由 50倍trc启动子启动的基因entC-pchB替换基因组上的假意义基因mbhA和yncI,具体的实施方法:
(1)电转化法将载体pCas 9导入大肠杆菌BW25113中,在壮观霉素平板上30度培养20小时,将阳性克隆转化子命名为BW-pCas 9,挑取平板上所长的BW-pCas 9单克隆,接种到1.5μL/mL壮观霉素的LB液体培养基中30度培养。
(2)构建基因ptsHI crr位点sgRNA质粒
本研究中sgRNA使用的靶向序列,如表1所示
表1.基因ptsHI crr位点sgRNA使用的靶向序列
本研究中应用的引物序列,如表2所示
表2.引物序列表
P1与P2为一组做引物,pTarget质粒为模板,PCR获得含有sgRNA的核苷酸序列长度为2200kbp,琼脂糖凝胶电泳后,采用凝胶回收试剂盒对PCR产物进行纯化和回收,PCR 纯化液化学转化法到大肠杆菌DH5α感受态细胞中,在其中重组自连,形成带有氨苄青霉素抗性的pTarget-ptsHI-crr质粒。
(3)构建ptsHI crr位点整合片段
本研究中所应用的引物序列,如表3所示
表3.引物序列表
以大肠杆菌BW25113为模板,P3与P4为一组和P7与P8为一组分别做引物,PCR 扩增得到基因ptsHI crr两段同源臂,以质粒pCs-Glf-Glk为模板,以P5与P6为一组做引物,PCR扩增得到基因Glf片段;接着以这四段片段为模板,P3与P8为一组做引物,应用PCR重叠延伸的方法得到一个整合片段,进行琼脂糖凝胶电泳,然后采用凝胶回收试剂盒对PCR产物进行纯化和回收。
(4)电转化法将pTarget-ptsHI-crr质粒和ptsHI crr位点整合片段导入实施例2的(1) 中BW Cas 9菌株中,在菌体中sgRNA指引cas9蛋白识别整合位点序列对其切割,菌体自身的修复功能使整合片段同源重组替换假义位点,得到将基因glfzm替换基因组上的ptsHI crr的菌,在含壮观霉素和氨苄青霉素抗性抗性的平板上30度培养24小时。
(5)在带有壮观霉素液体LB培养基中培养上述基因工程菌株,同时加入10mmol/L的阿拉伯糖,30度培养24小时,诱导cas9蛋白表达降解pTarget-ptsHI-crr质粒。得到只含pCas质粒的菌,于带有壮观霉素的LB液体培养基中培养。
(6)构建基因yghX位点sgRNA质粒
本研究中sgRNA使用的靶向序列,如表4所示
表4.基因yghX位点sgRNA使用的靶向序列
本研究中应用的引物序列,如表5所示
表5引物序列表
P9与P2为一组做引物,pTarget质粒为模板,PCR获得含有sgRNA的核苷酸序列长度为2200kbp,琼脂糖凝胶电泳后,采用凝胶回收试剂盒对PCR产物进行纯化和回收,PCR 纯化液化学转化法到大肠杆菌DH5α感受态细胞中,在其中重组自连,形成带有氨苄青霉素的pTarget-yghX质粒。
(7)构建yghX位点整合片段
本研究中所应用的引物序列,如表6所示
表6引物序列表
以大肠杆菌BW25113为模板,P10与P11为一组和P14与P15为一组分别做引物, PCR扩增得到基因ptsHI crr两段同源臂,以质粒pCs-Glf-Glk为模板,以P12与P13为一组做引物,PCR扩增得到基因Glk片段;接着以这四段片段为模板,P10与P15为一组做引物,应用PCR重叠延伸的方法得到一个整合片段,进行琼脂糖凝胶电泳,然后采用凝胶回收试剂盒对PCR产物进行纯化和回收。
(8)电转化法将pTarget-yghX质粒和yghX位点整合片段导入实施例2的(5)中消除pTarget-ptsHI-crr后的菌株中,在菌体中sgRNA指引cas9蛋白识别整合位点序列对其切割,菌体自身的修复功能使整合片段同源重组替换假义位点,得到将基因glkEC替换基因组上的yghX的菌,在含壮观霉素和氨苄青霉素抗性的平板上30度培养24小时。
(9)将pTarget-yghX质粒消掉以得到只含的菌,其方法同实施例2的(5)所述。得到消除质粒,只含pCas质粒的菌。
(10)构建基因yeeP位点sgRNA质粒
本研究中sgRNA使用的靶向序列,如表7所示
表7.基因yeeP位点sgRNA使用的靶向序列
本研究中应用的引物序列,如表8所示
表8引物序列表
基因yeeP位点sgRNA质粒的构建
P16与P2为一组做引物,pTarget质粒为模板,PCR获得含有sgRNA的核苷酸序列长度为2200kbp,琼脂糖凝胶电泳后,采用凝胶回收试剂盒对PCR产物进行纯化和回收,PCR 纯化液化学转化法到大肠杆菌DH5α感受态细胞中,在其中重组自连,形成带有氨苄青霉素抗性的pTarget-yeeP质粒。
