CN116790647B - 一种低背景、高信号强度的2-吡咯烷酮生物传感器及应用 - Google Patents
一种低背景、高信号强度的2-吡咯烷酮生物传感器及应用 Download PDFInfo
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Abstract
本发明公开了一种低背景、高信号强度的2‑吡咯烷酮生物传感器及应用，属于基因工程技术领域。本发明提供了一种改进的2‑吡咯烷酮的生物传感器，在原有基础上将报告基因mCherry优化为sfGFP，同时对调控因子chnR进行改造，减少了原信号蛋白的泄露表达，增强了该传感器对不同浓度的2‑吡咯烷酮的信号响应的差异性，可更好用于对2‑吡咯烷酮合成相关酶和菌的定向进化。
Description
技术领域
本发明涉及一种低背景、高信号强度的2-吡咯烷酮生物传感器及应用，属于基因工程技术领域。
背景技术
2-吡咯烷酮(2-Pyrrolidone,2P)，又称2-氧代吡咯烷、γ-丁内酰胺，是一类具有一个五元内酰胺环的吡咯烷类化合物，广泛存在于天然产物和各类人工合成的化合物当中。2-吡咯烷酮是生产聚乙烯吡咯烷酮、锦纶-4和脑复康(酰胺吡咯烷酮)等多种化合物的前体，在医药领域和工业领域具有广泛且重要的用途。
2-吡咯烷酮可由生物法合成。近年来已经有一些国内外的文献报道了关于使用微生物生产2-吡咯烷酮。目前产量最高的是，Tong Un Chae通过对大肠杆菌的改造，产量可达54g/L。Chae T U,Ko Y S,Hwang K S,et al.Metabolic engineering of Escherichiacoli for the production of four-,five-and six-carbon lactams.Metab Eng，2017,41:82-91)β-丙氨酸CoA转移酶(CoAtransferase,Act)催化的ω-氨基酸的活化，然后自发环化合成2-吡咯烷酮，其中Act是生产关键限速酶。Jay D.Keasling等基于调控蛋白开发了内酰胺生物传感器，可特异性、以浓度依赖性检测ε-己内酰胺、δ-戊内酰胺和丁内酰胺，从而筛选定向进化后的菌株或酶。
目前还没有利用生物传感器筛选定向改造关键限速酶高产2-吡咯烷酮菌株的报道，由于现有的生物传感器对2-吡咯烷酮的灵敏性较低，导致在实际应用中荧光弱、可能出现假阳性，难以真正应用。
发明内容
本发明提供一种2-吡咯烷酮的生物传感器，含有启动子Pcon、ChnR编码基因，启动子Pb和荧光蛋白编码基因sfGFP；所述启动子Pb上具有转录因子结合位点，所述启动子Pb调控荧光蛋白编码基因的表达；所述启动子Pcon调控ChnR基因的表达；所述启动子Pcon和启动子Pb的转录方向相反；所述转录因子结合位点的核苷酸序列如SEQ ID NO.5所示。
在一种实施方式中，所述启动子Pcon的核苷酸序列如SEQ ID NO.1所示；所述ChnR编码基因的核苷酸序列如SEQ ID NO.2或SEQ ID NO.6所示；所述启动子Pb的核苷酸序列如SEQ ID NO.3所示；所述荧光蛋白编码基因sfGFP的核苷酸序列如SEQ ID NO.4所示。
在一种实施方式中，所述生物传感器以pBbS5C-RFP为质粒骨架。
本发明还提供了含有所述生物传感器的重组微生物细胞。
在一种实施方式中，所述微生物包括但不限于大肠杆菌或谷氨酸棒杆菌。
在一种实施方式中，所述大肠杆菌为大肠杆菌BW25113。
在一种实施方式中，所述ChnR编码基因是在SEQ ID NO.2所示基因编码的氨基酸的基础上，将第121位缬氨酸突变为丙氨酸，将第230位甲硫氨酸突变为异亮氨酸，并将第236位天冬氨酸突变为天冬酰胺。
