CN116284276B - 一种大肠杆菌调节蛋白AraC突变体蛋白AraCm及其应用 - Google Patents
一种大肠杆菌调节蛋白AraC突变体蛋白AraCm及其应用 Download PDFInfo
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Abstract
本发明公开了一种大肠杆菌调节蛋白AraC突变体蛋白AraCm及其应用，属于生物医药技术领域。本申请通过随机突变，获得能与戊基二乙酸内酯相结合的突变体AraCm。在此基础上，对突变体AraCm序列进行表征，通过串联BAD启动子介导的抗性基因表达框，得到戊基二乙酸内酯浓度与大肠杆菌菌株生长的关系；或者通过结果表明，通过串联BAD启动子介导的荧光蛋白基因表达框，得到戊基二乙酸内酯浓度与荧光蛋白强度的关系。AraCm的核苷酸序列如SEQ ID NO.1所示，AraCm的氨基酸序列如SEQ ID NO.2所示。这对检测聚酮合酶突变体催化过程合成副产物戊基二乙酸内酯水平的高通量检测具有重要的应用价值。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种大肠杆菌调节蛋白AraC突变体蛋白AraCm及其应用。
背景技术
大麻素类化合物具有多种医药价值，在大麻素的生物合成途径中，大麻聚酮合酶（tetraketide synthase，TKS）是第一个关键酶，其催化己酰辅酶A和丙二酰辅酶A合成一种线性四酮中间体，然后经过橄榄酸环化酶（olivetolic acid cyclase，OAC）催化形成橄榄酸。然而，TKS的催化产物绝大部分为副产物戊基二乙酸内酯和己酰三乙酸内酯，这严重限制了橄榄酸的合成，进而影响大麻素的产量。随着蛋白质工程技术的发展，通过理性设计或随机突变TKS的氨基酸序列，可以改变TKS的催化产物的各组分含量分布。然而，目前还没有针对戊基二乙酸内酯和己酰三乙酸内酯的快速检测方法，而常规手段所用的高效液相色谱-质谱联用在检测上述产物过程中费时、费力且通量较低，因此不适合大规模的高通量筛选。这也严重的阻碍了TKS催化机制的解析及其突变体酶的构建和高产橄榄酸的TKS突变体酶的筛选，从而限制了大麻素类化合物的工业化生产及其应用。
发明内容
针对现有技术存在的上述问题，本发明所要解决的第一技术问题在于提供一种大肠杆菌调节蛋白AraC突变体蛋白AraCm，可以响应戊基二乙酸内酯（Pentyl diaceticlactone，PDAL）。本发明所要解决的第二技术问题在于提供上述突变体蛋白AraCm的高效、快速测定PDAL的方法。
为了解决上述技术问题，本发明所采用的技术方案如下：
一种大肠杆菌调控蛋白AraC的突变体蛋白AraCm，其氨基酸序列如SEQ ID NO. 2所示。
表达所述的大肠杆菌调控蛋白AraC的突变体蛋白AraCm的基因，其核苷酸序列如SEQ ID NO. 1所示。
含有所述的如SEQ ID NO. 1所示的核苷酸序列的表达载体或宿主菌。
所述的表达载体，为pET28a(+)-T 7 -AraCm-TAA-P BAD -gfp-TAA或pET28a(+)-T 7 -AraCm-TAA-P BAD -Amp-TAA。
所述的宿主菌，为含有pET28a(+)-T 7 -AraCm-TAA-P BAD -gfp-TAA或pET28a(+)-T 7 -AraCm-TAA-P BAD -Amp-TAA的大肠杆菌。
所述的突变体蛋白AraCm在检测PDAL中的应用。
所述的应用，包括以下步骤：
1）构建携带有编码突变体蛋白AraCm的基因序列、BAD启动子和绿色荧光蛋白基因gfp的表达载体；
2）构建携带有编码突变体蛋白AraCm的基因序列、BAD启动子和氨苄青霉素基因amp的表达载体；
3）以大肠杆菌为宿主，转化步骤1）和2）得到的表达载体，获得PDAL的生物传感器工程菌株；
4）培养生物传感器工程菌株，获得PDAL浓度与大肠杆菌菌株生长的线性关系；或者获得PDAL与荧光蛋白强度的线性关系；
5）利用所述的线性关系，检测出TKS合成PDAL的含量。
所述的PDAL浓度与大肠杆菌菌株生长的线性关系为：，式中，x为PDAL浓度，y为OD 600 nm。
