Nonpathogenicity are prerequisites for the selection of engineering bacteria chassis for diagnosis and treatment. Therefore, safe non pathogenic probiotics should be selected as chassis strains as far as possible. For example, Escherichia coli Nissle 1917 (EcN) is a gram negative probiotic that is sensitive to serum and does not produce enterotoxins or cytotoxins associated with pathogenic Escherichia coli (Escherichia coli) strains (Yu et al., 2023). However, the virulence of some chassis strains is inevitable, and the virulence of bacteria can be reduced by modifying the relevant genes of bacteria, so as to enhance its safety. For example, attenuated Salmonella strain ΔppGpp was engineered by regulating endotoxin gene expression. This strain is a double mutant (rela-, spot-), which has defects in ppGpp synthesis, resulting in downregulation of endotoxin gene expression (Na et al., 2006). Biosafety is a key factor in the approval of clinical trials and the use of engineered bacterial therapies. Preventing engineered bacteria from surviving after leaving the experimental and clinical environment is an important method to improve biosafety. Scientists have designed a killing switch in response to temperature changes. When the engineered bacteria leave the host, the cold induced promoter will express the toxin, thus reducing the survival rate of the strain (Stirling et al., 2017).

Gene modification technology plays a pivotal role in the in-depth exploration of biotechnology. The precise gene modification is essential for accurately regulating the functions of organisms and serves as an indispensable cornerstone in the construction of “intelligent” diagnostic engineering bacteria. Successful gene modification relies on reliable DNA transformation tools, effective control of gene expression, and post-translational control of protein processes, such as secretion (Arnold et al., 2023). In addition to these fundamental elements, the design of engineering bacteria also necessitates the integration of various advanced functional components, including sensors, circuit components, and actuators. E. coli, due to its clear genetic background, rapid propagation, low cost, and robust ability to express foreign proteins, has been employed as the primary strain for engineering development (Rosano et al., 2019). Lactobacillus, being easy to cultivate and cost-effective, possesses a variety of mature gene tools and vector systems, and can effectively target single membrane proteins of eukaryotic cells, making it a promising candidate chassis (Frelet-Barrand, 2022). Furthermore, thanks to the successful development of supportive genetic tools, including promoters, ribosome binding sites (RBS), vector backbones, and genome integration systems, the intestinal commensal bacterium Bacteroides polymorphus has also been gradually utilized as a new probiotic chassis (Lai et al., 2022).

The survival and colonization capabilities of chassis strains at the lesion site are crucial for disease diagnosis and treatment. Taking ulcerative colitis, a challenging condition in gastroenterology, as an example, if the chassis strain can effectively colonize the ileocolonic region, it may provide significant assistance in the treatment of such diseases. Given that Bacteroides and other intestinal bacteria have a higher distribution in the colon, they can be considered as potential strains for the treatment of ulcerative colitis (Aitken et al., 2024). In contrast, since the pathological changes in Crohn’s disease can extend from the mouth to the anus, implying that the corresponding treatment strategies need to cover a broader intestinal area, the use of Lactobacillus, which can colonize both the small intestine and the colon, is likely to enhance the therapeutic effect on Crohn’s disease (Li et al., 2023). Furthermore, the nanoscale outer membrane vesicles (OMVs) produced by symbiotic gut bacteria Bacteroides thetaiotaomicron can penetrate the intestinal mucosa and directly interact with the innate layer immune cells, making it highly suitable as an engineered bacterial chassis for the diagnosis and treatment of intestinal diseases (Durant et al., 2020; Taketani et al., 2020).