(11)构建yeeP位点整合片段
本研究中所应用的引物序列,如表9所示
表9引物序列表
以大肠杆菌BW25113为模板,P17与P18为一组和P21与P22为一组分别做引物, PCR扩增得到基因yeeP两段同源臂,以质粒pCS-aroGfbr为模板,以P19与P20为一组做引物,PCR扩增得到基因aroGfbr片段;接着以这三段片段为模板,P17与P22为一组做引物,应用PCR重叠延伸的方法得到一个整合片段,进行琼脂糖凝胶电泳,然后采用凝胶回收试剂盒对PCR产物进行纯化和回收。
(12)电转化法将pTarget-yeeP质粒和yeeP位点整合片段导入实施例2(9)中带有pCas质粒的工程菌中,在菌体中sgRNA指引cas9蛋白识别整合位点序列对其切割,菌体自身的修复功能使整合片段同源重组替换假义位点,得到由10倍trc启动子启动的基因aroGfbr替换基因组上假意义基因yeeP的菌,在含壮观霉素和氨苄青霉素抗性的平板上30 度培养24小时。
(13)将pTarget-yeeP质粒消掉以得到只含pCas质粒的菌,其方法同实施例2的(5)所述。
(14)基因mbhA和yncI位点sgRNA质粒的构建
本研究中sgRNA使用的靶向序列,如表10所示
表10.基因mbhA和yncI位点sgRNA使用的靶向序列
本研究中应用的引物序列,如表11所示
表11.引物序列表
基因mbhA和yncI位点sgRNA质粒的构建
P23与P2为一组做引物,pTarget质粒为模板,PCR获得含有sgRNA的核苷酸序列长度为2200kbp,琼脂糖凝胶电泳后,采用凝胶回收试剂盒对PCR产物进行纯化和回收,PCR 纯化液化学转化法到大肠杆菌DH5α感受态细胞中,在其中重组自连,形成带有氨苄青霉素的pTarget-mbhA质粒。P24与P2为一组做引物,pTarget质粒为模板,PCR获得含有sgRNA 的核苷酸序列长度为2200kbp,琼脂糖凝胶电泳后,采用凝胶回收试剂盒对PCR产物进行纯化和回收,PCR纯化液化学转化法到大肠杆菌DH5α感受态细胞中,在其中重组自连,形成带有氨苄青霉素的pTarget-yncI质粒。
(15)mbhA和yncI位点整合片段的构建
本研究中所应用的引物序列,如表12所示
表12.引物序列表
整合片段的构建
以大肠杆菌BW25113为模板,P25与P26为一组和P29与P30为一组分别做引物, PCR扩增得到基因mbhA两段同源臂,以质粒pCS-PchB-EntC为模板,以P27与P28为引物,PCR扩增得到基因mbhA-EP片段;接着以这三段片段为模板,P25与P30为一组做引物,应用PCR重叠延伸的方法得到一个整合片段;以大肠杆菌BW25113为模板,P31与 P32为一组和P34与P35为一组分别做引物,PCR扩增得到基因yncI两段同源臂,以质粒 pCS-PchB-EntC为模板,以P27与P33为一组做引物,PCR扩增得到基因yncI-EP片段;接着以这三段片段为模板,P31与P35为一组做引物,应用PCR重叠延伸的方法得到一个整合片段,进行琼脂糖凝胶电泳,然后采用凝胶回收试剂盒对PCR产物进行纯化和回收。
(16)电转化法将pTarget-mbhA质粒和mbhA-EP整合片段导入实列2的(13)中只带有pCas 9质粒的菌株中,在菌体中sgRNA指引cas9蛋白识别整合位点序列对其切割,菌体自身的修复功能使整合片段同源重组替换假义位点,得到一倍EP基因整合的菌株,在含壮观霉素与氨苄青霉素抗性的平板上30度培养24小时。
(17)在带有壮观霉素液体LB中培养上述菌株,同时加入10mmol/L的阿拉伯糖,30度培养24小时,诱导cas9蛋白表达降解pTarget-mbhA质粒。
(18)电转化法将yncI位点的pTarget-yncI质粒和整合片段导入实例2的(16)中只带有pCas 9质粒的菌株中,在菌体中sgRNA指引cas9蛋白识别整合位点序列对其切割,菌体自身的修复功能使整合片段同源重组替换假义位点,得到两倍EP基因整合的菌株,在含壮观霉素与氨苄青霉素抗性的平板上30度培养24小时。
(19)消除pTarget-yncI质粒,在带有壮观霉素液体LB中培养上述基因工程菌株,同时加入10mmol/L的阿拉伯糖,30度培养24小时,诱导cas9蛋白表达降解sgRNA质粒。