本发明还提供了一种提高生物传感器检测灵敏度的方法，所述方法是将所述生物传感器的ChnR基因编码的氨基酸序列的第121位缬氨酸突变为丙氨酸，将第230位甲硫氨酸突变为异亮氨酸，并将第236位天冬氨酸突变为天冬酰胺，获得SEQ ID NO.6所示的序列。
本发明还提供了所述生物传感器在筛选2-吡咯烷酮高产菌株、高活力的2-吡咯烷酮合成关键性酶等方面的应用。
在一种实施方式中，所述筛选2-吡咯烷酮高产菌株是将所述生物传感器转入目的菌株细胞内，将待筛选的菌株在一定条件下培养一段时间，根据菌株发酵液的荧光强度筛选2-吡咯烷酮高产菌株。
有益效果：本发明提供了一种改进的2-吡咯烷酮的生物传感器，在原有基础上将报告基因mCherry优化为sfGFP，同时对调控因子chnR进行改造，减少了原信号蛋白的泄漏表达，增强了该传感器对不同浓度的2-吡咯烷酮的信号响应的差异性，可更好用于对2-吡咯烷酮合成相关酶和菌的定向进化。
附图说明
图1为报告基因优化前后的荧光响应对比。
图2为2-吡咯烷酮生物传感器调控蛋白ChnR位点优化。
图3为调控蛋白ChnR优化前后的荧光响应对比。
具体实施方式
培养基：
M9培养基：葡萄糖4.0g/L，Na2HPO4 6.78g/L，KH2PO4 3.0g/L，NaCl 0.5g/L，NH4Cl1.0g/L，MgSO4·7H2O 0.493g/L，CaCl2 11mg/L。
实施例1生物传感器的荧光蛋白的优化
生物传感器的构建：生物传感器以pBbS5C-RFP质粒(公开于论文《Development ofa Transcription Factor-Based Lactam Biosensor》)为骨架，含有启动子Pcon、调控蛋白的编码基因ChnR，启动子Pb和荧光蛋白编码基因sfGFP；所述启动子Pcon、调控蛋白的编码基因ChnR和启动子Pb以及报告基因sfGFP位于同一载体或基因组上；所述启动子Pb上具有转录因子结合位点(SEQ ID NO.5所示)，所述启动子Pb调控荧光蛋白编码基因sfGFP的表达；所述启动子Pcon调控ChnR基因的表达；所述启动子Pcon和启动子Pb的转录方向相反。所述启动子Pcon的核苷酸序列如SEQ ID NO.1所示；所述ChnR编码基因的核苷酸序列如SEQ ID NO.2所示；所述启动子Pb的核苷酸序列如SEQ ID NO.3所示；所述荧光蛋白编码基因sfGFP的核苷酸序列如SEQ ID NO.4所示。。
本发明构建的生物传感器的工作原理为：在环境中含有2-吡咯烷酮的情况下，受Pcon启动子调控表达的转录因子ChnR结合环境中的2-吡咯烷酮诱发DNA结合域的构象变化，并结合在位于启动子Pb上的转录因子结合位点ttgtttggatc(SEQ ID NO.5所示)，激活转录，从而调控启动子Pb表达荧光蛋白。2-吡咯烷酮浓度越高，Pb调控转录的强度越强，从而荧光信号越强。
以pBbSLactamC-mCherry为质粒模板，将基因mCherry替换成sfGFP，构建质粒pBb-ChnR-sfGFP。具体操作如下：
以pBbSLactamC-mCherry(pBb-mCherry)质粒(公开于论文《Development of aTranscription Factor-Based Lactam Biosensor》)为模板，使用引物扩增质粒主片段，并以合成sfGFP基因为模板扩增sfGFP基因：
pBbBBF：ggtaccctccattacgacatg；
pBbBBR：aggatccaaactcgagtaagg；
sfGFPpBbF：ccttactcgagtttggatcctcatttgtacagttcatccatac；
