CN114107354A - 构建稳定遗传的β-熊果苷高效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β-熊果苷高效生物合成的基因工程菌株的方法及其应用 Download PDFInfo
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- CN114107354A CN114107354A CN202111387603.8A CN202111387603A CN114107354A CN 114107354 A CN114107354 A CN 114107354A CN 202111387603 A CN202111387603 A CN 202111387603A CN 114107354 A CN114107354 A CN 114107354A
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Abstract
本发明提供了构建稳定遗传的β‑熊果苷高效生物合成的基因工程菌株的方法及其应用。首先,在宿主内整合编码酪氨酸分解酶、香豆酸辅酶A连接酶、β‑肉桂酰羟基化酶、β‑肉桂酰氧化酶、β‑肉桂酰脱酰酶(phdC)的基因,构建能够高产对羟基苯甲酸的宿主菌株。其次,在该宿主内整合编码4‑羟基苯甲酸羟化酶和葡萄糖基转移酶的基因,构建能够生产β‑熊果苷的菌株。最后将编码核心途径的基因莽草酸途径基因整合到上述工程菌的基因组上,同时敲除竞争途径基因构建了稳定遗传的β‑熊果苷高效生物合成的基因工程菌株,摆脱了应用质粒对工程菌生产造成不稳定的影响,对β‑熊果苷的工业化生产极具有应用前景。
Description
技术领域
本发明涉及生物工程技术领域,具体涉及到β-熊果苷高产菌株的构建及其方法和应用。
背景技术
β-熊果苷又称对苯二酚葡萄糖苷、4-羟基苯基-β-D-吡喃葡糖苷,存在于熊果树、越橘树、梨树、冬青树等的植物叶子中,具有抑制酪氨酸酶阻止黑色素形成的能力,具有美白护肤的功效,广泛的应用在医疗和化妆品行业中。获取熊果苷的方法主要有四种:植物提取法、化学合成法、酶转化法、生物合成法,植物提取法的生产过程复杂,同时受环境季节的影响,使其产量较低;由于低催化效率和低选择性,化学合成熊果苷的方法也不是首选;由于氢醌的毒性,抑制酶的活性,并且底物和产物的分离困难,造成能源上的浪费;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熊果苷,但是质粒的不稳定性遗传影响生产菌株的活性,使工程化的大肠杆菌维持系数降低。
本发明将部分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β-熊果苷,提高工程菌株生产功能的稳定性,节约生产成本,同时过表达莽草酸途径和敲除竞争途径基因,提高生产熊果苷途径的碳通量,进而使熊果苷的产量提高原来的1.5倍,生产熊果苷的产量为9.3±0.03g/L。
发明内容
1.本发明的一个目的是提供高产β-熊果苷的基因工程菌。
2.上述基因工程菌,所述的目的菌为大肠杆菌。
3.本发明构建的基因工程菌,通过过表达莽草酸途径、敲除竞争途径基因以及整合生产对羟基苯甲酸的途径基因,构建了一种高产对羟基苯甲酸的基因工程菌株,然后,在该工程菌中整合编码4-羟基苯甲酸羟化酶(4HB1H)和葡萄糖基转移酶(TGS)的基因,进而构建了一株高产β-熊果苷的菌株。实验结果表明,该工程菌株利用葡萄糖、甘油等简单碳源来生产β-熊果苷能达到9.3±0.03g/L的最终产量。
4.上述高产β-熊果苷的基因工程菌:在宿主内整合编码酪氨酸分解酶(TAL)、香豆酸辅酶A连接酶(4CL2)、β-肉桂酰羟基化酶(phdE)、β-肉桂酰氧化酶(phdB)、β-肉桂酰脱酰酶(phdC)的基因,然后在宿主内整合编码4-羟基苯甲酸羟化酶(4HB1H)和葡萄糖基转移酶(TGS)的基因,最后在宿主内整合编码3-脱氧-7-磷酸庚酸合酶(AroG)、3-磷酸莽草酸酯-1-羧基乙烯基转移酶(AroA)、莽草酸激酶(AroL)、分支酸合成酶(AroC)的基因。
