
Construction and application of T7-like expression system in Escherichia coli Nissle 1917
ZHENG Ye, DENG Ruizhe, YANG Furen, LIN Yina, WANG Hui, YE Jianwen
Journal of Jinan University Natural Science & Medicine Edition ›› 2025, Vol. 46 ›› Issue (2) : 151-162.
Construction and application of T7-like expression system in Escherichia coli Nissle 1917
Objective: To construct a strong inducible expression system based on T7-like RNA polymerase MmP1 in Escherichia coli Nissle 1917 (EcN) to achieve efficient expression of the target protein. Methods: Constructing different inducible expression systems in EcN for “dose-response” characterization to screen out the best-performing induced system with low leakage and high induction expression level. Optimizing the expression output and dynamic range of the T7-like system by integrating the T7-like RNA polymerase (MmP1) expression on the chromosome of EcN, namely strain LM01, thereby constructing promoter mutation library of PMmP1 for tunable induced expression output. T7-like induced system based on MmP1 was used to control the expression of superoxide dismutase (SOD) of high yield, giving strong evidence for protein expression of T7-like system in EcN. Results: Several inducible systems were successfully constructed and characterized in EcN. Of which, the T7-like induction system based on MmP1 RNA polymerase displayed the best-performing saturated induction output with maximum dynamic range reaching up to 909-fold. After the MmP1 polymerase expression module was integrated into the genome of EcN, the saturated induction level was increased by 66.8%. The construction of PMmP1 promoter library provides a wide range of inducible expression output (dynamic range), spanning 65 to 1 097 fold. The T7-like (MmP1) system demonstrated the highest soluble SOD protein production yield of 435.7 mg/L, which was 4.02-fold higher than vanillic acid induction system. In addition, the maximum enzymatic activity of SOD achieves 391.3 U/mL. Conclusion: T7-like (MmP1) induction system can be successfully constructed in EcN with high saturated expression level, low basal leakiness, as well as controllable dynamic range of tunable gene expression control for efficient protein synthesis in the coming future.
Escherichia coli Nissle 1917 / inducible expression system / T7-like expression system / protein expression / synthetic biology {{custom_keyword}} /
Table 1 Strains used in this study表1 实验使用菌株 |
菌株名称 | 描述 | 来源 |
---|---|---|
EcN | 大肠杆菌Nissle 1917的野生型菌株 | 实验室保存 |
E. coli S17-1 | 用于重组质粒的构建 | 实验室保存 |
EcN LM01 | EcN的衍生菌株,在基因组整合了lacI-RNAPMmP1模块 | 本研究 |
Table 2 Plasmids used in this study表2 实验使用质粒 |
质粒名称 | 描述 | 来源 |
---|---|---|
pSEVA321-araC-araE-PBAD-sfgfp | pSEVA321载体,PBAD启动子控制sfgfp表达 | 文献[18] |
pSEVA321-LuxR-PLux-sfgfp | pSEVA321载体,PLux启动子控制sfgfp表达 | 文献[18] |
pSEVA321-lacI-Ptac-sfgfp | pSEVA321载体,Ptac启动子控制sfgfp表达 | 文献[18] |
pSEVA321-vanR-PVan-sfgfp | pSEVA321载体,PVan启动子控制sfgfp表达 | 文献[18] |
pSEVA321-lacI-RNAPMmP1-PMmP1-sfgfp | pSEVA321载体,包含lacI-RNAPMmP1模块,PMmP1启动子控制sfgfp表达 | 本研究 |
pSEVA321-lacI-RNAPT7-PT7-sfgfp | pSEVA321载体,包含lacI-RNAPT7模块,PT7启动子控制sfgfp表达 | 本研究 |
pSEVA321-PMmP1-sfgfp | pSEVA321载体,PMmP1启动子控制sfgfp表达 | 文献[23] |
pSEVA321-PMmP1 library-sfgfp | pSEVA321-PMmP1-sfgfp衍生,PMmP1部分序列进行了定点突变 | 文献[23] |
pSEVA321-PMmP1-sod | pSEVA321-PMmP1-sfgfp衍生,用sod替换sfgfp | 本研究 |
pSEVA321-vanR-PVan-sod | pSEVA321-vanR-PVan-sfgfp衍生,用sod替换sfgfp | 本研究 |
p46cas9 | 表达λ-Red和SpCas9,用于EcN的遗传改造 | 文献[26] |
Ts-PG-LM | 供体质粒,用于EcN的遗传改造 | 本研究 |
Table 3 Primers used in this study表3 实验使用引物 |
引物名称 | 引物序列(5'→3') |
---|---|
321B-F | TCTAGAGCTCGGTACCAAATTCCA |
321B-R | TCCTGTGTGAAATTGTTATCCGCT |