所述的PDAL浓度与荧光蛋白强度的线性关系为：y=136309+16728*lnx，式中，x为PDAL浓度，y为荧光蛋白的荧光强度。
有益效果：相比于现有技术，本发明的技术优势为：
1）本申请从大肠杆菌调控蛋白AraC的突变体AraCm出发，以pET28a(+)为表达载体，通过构建AraCm基因、BAD启动子序列和绿色荧光蛋白基因的串联表达框，获得大肠杆菌调控蛋白AraC的突变体AraCm和绿色荧光蛋白，在此基础上，添加不同浓度的PDAL作为效应剂，经一步法发酵调控绿色荧光蛋白的表达量，以此来建立PDAL与绿色荧光蛋白的函数关系，从而达到构建PDAL生物传感器的目的；
2）本申请构建的PDAL生物传感器，相较于其他无机二乙酸内酯的测定方法，价格更便宜，通量更大，省时、省力，适合大规模的检测。
附图说明
图1是PDAL生物传感器的重组表达载体pET28a(+)-T 7 -AraCm-TAA-P BAD - gfp-TAA构建示意图；
图2是PDAL生物传感器的重组表达载体pET28a(+)-T 7 -AraCm-TAA-P BAD - Amp-TAA构建示意图；
图3是PDAL线性检测范围结果图；
图4是TKS催化合成PDAL产量结果图；
图5是TKS催化合成产物对绿色荧光蛋白表达的影响结果图；
图6是TKS催化合成产物对重组大肠杆菌生长的影响结果图。
实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1 基于绿色荧光蛋白构建PDAL的生物传感器
1）PDAL生物传感器重组表达载体的构建
根据备选基因大肠杆菌调控蛋白AraC的晶体结构（PDB：2ARA）及待测化合物PDAL化学结构，利用生物信息学软件Discovery Studio进行分子对接、虚拟突变后，获得能够与PDAL结合的AraC突变体AraCm的氨基酸序列SEQ ID NO. 2；经密码子优化合成AraCm的DNA序列SEQ ID NO. 1；分别设计引物F1（CCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATGGATGTATTACTACCCGGATATTC）和R1（GATGCAATATGGACAATTGGTTTCTTTTAGGACTCGTTAATCGCTTCCATAC），F2（AAGAAACCAATTGTCCATATTGCATC）和R2（GGTTAATTCCTCCTGTTAGCCC），F3（GGGCTAACAGGAGGAATTAACCatggtgagcaagggcgag）和R3（GTCGACGGAGCTCGAATTCGttacttgtacagctcgtccatgcc），利用PCR高保真聚合酶Prime Star分别以SEQ ID NO. 1、pBAD/HisA质粒（Invitrogen）、pLac-EGFP质粒为模版，扩增AraCm基因序列、BAD启动子和绿色荧光蛋白的基因片段，经Gibbson组装，同源重组整合到pET28a(+)的Nco I和BamH I位点，构建出PDAL生物传感器的重组表达载体pET28a(+)-T 7 -AraCm-TAA-P BAD -gfp- TAA（图1）。
2）大肠杆菌细胞工厂生物传感器的构建
取重组表达载体pET28a(+)-T 7 -AraCm-TAA-P BAD -gfp-TAA 50 ng，与大肠杆菌E.coli BL21感受态冰上孵育30 min后，42℃热击90 s转化E. coli BL21获得表达绿色荧光蛋白的重组大肠杆菌E. coli BL21 pET28a(+)-T 7 -AraCm- TAA-P BAD -gfp-TAA。
3）大肠杆菌细胞工厂的培养
挑取重组大肠杆菌表达菌株E. coli BL21 pET28a(+)-T 7 -AraCm-TAA-P BAD -gfp- TAA单菌落，接种于3 mL LB培养基（加入终浓度为50 mg/L的卡那霉素）中，37℃，200 rpm培养过夜。按1%（v/v）的比例转接于50 mL 发酵培养基（含有终浓度为0.01-10 mM戊基二乙酸内酯和50 mM卡那霉素的LB培养基）中，37℃，培养至OD 600 nm为0.6-0.8，加入终浓度为0.1mM的IPTG，37℃，诱导培养5 h后，测定戊基二乙酸内酯浓度为0、0.05 mM、0.1 mM、0.2 mM、0.5 mM、1 mM、2 mM、3 mM、4 mM、5 mM和10 mM时的重组大肠杆菌细胞荧光强度（图3），建立荧光强度与PDAL浓度之间满足的函数方程。