5.本发明还提供了一种上述高产β-熊果苷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技术在宿主内整合编码酪氨酸分解酶(TAL)、香豆酸辅酶A连接酶(4CL2)、β-肉桂酰羟基化酶(phdE)、β-肉桂酰氧化酶(phdB)、β-肉桂酰脱酰酶(phdC)的基因构建能够生产对羟基苯甲酸的菌株,然后在宿主内整合编码4-羟基苯甲酸羟化酶(4HB1H)和葡萄糖基转移酶(TGS)的基因构建能够生产β-熊果苷的工程菌的菌株,最后在宿主内整合3-脱氧-7-磷酸庚酸合酶(AroG)、3-磷酸莽草酸酯-1-羧基乙烯基转移酶(AroA)、莽草酸激酶(AroL)、分支酸合成酶(AroC)的基因,同时敲除竞争途径基因trpE和pheA基因,构建一株稳定高产β-熊果苷的工程菌。
6.上述基因工程菌所述的整合位点,基因TAL、4Cl2、phdE、phdB、phdC插入到基因pgi的后面,基因4HB1H、TGS替换基因组上的假意义基因yneO,基因aroG、aroA、aroL、aroC插入到基因ack的后面。
7.上述基因工程菌所述的整合位点,选取的原因是因为基因yneO是大肠杆菌的无意义位点,所以将其换为其他基因不影响大肠杆菌的正常生长;而基因pgi和ack是大肠杆菌进入糖酵解途径和三羧酸循环的关键基因,所以这两个基因是大肠杆菌生长过程中最为活跃的基因,因此在这两个基因后面插入其他基因,可以提高插入基因的表达强度,进而提高相应酶的表达量,提高目标产物的产量。
8.本发明还提供一种高产β-熊果苷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,得到高产β-熊果苷的基因工程菌株,将其接种到发酵培养基中,37℃进行发酵从头合成生产熊果苷。
9.基于上述,所述的发酵培养基包括20g/L葡萄糖、10g/L甘油,3g/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。
10.本发明提供的基因工程菌是通过将途径基因整合到宿主菌的基因组,过表达莽草酸途径基因敲除竞争途径基因,首先构建了一株能够高产对羟基苯甲酸的基因工程菌株,然后在上述工程菌中共表达编码4-羟基苯甲酸羟化酶(4HB1H)和葡萄糖基转移酶(TGS)的基因,从头合成熊果苷,最终β-熊果苷产量可达到9.3±0.03g/L。
11.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β-熊果苷重组工程菌相比,其优点在于(1)该基因工程菌的生产更稳定,这是因为质粒的不稳定性遗传影响生产菌株的活性,使工程化的大肠杆菌维持系数降低,这是由于1)质粒的丢失问题,即质粒不均匀的分布到子细胞导致无质粒细胞;2)质粒的结构不稳定,其中一些质粒的DNA序列容易发生突变,导致所需蛋白质的不正确表达;上述两种原因使发酵体系中存在大量不工作的菌体,造成碳源的浪费,使目标产物的产率降低。(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β-熊果苷的菌株,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β-熊果苷高效生物合成。
12.本发明提供的上述基因工程菌实现β-熊果苷的高产,避免了对苯二酚的添加,缓解了氢醌的毒性对生物酶活性的抑制问题;而且应用构建的整合菌株,无需表达质粒就可以实现β-熊果苷的高产,缓解了多个质粒表达对工程菌株生产不稳定的影响,而且不需要添加抗生素来维持菌株生长,也不需要添加诱导剂来诱导基因表达,使β-熊果苷生产成本降低,更有利于β-熊果苷的工业化生产。
附图说明
图1是本发明提供的生物合成β-熊果苷的路径图。
图2是本发明实施例1提供的BW生产β-熊果苷的发酵结果图。
图3A是标品β-熊果苷的HPLC分析结果图;图3B实施例1提供的菌株的发酵产物HPLC检测图。
图4是本发明实施例3提供的工程菌BW2生产β-熊果苷的发酵结果图。
图5是本发明实施例5提供的工程菌BW3生产β-熊果苷的发酵结果图。
具体实施方式
以下结合附图与实施实例对本发明做进一步说明
1.构建应用质粒生产β-熊果苷的对照菌株pZE-pCS/BW
(1)构建重组质粒:对照菌株