sfGFPpBbR：catgtcgtaatggagggtaccatgcgtaaaggcgaagagc；
sfGFPCF:cacccgaaggtgagccagtgtgactc；
sfGFPCR：catccaagccttgtgattgcattcctgcg；
分别克隆质粒载体和sfGFP片段，37℃下使用DPN1酶消化2小时消除模板质粒，经DNA纯化试剂盒纯化后，通过Gibson方法将载体片段相连接，使用引物将sfGFPCF与sfGFPCR通过PCR验证并测序比对，确定pBb-ChnR-sfGFP质粒构建成功。
实施例2含生物传感器的大肠杆菌工程菌株的构建
将实施例1构建的重组质粒pBb-ChnR-sfGFP转化至大肠杆菌BW25113中，得到的菌株命名为pBb-sfGFP。并将质粒pBbSLactamC-mCherry转化至大肠杆菌DW25113中，得到的菌株命名为pBb-mCherry，作为对照。将pBb-sfGFP和pBb-mCherry在LB培养基中用试管于37℃培养12小时，获得种子液；将50mL M9培养基加到500mL摇瓶中，在加入0、0.3、0.6、0.9、1.2、1.5mM终浓度2-吡咯烷酮情况下，按10％的接种量接种，使接种后的初始OD为0.5，发酵温度为37℃，摇床转速200rpm，发酵24小时。用酶标仪(TECON INFINITE E PLEX)以激发波长488nm，发射波长507nm检测确定sfGFP荧光强度；以激发波长575nm，发射波长620nm检测确定发酵液中的mCherry荧光强度。
图1显示了摇瓶发酵过程随着加入底物2-吡咯烷酮产量的变化两种荧光蛋白的变化。随着产生的2-吡咯烷酮增加，sfGFP和mCherry荧光蛋白产生的荧光强度增加，并呈现浓度相关性。sfGFP荧光泄漏低、对不同浓度的2-吡咯烷酮的荧光信号的差异性大，说明sfGFP较mCherry作为报告基因更优。
实施例3 2-吡咯烷酮的大肠杆菌工程菌株关键酶进化生物传感器的调控蛋白优化
图2为生物传感器调控蛋白ChnR基因优化回路构建示意图，以实施例1构建的pBb-sfGFP为质粒模板，将调控蛋白ChnR第121位缬氨酸突变为丙氨酸，并将第230位甲硫氨酸突变为异亮氨酸，并将第236位天冬氨酸突变为天冬酰胺。
具体操作如下：
以实施例1构建的pBb-sfGFP为模板设计扩增引物实行点突变：
ChnR121F：ctccatcacctcaatggcgaataagGCTtttgattatgatatcgcttcgatccgaatc；
ChnR236R：gcagcaggggaattaccaaaATTgactttaaattttctGATtaaatgaggcacgctcatcttgac；
ChnR121BBF：gtcaagatgagcgtgcctcatttaATCagaaaatttaaagtcAATtttggtaattcccctgctgc；
ChnR121BBR：gattcggatcgaagcgatatcataatcaaaAGCcttattcgccattgaggtgatggag；
ChnRCF：gcgatgcctcttgggatacccaagtg；
ChnRCR：ctcgggtcatatggctctgatcgc；
以质粒pBb-sfGFP作为调控蛋白ChnR扩增模板，使用三位点点突变引物ChnR121F与ChnR236R通过PCR扩增得到第121位缬氨酸突变为丙氨酸、第230位甲硫氨酸突变为异亮氨酸、第236位天冬氨酸突变为天冬酰胺的目的片段。37℃下使用DPN1酶消化2小时消除模板质粒，经DNA纯化试剂盒纯化后，通过Gibson将PCR扩增的目的片段与质粒pBb-sfGFP的主干片段相连，使用引物ChnRCF与ChnRCR PCR验证并测序比对，确定质粒pBb-ChnR-M1-sfGFP构建成功。将重组质粒转化至大肠杆菌DW25113中，得到的菌株命名为pBb-ChnR。