M-F | CAATTTCACACAGGAAAATTAATATCTGAAAGAATCATAGGCTTGGA |
I-R | GTACCGAGCTCTAGAGCAGCCTGCCAGATTCT |
T-F | CAATTTCACACAGGATTACGCGAACGCGAAGTC |
S-F | GAGGAGAAATACTAGATGTCATTCGAATTACCTGCACT |
S-R | GGAGTGACGATTATGCAGCGAGATTTTTCGCTACG |
g1-F | TTGTCTGGAAGCGGCGATGGACTAGTATTATACCTAGGACTGAGCTAGCT |
g2-R | CCATCGCCGCTTCCAGACAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT |
R-F | CTAGTATTTCTCCTCTTTCTCTAGTATTAAACAAAA |
R-R | TCGTCACTCCACCGGTGCTTAATAA |
Figure 1 Construction and characterization of different inducible systems in EcN(A)Schematic of the inducible systems engineered in EcN. Left: Conventional inducible systems based on ligand-responsive promoters. Right: T7 expression system and T7-like MmP1 expression system. The systems were constructed on the pSEVA321 vector, respectively; (B)Characterization of expression levels driven by the above inducible systems in EcN. Inducer concentrations uesd: L-Ara (10-3 mol/L), OHC6 (10-5 mol/L), IPTG (10-3 mol/L), and Van (10-3 mol/L); (C)SDS-PAGE analysis of sfGFP expression under different induction systems.图1 EcN中不同诱导调控系统的构建及表征 (A)EcN中不同诱导系统的构建示意图。左侧为基于配体响应的常规正交诱导系统,右侧为T7表达系统及类T7 MmP1表达系统,以上系统分别构建在pSEVA321骨架上;(B)不同诱导系统在EcN中的表达强度表征,其中,不同诱导剂的饱和诱导浓度使用如下:L-Ara(10-3 mol/L),OHC6(10-5 mol/L),IPTG(10-3 mol/L),Van(10-3 mol/L);(C)不同诱导系统调控下sfGFP蛋白表达的SDS-PAGE电泳结果。 |
Figure 2 Robustness improvement and characterization of the T7-like inducible system in EcN(A)Schematic for integrating the RNAPMmP1 module into the EcN genome. The lacI-RNAPMmP1 module was integrated into the genome, while the PMmP1 promoter expression module was constructed on the pSEVA321 vector; (B)Inducible expression levels of the PMmP1 promoter of above modules. ****P<0.000 1; (C)Dose-response functions of the MmP1 system in strain LM01 under varying IPTG concentrations.图2 EcN中类T7 MmP1系统的遗传稳定性优化及表征 (A)RNAPMmP1模块整合EcN基因组流程。将lacI-RNAPMmP1模块整合至EcN的基因组,PMmP1启动子表达模块构建在pSEVA321载体上;(B)PMmP1启动子在上述模块构建方式下的诱导响应表达水平表征,****P<0.000 1;(C)LM01中MmP1系统对IPTG的剂量-响应曲线。 |
Figure 3 Constructing PMmP1 promoter library to achieve broad and precise gene expression control(A)Schematic of constructing the PMmP1 promoter library; (B)Inducible expression levels of PMmP1 promoter mutants in LM01; (C)Dose-response functions of promoter mutants under varying IPTG concentrations.图3 构建PMmP1启动子文库以实现不同表达调控水平的精确调控 (A)PMmP1启动子变体库构建示意图;(B)PMmP1启动子变体LM01中的诱导响应表达水平表征;(C)不同动态范围启动子变体对IPTG的剂量-响应曲线。 |
Figure 4 Therapeutic protein production of soluble SOD by engineered EcN using T7-like MmP1 system(A)Schematic of SOD expression modules regulated by the T7-like MmP1 system(upper) and vanillic acid-inducible(lower) control; (B)SDS-PAGE analysis of soluble SOD expression under different induction systems; (C)Characterization of SOD protein production under above inducible systems; (D)Enzymatic activity assessment of SOD protein expressed by different inducible systems. ****P<0.000 1. Inducer concentration unit: mol/L图4 类T7 MmP1系统应用于治疗蛋白SOD的可溶性表达 (A)类T7 MmP1系统(上)及Van诱导系统(下)的SOD表达模块构建示意图;(B)不同诱导系统调控下可溶性SOD表达的SDS-PAGE电泳结果;(C)不同诱导系统调控下表达可溶性SOD蛋白的产量及工程菌生物量;(D)不同诱导系统调控下表达可溶性SOD的酶活性。****P<0.000 1。诱导剂浓度单位:mol/L |
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Reactive oxygen species (ROS) play vital roles in intestinal inflammation. Therefore, eliminating ROS in the inflammatory site by antioxidant enzymes such as catalase and superoxide dismutase may effectively curb inflammatory bowel disease (IBD). Here, Escherichia coli Nissle 1917 (ECN), a kind of oral probiotic, was genetically engineered to overexpress catalase and superoxide dismutase (ECN-pE) for the treatment of intestinal inflammation. To improve the bioavailability of ECN-pE in the gastrointestinal tract, chitosan