i.重组质粒pZE-TAL-4CL-phdE/B/C-4HB1H-TGS,主要是将基因-TAL-4CL-phdE/B/C替换pZE载体kpnI和XbaI酶切位点间的DNA片段得到的重组载体,基因4HB1H-TGS替换pZE载体BcuI和SacI酶切位点间的DNA片段得到的重组载体,最终构建为pZE-TAL-4CL-phdE/B/C-4HB1H-TGS。
ii.重组质粒pCS-aroG-aroA-aroC-aroL,主要是将基因-aroG-aroA-aroC-aroL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-aroG-aroA-aroC-aroL。
(2)电转化法将重组载体pZE-TAL-4CL-phdE/B/C-4HB1H-TGS和pCS-aroG-aroA-aroC-aroL导入基因工程菌中,在氨苄青霉素和卡纳青霉素的平板上筛选阳性克隆转化子命名为pZE-pCS/BW,37度过夜培养。制备生产熊果苷的重组菌株。
(3)在上述重组菌株的平板上挑取单菌落,接到4ml的带有氨苄青霉素和卡纳青霉素的液体LB培养基中,37度培养12小时,将菌液转接到50ml的发酵培养基中,加入的IPTG进行诱导。之后在12,24,36,48,60h时取样并且用高效液相色谱测定目标产物熊果苷的浓度。最终产量图如图2所述。
(4)采用高效液相色谱(HPLC)分析方法对生成产物β-熊果苷进行检测,检测条件如下:
色谱柱:分离柱:Diamonsil C18,ID 5μm,250×4.6mm;
流动相:有机相为乙腈,流动相为千分之一三氟乙酸水溶液,柱温40℃,流速1mL/min检测波长为282nm。梯度洗脱程序如下表所示:
时间(min) | 有机相A% | 流动相B% |
0 | 5 | 95 |
17 | 15 | 85 |
18 | 10 | 90 |
20 | 5 | 95 |
取样品上述发酵液1000μL,过滤膜,取过膜后液体采用上述方法进行高效液相色谱分析,分析结果如图3中的图3B所示。采用上述方法取含有β-熊果苷的标品水溶液进行高效液相色谱分析,分析结果如图3中的图3A所示,图3A为标品图。从图3A中可以看出:β-熊果苷的特征峰保留时间为4.849min;从图3B中可以看出在4.768min也有一个特征峰,由此可以判断图3B中保留时间为4.768min的特征峰为β-熊果苷,因此,本实施例提供的方法可以制备出β-熊果苷。
2.实施例2构建基因工程大肠杆菌BW1
应用crispr cas9技术将基因TAL、4Cl2、phdE/B/C插入到基因pgi的后面,具体的实施方法
(1)电转化法将载体pCas 9导入大肠杆菌BW中,在壮观霉素平板上30度培养20小时筛选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1.5ul/mL壮观霉素的LB液体培养基中30度培养。
(2)基因pgi位点sgRNA质粒的构建
i.本研究中sgRNA使用的靶向序列,如表1所示
表1.基因pgi位点sgRNA使用的靶向序列
ii.本研究中应用的引物序列,如表2所示
表2引物序列表
iii.基因pgi位点sgRNA质粒的构建
P1/P2为引物,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质粒。
(3)整合片段的构建
i.本研究中所应用的引物序列,如表3所示
表3引物序列表
ii.整合片段的构建
iii.以大肠杆菌为模板,P3/P4和P7/P8为引物,PCR扩增得到基因pgi两段同源臂,以基因TAL-4Cl2-phdEBC为模板,以P5/P6为引物,PCR扩增得到基因TAL-4Cl2-phdEBC片段;接着以这三段片段为模板,P3/P8为引物,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sgRNA质粒和整合片段导入(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,得到带有壮观霉素和氨苄青霉素的基因工程菌株,在相应抗性的平板上30度培养24小时。
(5)将sgRNA质粒和pCas质粒消掉以得到不需要抗生素地基因工程菌