图3结果表明，微生物诱导表达获得的荧光蛋白的荧光强度与PDAL浓度之间满足的函数方程为y=136309+16728*lnx，其中x为PDAL浓度，y为荧光蛋白的荧光强度。
实施例2 基于抗性基因构建戊基二乙酸内酯的生物传感器
1）戊基二乙酸内酯生物传感器重组表达载体的构建
根据备选基因大肠杆菌调控蛋白AraC的晶体结构（PDB：2ARA）及待测化合物PDAL的化学结构，利用生物信息学软件Discovery Studio进行分子对接、虚拟突变后，获得与PDAL结合的突变体AraCm的氨基酸序列SEQ ID NO. 2；经密码子优化合成AraCm的基因序列SEQ ID NO. 1；分别设计引物F1（CCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATGGATGTATTACTACCCGGATATTC）和R1（GATGCAATATGGACAATTGGTTTCTTTTAGGACTCGTTAATCGCTTCCATAC），F2（AAGAAACCAATTGTCCATATTGCATC）和R2（GGTTAATTCCTCCTGTTAGCCC），F4（GGGCTAACAGGAGGAATTAACCatgagtattcaacatttccgtgtcgc）和R4（GTCGACGGAGCTCGAATTCGttaccaatgcttaatcagtgaggcacctatc），利用高保真聚合酶Prime Star分别以SEQ ID NO. 1、pBAD/HisA质粒（Invitrogen）、pET20b(+)（Novagen）为模版，扩增AraCm、BAD启动子和氨苄青霉素的基因片段，经Gibbson组装，同源重组整合到pET28a(+)的Nco I和BamH I位点，构建出PDAL生物传感器的重组表达载体pET28a(+)-T 7 -AraCm-TAA-P BAD -amp-TAA（图2）。
2）大肠杆菌细胞工厂生物传感器的构建
取重组表达载体pET28a(+)-T 7 -AraCm-TAA-P BAD -gfp-TAA 50 ng，与大肠杆菌E.coli BL21感受态冰上孵育30 min后，42℃热击90 s转化E. coli BL21获得表达绿色荧光蛋白的重组大肠杆菌E. coli BL21 pET28a(+)-T 7 -AraCm-TAA -P BAD -amp-TAA。
3）大肠杆菌细胞工厂的培养
挑取重组大肠杆菌表达菌株E. coli BL21 pET28a(+)-T 7 -AraCm-TAA-P BAD -amp- TAA单菌落，接种于3 mL LB培养基（加入终浓度50 mg/L的卡那霉素）中，37℃，200 rpm培养过夜。按1%（v/v）的比例转接50 mL LB培养基（加入终浓度为50 mg/L的卡那霉素、50 mg/L的氨苄青霉素和0-1 mM的PDAL）中，37℃，培养至OD 600 nm为0.6-0.8，加入终浓度为0.1 mM的IPTG，37℃，诱导5 h，测定PDAL浓度为0、0.02 mM、0.04 mM、0.06 mM、0.08 mM、0.1 mM、0.2mM、0.4 mM、0.6 mM、0.8 mM时的重组大肠杆菌E. coli BL21 pET28a(+)-T 7 -AraCm-TAA- P BAD -amp-TAA的OD 600 nm（图4），建立微生物菌体OD 600 nm和PDAL浓度之间满足函数方程。
由图4表明，微生物菌体OD 600 nm和PDAL浓度之间满足函数方程，其中x为PDAL浓度，y为OD 600 nm。
实施例3 TKS合成PDAL含量的测定
1、TKS催化体系
在20 mM Hepes缓冲液（pH 7.0）中，加入终浓度为5 mM DTT，0.2 mM己酰CoA，0.6mM丙二酰CoA，1 g/L TKS纯酶后10℃催化24 h后，加入等体积的甲醇溶液终止反应，待测PDAL含量。
2、TKS催化合成产物对绿色荧光蛋白表达的影响