(20)消除pCas质粒,在无抗生素液体LB中培养上述已消掉pTarget-yncI质粒的基因工程菌,42度培养48小时,使温敏型pCas质粒降解。
(21)得到不需抗生素实现稳定遗传生产水杨酸的基因工程菌株BW1。
实施例3基因工程菌BW1的应用:发酵培养从头合成水杨酸
将基因工程菌株BW1涂于无抗生素的平板上,37℃过夜培养,挑取阳性单克隆转化子到4ml LB试管中,7℃培养10h,转入到无抗生素的50ml的发酵培养基中,接种量为体积比的2%,发酵温度为37℃,转速为220rpm,所述培养基包括20g/L葡萄糖,5g/L 酵母粉,1g/LMOPS,5g/L NaHPO4,1g/L NaCl,3g/L KH2PO4,1g/L NH4Cl,250mg/L MgSO4,15mg/L CaCl2,溶剂为水。
发酵每隔12小时取出部分发酵液用以测定菌体生长状况及目标产物水杨酸的产量。采用HPLC分析方法对目标产物水杨酸进行检测,检测条件如下:
色谱柱:分离柱:Diamonsil C18,ID 5μm,250×4.6mm;
流动相:A为甲醇,B为0.1‰三氟乙酸水溶液,柱温35℃,总流速为0.8mL/min。检测波长为290nm。梯度洗脱程序如表13所示:
表13.梯度洗脱
最终水杨酸产量可达到1.07g/L。
实施例4基因工程菌株BW1的应用:发酵培养从头合成水杨醇
电化学转入法将pET1-Car-Sfp转入基因工程菌03中,得到水杨醇生产菌株,将水杨醇生产菌株涂于无抗生素的平板上,37℃过夜培养,挑取阳性单克隆转化子到4ml LB试管中,7℃培养10h,转入到无抗生素的50ml的发酵培养基中,接种量为体积比的2%,发酵温度为37℃,转速为220rpm,所述培养基包括20g/L葡萄糖,5g/L酵母粉,1g/L MOPS,5g/LNaHPO4,1g/L NaCl,3g/L KH2PO4,1g/L NH4Cl,250mg/L MgSO4,15mg/L CaCl2,溶剂为水。
发酵每隔12小时取出部分发酵液用以测定菌体生长状况及目标产物水杨醇的产量。 HPLC检测方法同实例5中HPLC检测方法,检测波长为290nm。最终水杨醇产量可达到1.60g/L。
Claims (3)
1.构建稳定遗传的水杨酸生物合成的基因工程菌株的方法,其特征在于包括以下步骤:
构建基因工程菌株:应用CRISPR/Cas9技术在宿主内敲除糖酵解途径基因pykA和pykF、敲除编码磷酸烯醇式丙酮酸-糖转移酶系统相关蛋白质的基因PtsH-I-crr、整合促葡萄糖转运酶即基因Glf编码、葡萄糖激酶即基因Glk编码、解除反馈抑制的3-脱氧-D-阿拉伯庚糖酮酸-7-磷酸合酶即基因AroGfbr编码,然后在宿主内整合异分支酸丙酮酸裂解酶PchB和异分支酸合酶EntC的基因构建遗传稳定的产水杨酸工程菌;
上述基因工程菌所述的整合位点,基因glfzm替换基因组上的基因ptsHI crr、基因glkEc替换基因组上的假意义基因yghX、10倍强度trc启动子启动的基因aroGfbr替换基因组上的假意义基因yeeP、基因entC-pchB替换基因组上的假意义基因mbhA和yncI。
2.按照权利要求1所述方法得到的基因工程菌株的应用,其特征在于:将上述工程菌划线于无抗生素的平板上,37℃过夜培养,挑取阳性单克隆转化子,得到水杨酸的基因工程菌株,将其接种到发酵培养基中,30℃进行发酵从头合成生产水杨酸;
所述的发酵培养基包括20g/L葡萄糖,5g/L酵母粉,1g/L MOPS,5g/L NaHPO4,1g/LNaCl,3g/L KH2PO4,1g/L NH4Cl,250mg/L MgSO4,15mg/L CaCl2,溶剂为水。
3.按照权利要求1所述方法得到的基因工程菌株的应用,其特征在于:将上述工程菌划线于无抗生素的平板上,37℃过夜培养,挑取阳性单克隆转化子,得到水杨酸的基因工程菌株,电化学转入水杨醇生产质粒pET1-Car-Sfp,其携带来源于海洋分枝杆菌的羧酸还原酶的基因car、来源于枯草芽孢杆菌的磷酰转移酶基因sfp,将其接种到发酵培养基中,30℃进行发酵从头合成生产水杨醇;
所述的发酵培养基包括20g/L葡萄糖,5g/L酵母粉,1g/L MOPS,5g/L NaHPO4,1g/LNaCl,3g/L KH2PO4,1g/L NH4Cl,250mg/L MgSO4,15mg/L CaCl2,溶剂为水。
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