i.消除sgRNA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。
ii.消除pCas质粒,在无抗生素液体LB中培养上述已消掉sgRNA质粒的基因工程菌,42度培养48小时,使温敏型pCas质粒降解。
(6)得到不需抗生素实现稳定遗传生产对羟基苯甲酸的基因工程菌株BW1。
3.实施例3构建重组大肠杆菌BW2
应用crispr cas9技术,基因4HB1H、TGS替换基因组上的假意义基因yneO,具体的实施方法
(1)基因yneO位点sgRNA质粒的构建
i.本研究中sgRNA使用的靶向序列,如表1所示
表4.基因pgi位点sgRNA使用的靶向序列
ii.本研究中应用的引物序列,如表5所示
表5引物序列表
iii.基因yneO位点sgRNA质粒的构建
P9/P2为引物,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质粒。
(2)整合片段的构建
i.本研究中所应用的引物序列,如表6所示
表6引物序列表
ii.整合片段的构建
iii.以大肠杆菌为模板,P11/P12和P15/P16为引物,PCR扩增得到基因yneO两段同源臂,以基因TAL-4Cl2-phdEBC为模板,以P13/P14为引物,PCR扩增得到基因TAL-4Cl2-phdE/B/C片段;接着以这三段片段为模板,P11/P16为引物,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。
(3)电转化法将sgRNA质粒和整合片段导入实列1中带有Cas 9质粒的BW1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,得到带有壮观霉素和氨苄青霉素的基因工程菌株,在相应抗性的平板上30度培养24小时。
(4)将sgRNA质粒和pCas质粒消掉以得到不需要抗生素地基因工程菌,其方法同实施例2的(5)所述。
(5)得到不需抗生素实现稳定遗传生产β-熊果苷的基因工程菌株BW2。
4.实施例4基因工程菌株BW2的应用:发酵培养从头合成β-熊果苷
(6)将基因工程菌株BW2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、10g/L甘油,3g/L酵母粉,1g/L MOPS,5g/L NaHPO4,1g/L NaCl,3g/L KH2PO4,1g/L NH4Cl,250mg/L MgSO4,15mg/L CaCl2。
(7)发酵每隔12h取出部分发酵液用以测定菌体生长状况及目标产物β-熊果苷的产量,HPLC发酵结果如图4所示。
5.实施例5高产β-熊果苷的工程菌:重组大肠杆菌BW3。
应用crispr cas9技术将基因aroG、aroA、aroL、aroC插入到基因ack的后面,具体的实施方法。
(1)电转化法将载体pCas 9导入大肠杆菌BW1中,在壮观霉素平板上30度培养20小时筛选阳性克隆转化子命名为BW1-pCas 9,挑取平板上所长的BW1-pCas 9单克隆,接种到1.5ul/mL壮观霉素的LB液体培养基中30度培养。
(2)基因ack位点sgRNA质粒的构建
i.本研究中sgRNA使用的靶向序列,如表4所示
表4.基因ack位点sgRNA使用的靶向序列
ii.本研究中应用的引物序列,如表5所示
表5.引物序列
基因yneO位点sgRNA质粒的构建
P16/P2为引物,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质粒。
(3)整合片段的构建
i.本研究中所应用的引物序列,如表6所示
表6引物序列表
ii.整合片段的构建
以大肠杆菌为模板,P17/P18和P21/P22为引物,PCR扩增得到基因ack两段同源臂,以基因aroG/A/L/C为模板,以P19/P20为引物,PCR扩增得到基因aroG/A/L/C片段;接着以这三段片段为模板,P17/P22为引物,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sgRNA质粒和整合片段导入实列2中带有Cas 9质粒的BW2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,得到带有壮观霉素和氨苄青霉素的基因工程菌株,在相应抗性的平板上30度培养24小时。