Currently, the reception modules used in therapeutic engineered bacteria are divided into two types: intracellular reception modules and transmembrane reception modules (Figure 1). Intracellular reception modules typically refer to transcription factors that can sense signals within the cytoplasm of engineered bacteria. The physicochemical signals in the disease environment directly enter the cytoplasm, causing the transcription factors to undergo conformational changes. The conformationally changed transcription factors activate or inhibit the inducible promoters on the transmission control module or the output module, thereby controlling the expression of diagnostic reporting factors or therapeutic factors. For example, the zinc-responsive transcription factors Zur and ZntR can serve as reception modules that react with zinc ions entering the cell, and their integration into E. coli allows for the sensing of serum zinc levels and the production of visible pigments to identify zinc deficiency (McNerney et al., 2019). A protein, lasR, which reacts with acyl-homoserine lactone (AHL), has been used as an intracellular reception module in engineered E. coli to monitor the presence of pathogenic Pseudomonas aeruginosa (P.aeruginosa) in solid agar (Gupta et al., 2013), biofilms (Saeidi et al., 2011), and animal models (Hwang et al., 2017). Another transcriptional regulator, NorR, which is activated by nitric oxide (NO), has also become an intracellular reception module for engineered bacteria to detect inflammatory signals (Archer et al., 2012; Chen et al., 2021). Finally, various orally administered gut-adapted bacteria have been implanted with intracellular reception modules to sense lactose (Drouault et al., 2002), xylan (Hamady et al., 2010), and rhamnose (Mimee et al., 2015) in the diet, as well as chemical substances produced by the gut microbiota in mice (Pickard et al., 2014). Although intracellular reception modules play an important role in disease diagnosis, they also have limitations because they cannot sense extracellular disease signals.

Transmembrane reception modules are the primary means for engineered bacteria to sense extracellular stimuli (Gao and Stock, 2009; Galperin, 2010). The classic transmembrane reception module features a histidine kinase transmembrane protein that, upon stimulation by specific signals in the environment, phosphorylates histidine on the intracellular side of the membrane. It then phosphorylates a transcriptional regulatory protein, which can bind to the promoters of the transmission control module or the output module, activating or inhibiting the transcription of the encoded genes (Beier and Gross, 2006). Many transmembrane reception modules have been utilized for disease diagnosis and treatment (Lazar and Tabor, 2021). Two early-developed transmembrane reception modules from the marine bacterium Shewanella, thiosulfate sensor ThsS-ThsR and tetrathionate sensor TtrS-TtrR, respectively receive thiosulfate and tetrathionate signals across the membrane, and these modules have been designed to be installed in E. coli as a potential therapeutic means for intestinal inflammation (Daeffler et al., 2017; Riglar et al., 2017). Recently, researchers designed a NarX-NarL-based transmembrane reception module to detect nitrate levels, and in EcN, the combination of NarX-NarL with ThsS-ThsR modules across the membrane receives nitrate and thiosulfate, enhancing the specificity of the engineered bacteria in diagnosing intestinal inflammation (Woo et al., 2020). Another study integrated the transmembrane reception elements CqsS-LuxU-LuxO derived from Vibrio cholerae with a dCAS9-based green fluorescent protein (GFP) reporter gene system into E. coli, constructing a biosensor with high sensitivity to quorum sensing (QS) ligand cholera CAI-1, indicating the presence and proliferation of Vibrio cholerae (Holowko et al., 2016). The reception module can act directly on the output module or can be combined with the output module through the transmission control module to form a more intelligent biosensing system (Anderson et al., 2007).

The transmission control module is located between the reception module and the output module, and it is composed of several serial or parallel transcriptional units (Figure 1). The efficiency of the transmission control module is equal to the sum of the rates of all transcriptional units. The efficiency of each transcriptional unit depends on its promoter, terminator, and ribosome binding site (RBS) components. The promoter marks the starting point of transcription and can ensure the transcription rate of mRNA through a series of well-designed constitutive promoter libraries or chemically inducible promoters (Kelly et al., 2009; Mutalik et al., 2013; Yao et al., 2013; Lutz and Bujard, 1997; Cox et al., 2007; Armetta et al., 2021). Terminating the transcription process is crucial to avoid unintended interactions between different transcriptional units in the engineered DNA sequence. Previous studies have established numerous high-efficiency transcription terminator resource libraries, which include a large number of high-performing terminators (Chen et al., 2013; Cambray et al., 2013). The RBS determines the efficiency and location of ribosome binding, and it can regulate the rate of translation initiation, thereby controlling the expression level of proteins. By using RBS calculation tools to design specific ribosome binding sequences (Salis et al., 2009; Espah Borujeni et al., 2014; Farasat et al., 2014), or employing a more flexible “dual-cistron design” method with RBS (Mutalik et al., 2013), the translation efficiency of target genes in engineered bacteria can be conveniently adjusted.