and sodium alginate, effective biofilms, were used to coat ECN-pE via a layer-by-layer electrostatic self-assembly strategy. In a mouse IBD model induced by different chemical drugs, chitosan/sodium alginate coating ECN-pE (ECN-pE(C/A)) effectively relieved inflammation and repaired epithelial barriers in the colon. Unexpectedly, such engineered EcN-pE(C/A) could also regulate the intestinal microbial communities and improve the abundance of Lachnospiraceae_NK4A136 and Odoribacter in the intestinal flora, which are important microbes to maintain intestinal homeostasis. Thus, this study lays a foundation for the development of living therapeutic proteins using probiotics to treat intestinal-related diseases.© 2022. The Author(s).
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The scavenging of the excess reactive oxygen species (ROS) induced by radiation is fundamental for radiation protection. However, directly applying antioxidants results in low bioavailability and side effects. Superoxide dismutase (SOD) and catalase (CAT) have high ROS clearance efficiency, whereas their application is limited by the enzyme inactivation, making it difficult to exhibit significant therapeutic effects. Here, we engineered a probiotic Escherichia coli Nissle 1917 (EcN), i.e., AAEcN, serving as a SOD/CAT vehicle to scavenge ROS for the prevention and treatment of radiation enteritis (RE). The overexpressed Drsod and katE in AAEcN showed 5-fold ROS elimination efficiency compared to the wild EcN. Furthermore, the intestinal retention time of engineered EcN was prolonged through trefoil factor 3 gene (TFF3) modification of curli fibers on the bacterial surface, which contributed to the persistence of antioxidant enzyme activity. We found that AAEcN rapidly eliminated the intracellular ROS induced by radiation. Only a single oral dosing of AAEcN was satisfied to alleviate the radiation damage to the small intestine, colon, and spleen. Moreover, the homeostasis of pro-/anti-inflammatory cytokines was realized. The proliferation of the intestinal stem cells and spleen hematopoietic stem cells was enhanced, while the apoptosis of mucosal cells was inhibited. Our findings suggest valuable insights into the ROS scavenging way in RE, and establish an empirical basis for developing probiotics as an antioxidant enzyme vehicle for the bacteriotherapy of RE.Copyright © 2024 Elsevier Inc. All rights reserved.
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癌症作为一种威胁人类健康的疾病,其病因极其复杂,人类至今尚无法完全治愈。在肿瘤治疗方法的探索中,研究者发现某些细菌可以有效靶向肿瘤细胞并抑制其生长。相比于传统的肿瘤治疗方法,细菌具备特异性定植于肿瘤微环境的能力,极大地避免了癌症治疗中对于正常组织的伤害,提高了治疗的靶向性和安全性。另一方面,合成生物学工具的发展使研究人员可依据肿瘤特征对细菌的性状进行“设计”,进而提高细菌的靶向能力和肿瘤杀伤能力以及细菌治疗的安全性。近年来,针对人益生菌E. coli Nissle 1917进行细菌靶向治疗肿瘤的研究越来越多。通过合成生物学理念在E. coli Nissle 1917中添加基因电路,使其具备更强的感应与靶向和杀伤肿瘤的能力,更加“智能化”地应用于癌症的治疗中。本文主要综述利用细菌治疗癌症的发展,介绍利用合成生物学改造E. coli Nissle 1917的方法,探讨基因改造活细菌的靶向性、高效性与安全性,并展望以合成生物学为指导的细菌治疗癌症的未来发展方向。
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Since its discovery in the 1970s, the T7 RNA polymerase (T7 RNAP) transcription system has been applied extensively as an effective tool in molecular biology because of its robust function in various hosts, including prokaryotic, eukaryotic and cell free systems. Recently, the T7 RNAP transcription system has emerged as a critical component for synthetic biology. The present paper summarizes the advances of the T7 RNAP transcription system in synthetic biology, including the recent progress of T7 RNAP structure and its cognate promoter and terminator and its application in cell free systems, logic gates and orthogonal genetic circuits.Copyright © 2018 Elsevier Inc. All rights reserved.