挑取重组大肠杆菌表达菌株E. coli BL21 pET28a(+)-T 7 -AraCm-TAA-P BAD -gfp- TAA单菌落，接种于3 mL LB培养基（加入终浓度为50 mg/L的卡那霉素）中，37℃，200 rpm培养过夜。按1%（v/v）的比例转接10 mL LB培养基（加入终浓度为50 mg/L的卡那霉素）后，添加10 mL TKS催化后的反应液（以甲醇失活的TKS纯酶为空白对照），37℃，培养至OD 600 nm为0.6-0.8，加入终浓度为0.1 mM的IPTG，37℃，诱导5 h后，测定荧光强度。
结果如图5所示，表明对照样品无荧光合成，TKS催化反应液添加后的重组大肠杆菌E. coli BL21 pET28a(+)-T 7 -AraCm-TAA-P BAD -gfp-TAA的荧光强度为110834，由函数方程y=136309+16728*lnx计算得出TKS催化合成的PDAL的含量为0.06 mM。
3. TKS催化合成产物对重组大肠杆菌生长的影响
挑取重组大肠杆菌表达菌株E. coli BL21 pET28a(+)-T 7 -AraCm-TAA-P BAD -amp- TAA单菌落，接种于3 mL LB培养基（加入终浓度为50 mg/L的卡那霉素）中，37℃，200 rpm培养过夜。按1%（v/v）的比例转接于10 mL LB培养基（加入终浓度为50 mg/L的卡那霉素和50mg/L的氨苄青霉素）后，添加10 mL TKS催化后的反应液（以甲醇失活的TKS纯酶为空白对照），37℃，培养至OD 600 nm为0.6-0.8，加入终浓度为0.1 mM的IPTG，37℃，诱导5 h后，测定菌体生长情况。
结果如图6所示，表明对照样品菌体不生长，TKS催化反应液添加后的重组大肠杆菌E. coli BL21 pET28a(+)-T 7 -AraCm-TAA-P BAD -amp-TAA的OD 600 nm =0.34，由函数方程同样计算得出TKS催化合成的PDAL的含量为0.06 mM。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (9)
1.一种大肠杆菌调控蛋白AraC的突变体蛋白AraCm，其氨基酸序列如SEQ ID NO. 2所示。
2.表达权利要求1所述的大肠杆菌调控蛋白AraC的突变体蛋白AraCm的基因，其核苷酸序列如SEQ ID NO. 1所示。
3.含有权利要求2所述的如SEQ ID NO. 1所示的核苷酸序列的表达载体或宿主菌。
4.根据权利要求3所述的表达载体，其特征在于：为pET28a(+)-T 7 -AraCm-TAA-P BAD -gfp- TAA或pET28a(+)-T 7 -AraCm-TAA-P BAD -Amp-TAA。
5.根据权利要求3所述的宿主菌，其特征在于：为含有pET28a(+)-T 7 -AraCm-TAA-P BAD - gfp-TAA或pET28a(+)-T 7 -AraCm-TAA-P BAD -Amp-TAA的大肠杆菌。
6.权利要求1所述的突变体蛋白AraCm在检测戊基二乙酸内酯中的应用。
7.根据权利要求6所述的应用，其特征在于，包括以下步骤：
1）构建携带有编码突变体蛋白AraCm的基因序列、BAD启动子和绿色荧光蛋白基因gfp的表达载体；
2）构建携带有编码突变体蛋白AraCm的基因序列、BAD启动子和氨苄青霉素基因amp的表达载体；
3）以大肠杆菌为宿主，转化步骤1）或2）得到的表达载体，获得戊基二乙酸内酯的生物传感器工程菌株；
4）培养步骤3）得到的生物传感器工程菌株，获得戊基二乙酸内酯浓度与大肠杆菌菌株生长的线性关系；或者获得戊基二乙酸内酯浓度与荧光蛋白强度的线性关系；
5）利用所述的线性关系，检测戊基二乙酸内酯的含量。
8.根据权利要求7所述的应用，其特征在于：戊基二乙酸内酯浓度与大肠杆菌菌株生长的线性关系为：，式中，x为戊基二乙酸内酯浓度，y为OD 600 nm。
9.根据权利要求7所述的应用，其特征在于：戊基二乙酸内酯浓度与荧光蛋白强度的线性关系为：，式中，x为戊基二乙酸内酯浓度，y为荧光蛋白的荧光强度。
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