The transmission control module, based on Boolean logic, is divided into three basic modes: “AND gate transmission control” (functional factors are expressed only when both signals are present simultaneously) (Figure 2A) (Woo et al., 2020; Anderson et al., 2007), “OR gate transmission control” (functional factors are expressed as long as one of the two signals is present) (Figure 2B) (Wong et al., 2015), and “NOT gate transmission control” (functional factors are not expressed when a certain signal is present, otherwise they are expressed) (Figure 2C) (Bartoli et al., 2020). These three basic modes can also be combined to form advanced transmission control modules such as NAND (Wang et al., 2011), NOR (Tamsir et al., 2011), and XOR gates (Wong et al., 2015).

The output module of intelligent therapeutic engineered bacteria is the functional gene that ultimately exerts its effect, which can be divided into two types: diagnosis and treatment (Figure 1).

Diagnostic output modules often express pigments, luciferases, or fluorescent proteins because the intensity of this form of output is easily observable and measurable. The advantage of pigments as diagnostic outputs is that they are visible to the naked eye without the need for special equipment. The earliest example is the enzymatic activity of the lacZ gene on X-gal, which produces a blue pigment (Horwitz et al., 1964). Violacein, lycopene, and carotenoids are induced to express by Zur and ZntR zinc-responsive transcription factors (TFs) to sense serum zinc levels for identifying zinc deficiency (McNerney et al., 2019). Luciferase, due to its very low background luminescence in fecal samples, typically produces higher sensitivity than pigment reporter genes. Although the use of specific equipment and substrates is required, these limitations do not hinder their significant role in improving the reliability of detection (Corthier et al., 1998). Fluorescent proteins are also commonly used as output proteins for disease diagnosis. Researchers have combined GFP and its variants (such as RFP, YFP, CFP) with transmembrane reception modules for the diagnosis of inflammatory bowel disease (IBD) (Daeffler et al., 2017; Riglar et al., 2017; Woo et al., 2020). This allows researchers to more accurately observe the morphology, growth status, and interactions of engineered bacteria at the single-cell level.

Therapeutic output modules express functional factors such as interleukins, hormones, enzymes, and bacterial toxins. Lactobacillus expressing interleukin-27 (IL-27) can be used to treat inflammatory bowel disease (Hanson et al., 2014). Interleukin-10 (IL-10) and proinsulin are co-expressed in Lactobacillus for the treatment of type 1 diabetes (Takiishi et al., 2017). Engineering EcN can bind to heparin sulfate proteoglycans (HSPG) on the surface of cancer cells and secrete a substance called myrosinase, which converts glucosinolates in food into sulforaphanes with anticancer activity (Ho et al., 2018). E. coli loaded with the bacterial toxin antimicrobial protein (S5 pyocin) and biofilm-degrading enzyme (DspB) has shown a synergistic effect in eliminating P. aeruginosa, improving the efficiency of pathogen clearance (Hwang et al., 2017).