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Rapid advances in synthetic biology are driving the development of genetically engineered microbes as therapeutic agents for a multitude of human diseases, including cancer. The immunosuppressive microenvironment of solid tumors, in particular, creates a favorable niche for systemically administered bacteria to engraft and release therapeutic payloads. However, such payloads can be harmful if released outside the tumor in healthy tissues where the bacteria also engraft in smaller numbers. To address this limitation, we engineer therapeutic bacteria to be controlled by focused ultrasound, a form of energy that can be applied noninvasively to specific anatomical sites such as solid tumors. This control is provided by a temperature-actuated genetic state switch that produces lasting therapeutic output in response to briefly applied focused ultrasound hyperthermia. Using a combination of rational design and high-throughput screening we optimize the switching circuits of engineered cells and connect their activity to the release of immune checkpoint inhibitors. In a clinically relevant cancer model, ultrasound-activated therapeutic microbes successfully turn on in situ and induce a marked suppression of tumor growth. This technology provides a critical tool for the spatiotemporal targeting of potent bacterial therapeutics in a variety of biological and clinical scenarios.© 2022. The Author(s).
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Promoters adjust cellular gene expression in response to internal or external signals and are key elements for implementing dynamic metabolic engineering concepts in fermentation processes. One useful signal is the dissolved oxygen content of the culture medium, since production phases often proceed in anaerobic conditions. Although several oxygen-dependent promoters have been described, a comprehensive and comparative study is missing. The goal of this work is to systematically test and characterize 15 promoter candidates that have been previously reported to be induced upon oxygen depletion in Escherichia coli. For this purpose, we developed a microtiter plate-level screening using an algal oxygen-independent flavin-based fluorescent protein and additionally employed flow cytometry analysis for verification. Various expression levels and dynamic ranges could be observed, and six promoters (nar-strong, nar-medium, nar-weak, nirB-m, yfiD-m, and fnrF8) appear particularly suited for dynamic metabolic engineering applications. We demonstrate applicability of these candidates for dynamic induction of enforced ATP wasting, a metabolic engineering approach to increase productivity of microbial strains that requires a narrow level of ATPase expression for optimal function. The selected candidates exhibited sufficient tightness under aerobic conditions while, under complete anaerobiosis, driving expression of the cytosolic F-subunit of the ATPase from E. coli to levels that resulted in unprecedented specific glucose uptake rates. We finally utilized the nirB-m promoter to demonstrate the optimization of a two-stage lactate production process by dynamically enforcing ATP wasting, which is automatically turned on in the anaerobic (growth-arrested) production phase to boost the volumetric productivity. Our results are valuable for implementing metabolic control and bioprocess design concepts that use oxygen as signal for regulation and induction.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.
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The advancements in biotechnology over time have led to an increase in the demand of pure, soluble and functionally active proteins. Recombinant protein production has thus been employed to obtain high expression of purified proteins in bulk. E. coli is considered as the most desirable host for recombinant protein production due to its inexpensive and fast cultivation, simple nutritional requirements and known genetics. Despite all these benefits, recombinant protein production often comes with drawbacks, such as, the most common being the formation of inclusion bodies due to improper protein folding. Consequently, this can lead to the loss of the structure-function relationship of a protein. Apart from various strategies, one major strategy to resolve this issue is the use of molecular chaperones that act as folding modulators for proteins. Molecular chaperones assist newly synthesized, aggregated or misfolded proteins to fold into their native conformations. Chaperones have been widely used to improve the expression of various proteins which are otherwise difficult to produce in E. coli. Here, we discuss the structure, function, and role of major E. coli molecular chaperones in recombinant technology such as trigger factor, GroEL, DnaK and ClpB.
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