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).

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.

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。

Superoxide dismutases (SODs) are universal enzymes of organisms that live in the presence of oxygen. They catalyze the conversion of superoxide into oxygen and hydrogen peroxide. Superoxide anions are the intended product of dedicated signaling enzymes as well as the byproduct of several metabolic processes including mitochondrial respiration. Through their activity, SOD enzymes control the levels of a variety of reactive oxygen species (ROS) and reactive nitrogen species, thus both limiting the potential toxicity of these molecules and controlling broad aspects of cellular life that are regulated by their signaling functions. All aerobic organisms have multiple SOD proteins targeted to different cellular and subcellular locations, reflecting the slow diffusion and multiple sources of their substrate superoxide. This compartmentalization also points to the need for fine local control of ROS signaling and to the possibility for ROS to signal between compartments. In this review, we discuss studies in model organisms and humans, which reveal the dual roles of SOD enzymes in controlling damage and regulating signaling.© 2018 Wang et al.

动态调控作为代谢工程优化中最有效的策略之一,通常包含输入信号发生器、生物传感器和执行机构3个部分。输入信号可以是细胞代谢物和环境条件变化,如化学分子、核糖核酸、温度、光信号等。而生物传感器是能够响应输入信号变化,并转化成特定信号输出的基因元件,其输出信号可以直接调控基因表达,也可以作为其他感应元件的输入。重点介绍了动态调控的基本原理及分类及其在微生物细胞工厂工程化改造中的应用实例,主要包括动态调控的优势和研究进展、动态调控系统的构建与表征,同时,也着重讨论了动态调控在细胞工厂改造中潜在的机遇与可能面临的挑战。

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.

There is a groundswell of interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we computationally identify the first biological thiosulfate sensor and an improved tetrathionate sensor, both two-component systems from marine Shewanella species, and validate them in laboratory Escherichia coli. Then, we port these sensors into a gut-adapted probiotic E. coli strain, and develop a method based upon oral gavage and flow cytometry of colon and fecal samples to demonstrate that colon inflammation (colitis) activates the thiosulfate sensor in mice harboring native gut microbiota. Our thiosulfate sensor may have applications in bacterial diagnostics or therapeutics. Finally, our approach can be replicated for a wide range of bacterial sensors and should thus enable a new class of minimally invasive studies of gut microbiota pathways.

We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.

Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a 'controller' plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.

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).

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.

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.