实施例4利用调控蛋白ChnR优化系统在大肠杆菌中监测2-吡咯烷酮的浓度变化
将实施例2中构建的调控蛋白优化菌株pBb-ChnR和实施例1中构建的菌株pBb-sfGFP分别在加入不同浓度2-吡咯烷酮情况下，对比两株菌的sfGFP产生的荧光。将菌株pBb-ChnR和pBb-sfGFP分别在LB培养基中，用试管于37℃培养12小时，获得种子液；将50mLM9培养基加到500mL摇瓶中，在加入0、0.3、0.6、0.9、1.2、1.5mM终浓度2-吡咯烷酮情况下，按10％的接种率接种，发酵温度为37℃，摇床转速200rpm，发酵24小时。2-吡咯烷酮会与调控蛋白ChnR结合，形成的结合物能够与Pb位点结合，使启动子起始转录，激活报告基因sfGFP荧光蛋白的表达，当菌株的生长环境中加入2-吡咯烷酮时Pb启动子就会开始转录，进而生成绿色荧光蛋白。该生物传感器能够响应不同浓度的2-吡咯烷酮，进而调控不同强度的绿色荧光蛋白表达。酶标仪(TECON INFINITE E PLEX)以激发波长488nm，发射波长507nm检测荧光强度。
图3显示了摇瓶发酵过程随着加入底物2-吡咯烷酮产量的变化荧光蛋白的变化。随着2-吡咯烷酮浓度增加，sfGFP荧光蛋白产生的荧光强度增加，并呈现浓度依赖性。对比未优化调控蛋白菌株，ChnR优化后的生物传感器对不同浓度的2-吡咯烷酮的信号响应的差异性进一步增大，可以更好地区分开合成不同浓度的2-吡咯烷酮菌株。
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 (6)
1.一种2-吡咯烷酮的生物传感器，其特征在于，以pBbS5C-RFP为质粒骨架，含有启动子Pcon、ChnR编码基因，启动子Pb和荧光蛋白编码基因sfGFP；所述启动子Pb上具有转录因子结合位点，所述启动子Pb调控荧光蛋白编码基因的表达；所述启动子Pcon调控ChnR基因的表达；所述启动子Pcon和启动子Pb的转录方向相反；所述转录因子结合位点的核苷酸序列如SEQ ID NO.5所示；
所述ChnR编码基因是在SEQ ID NO.2所示基因编码的氨基酸的基础上，将第121位缬氨酸突变为丙氨酸，将第230位甲硫氨酸突变为异亮氨酸，并将第236位天冬氨酸突变为天冬酰胺，获得SEQ ID NO.6所示的基因；所述启动子Pcon的核苷酸序列如SEQ ID NO.1所示；所述启动子Pb的核苷酸序列如SEQ ID NO.3所示；所述荧光蛋白编码基因sfGFP的核苷酸序列如SEQ ID NO.4所示。
2.根据权利要求1所述的生物传感器，其特征在于，所述启动子Pcon、ChnR编码基因，启动子Pb和荧光蛋白编码基因sfGFP位于同一质粒或基因组DNA上。
3.含有权利要求1或2所述生物传感器的重组微生物细胞，其特征在于，所述微生物为大肠杆菌或谷氨酸棒杆菌。
4.根据权利要求3所述的重组微生物细胞，其特征在于，所述大肠杆菌为大肠杆菌BW25113。
5.权利要求1或2所述的生物传感器在筛选2-吡咯烷酮高产菌株、高活力的2-吡咯烷酮合成关键性酶中的应用，其特征在于，所述菌株为大肠杆菌或谷氨酸棒杆菌。
6.根据权利要求5所述的应用，其特征在于，所述筛选2-吡咯烷酮高产菌株是将所述生物传感器转入目的菌株细胞内，将待筛选的菌株在一定条件下培养一段时间，根据菌株发酵液的荧光强度筛选2-吡咯烷酮高产菌株。
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