Type 1 diabetes is an autoimmune disease that leads to insulin deficiency and ultimately the loss of function of pancreatic β-cells, necessitating lifelong treatment, particularly with long-term, scheduled insulin injections (Subramanian et al., 2024). Researchers have utilized Lactobacillus gasseri to express glucagon-like peptide 1 (GLP-1_7–37) and engineered it to be released via a type III secretion system. Data from diabetic rats treated orally provide evidence for the use of engineered commensal bacteria in the treatment of diabetes (Duan et al., 2015). Oral administration of engineered Lactococcus lactis expressing both human proinsulin (PINS) and human interleukin-10 (hIL-10), in combination with low-dose systemic anti-CD3 monoclonal antibodies, can stably restore blood glucose levels in non-obese diabetic (NOD) mice (Takiishi et al., 2017; Takiishi et al., 2012). Similarly, engineered Lactococcus lactis co-expressing human glutamic acid decarboxylase (GAD65) and human interleukin-10 (hIL-10) has shown the same therapeutic efficacy (Robert et al., 2014).

Type 2 diabetes is widely concerned because of its incidence rate and complications (Tao et al., 2020). GLP-1 is an incretin hormone produced by intestinal cells that stimulates insulin secretion from the pancreas in a glucose-dependent manner. Exogenous GLP-1 analogs are used for the treatment of type 2 diabetes. Lactobacillus lactis (LL-pUBGLP-1) containing a plasmid encoding GLP-1 was used as a delivery system for GLP-1, and its effectiveness was demonstrated by the reduction in plasma glucose levels and increment insulin concentrations in obese type 2 diabetic rats (Zucker Diabetic Fatty rats, ZDF) following oral administration (Agarwal et al., 2014). Researchers have designed a GLP-1 mutant pentamer (5 × GLP-1), which is expressed and extracellularly anchored on the cell wall of lactic acid bacteria, and retains its in vitro bioactivity after being digested by intestinal trypsin into GLP-1 monomers (Lin et al., 2016). To induce a significant insulinotropic effect in diabetic animal models, further enhancement of the expression level of GLP-1 by lactic acid bacteria is required. Consequently, researchers have further mutated GLP-1 and linked it to form a GLP-1 hexamer (6 × mGLP-1), and the hexamer expressed by E. coli has been shown to exert a long-lasting hypoglycemic effect in type 2 diabetic mice (Xu et al., 2017). Additionally, exendin-4, a GLP-1 receptor agonist with a more prolonged bioactivity compared to GLP-1, has been effectively expressed and secreted by Lactobacillus paracasei transformed with a plasmid encoding the exendin-4 gene. The secreted exendin-4 significantly enhanced insulin secretion from INS-1 β-cells, providing a novel strategy for the treatment of diabetes with engineered bacteria (Zeng et al., 2016). Recently, researchers have orally administered optogenetic engineered Lactococcus lactis in the gut, which secretes GLP-1 to regulate metabolism under the control of a wearable optical device, and this strategy has effectively controlled blood glucose, body weight, and other characteristics in rat and mouse models (Zhang X. et al., 2023).

Phenylketonuria (PKU; also known as phenylalanine hydroxylase deficiency) is an autosomal recessive genetic disorder of phenylalanine (Phe) metabolism, where excessive Phe levels can lead to brain dysfunction (van Spronsen et al., 2021). To reduce Phe levels in the body, Synlogic Corporation has designed a strain, EcN (SYNB1618), capable of expressing genes for phenylalanine transport protein (PheP), phenylalanine ammonia lyase (PAL), and L-amino acid deaminase (LAAD), which can convert Phe to trans-cinnamic acid (TCA) and phenylacetate (PP) in the gastrointestinal tract, effectively reducing blood Phe levels in mice and crab-eating macaques (Isabella et al., 2018). Subsequently, researchers conducted human trials with EcN (SYNB1618), which demonstrated safety and good tolerability (Puurunen et al., 2021). Building upon the success of EcN (SYNB1618), the entire cell PAL activity was optimized, resulting in a more effective strain, EcN (SYNB 1934), which showed approximately twice the PAL activity in vivo compared to its predecessor (Adolfsen et al., 2021).