(5)将sgRNA质粒消掉以得到带有pCas质粒基因工程菌,用于下一步的基因tyrA合pheA的敲除。
(6)应用crispr cas9技术敲除基因tyrA合pheA。
敲除基因tyrA
i.基因tyrA位点sgRNA质粒的构建
本研究中sgRNA使用的靶向序列,如表7所示
表7.基因tyrA位点sgRNA使用的靶向序列
ii.本研究中应用的引物序列,如表8所示
表8.引物序列
基因tyrA位点sgRNA质粒的构建
P23/P2为引物,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质粒。
iii.整合片段的构建:
本研究中所应用的引物序列,如表9所示
表9引物序列表
iv.以大肠杆菌为模板,P17/P18和P21/P22为引物,PCR扩增得到基因ack两段同源臂,以基因aroG/A/L/C为模板,以P19/P20为引物,PCR扩增得到基因aroG/A/L/C片段;接着以这三段片段为模板,P17/P22为引物,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。
v.电转化法将sgRNA质粒和整合片段导入(5)中带有Cas 9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,得到带有壮观霉素和氨苄青霉素的基因工程菌株,在相应抗性的平板上30度培养24小时,得到敲掉基因tyrA的菌株。
敲除基因pheA,方法同上述敲除基因tyrA,所需的靶向基因列表和引物列表如表10-12所示。得到高产β-熊果苷的基因工程菌BW3。
基因pheA的sgRNA使用的靶向序列,如表10所示
表10.基因pheA位点sgRNA使用的靶向序列
表11.基因pheA的sgRNA的引物序列
表11.基因pheA的sgRNA的引物序列
6.实施例5基因工程菌株BW3的应用
参照实施例4,将上述基因工程菌株BW3在所述培养基中,每隔12h取样1mL用以测定菌体生长状况及目标产物产量,结果如图5所示。
Claims (4)
1.构建稳定遗传的β-熊果苷高效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:首先,在宿主内整合编码酪氨酸分解酶(TAL)、香豆酸辅酶A连接酶(4CL2)以及β-肉桂酰羟基化酶(phdE)、β-肉桂酰氧化酶(phdB)、β-肉桂酰脱酰酶(phdC)的基因,构建熊果苷生产过程中的关键中间体对羟基苯甲酸的高产菌株;然后在该宿主内整合编码4-羟基苯甲酸羟化酶(4HB1H)和葡萄糖基转移酶(TGS)的基因,构建了能够稳定遗传生产β-熊果苷的基因工程菌株;最后,宿主内整合编码3-脱氧-7-磷酸庚酸合酶(aroG)、3-磷酸莽草酸酯-1-羧基乙烯基转移酶(AroA)、莽草酸激酶(AroL)、分支酸合成酶(AroC)的基因以及敲除trpE和pheA基因,获得了稳定遗传的β-熊果苷高效生物合成的基因工程菌株。
2.根据权利要求1所述的方法,其特征在于:将基因TAL、4Cl2、phdE、phdB、phdC插入到基因pgi之后,基因4HB1H、TGS替换基因组上的假意义基因yneO,基因aroG、aroA、aroL、aroC插入到基因ack的后面。
3.应用权利要求1所述方法所制备的基因工程菌的应用,其特征在于:将所构建的β-熊果苷高效生物合成的基因工程菌株涂于无抗生素的平板上,37℃过夜培养,挑取单克隆到液体LB中培养10h,接种到发酵培养基中,37℃进行发酵从头合成生产熊果苷。
4.根据权利要求3所述的高产β-熊果苷的基因工程菌的应用,其特征在于:所述发酵培养基包括20g/L葡萄糖、20g/L甘油,3g/L酵母粉,1g/L MOPS,5g/L NaHPO4,1g/L NaCl,3g/LKH2PO4,1g/L NH4Cl,250mg/L MgSO4,15mg/L CaCl2,溶剂为水。
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