Obesity is a major global issue that significantly impacts public health (Perdomo et al., 2023). In recent years, researchers have also made attempts to treat obesity using smart engineered bacteria. N-acylphosphatidylethanolamines (NAPEs) synthesized in the small intestine can enhance satiety and reduce food intake. Engineered EcN, through genetic modification, can synthesize NAPEs in the gut, thereby treating obesity. This approach is more effective and enduring than traditional dietary and lifestyle interventions (Chen et al., 2014). Butyrate can reduce appetite and alleviate fatty liver through gut-brain neural circuits. Transgenic Bacillus subtilis SCK6 can increase butyrate production, which has beneficial effects on obesity, fasting blood glucose, insulin resistance, hepatic steatosis, and fat accumulations (Bai et al., 2020). Engineered Lactobacillus expressing GLP-1 has an ameliorative effect on obesity in mice induced by a high-fat diet (HFD) (Wang et al., 2021).

Ammonia (NH₃) is a toxic metabolite, which is produced in the small intestine and colon. The main route of ammonia (NH₃) is to synthesize urea in the liver and excrete it in the urine from the kidney. Hyperammonemia occurs when there is a defect in ammonia metabolism. A large amount of ammonia that is not metabolized into urea by the liver enters the brain through the blood circulation, which can lead to brain injury and death (Ribas et al., 2022). Kurtz et al. Modified the metabolism of probiotic ECN, deleted the gene encoding arginine repressor ArgR, and then integrated the gene Arga215 encoding ArgAY19C into the intergenic region of malE and malK genes under the control of endogenous anaerobic inducible promoter fnrS (P _fnrs ) of Escherichia coli. ArgAY19C is the feedback resistant version of N-acetylglutamate synthase ArgA (argA ^fbr ). This engineered strain, SYNB1020, can overproduce arginine, thereby sequestering some ammonia produced by intestinal bacteria into amino acid molecules. The experiment proved that synb1020 could reduce blood ammonia, improve the survival rate of mouse hyperammonemia model, and showed repeated dose tolerance in non-human primates. A phase 1 dose escalation study in healthy human volunteers did not result in serious adverse events and showed that the bacterium was metabolically active in vivo, indicating that SYNB1020 needs further clinical development (Kurtz et al., 2019). Subsequently, the researchers conducted experiments using two engineered EcN strains, S-ARG (SYNB1020) and S-ARG + BUT (SYNB1536), in a rat model of chronic liver injury (CLD) and hepatic encephalopathy induced by bile duct ligation (BDL). The S-ARG (SYNB1020) strain showed positive benefits in reducing hyperammonemia, thereby alleviating memory impairment in BDL rats. The S-ARG + BUT (SYNB1536) strain exhibits similar therapeutic effects in hyperammonemia and shows additional positive effects in systemic inflammation and endotoxemia, leading to adequate protection against memory deterioration (Ochoa-Sanchez et al., 2021).

Uric acid is the final product of purine degradation in the human body, which can be excreted from the body through the kidneys and intestines. Excess uric acid will lead to hyperuricemia. The chronic form of arthritis caused by precipitation of sodium urate crystals is called gout. Gout afflicts hundreds of millions of people worldwide, with a high incidence rate in developed countries (Borghi et al., 2020). Yang et al. discovered a new method to eliminate purines from the diet by engineering gut bacteria, integrating a set of enzymes and transporters capable of degrading purines under anaerobic conditions into the EcN chromosome, which can bypass the formation of uric acid. By orally supplementing these engineered probiotics or purified recombinant enzymes, it is possible to effectively treat hyperuricemia fruit fly models, providing a new approach for developing novel probiotic therapies for the treatment of hyperuricemia and gout (Tong et al., 2023). Zou et al. also developed a probiotic EcN called YES301, which significantly improved the ability of bacteria to uptake xanthine and hypoxanthine by rationally designing overexpression of the xanthine transporter XanQ. As a result, serum uric acid levels were reduced and kidney damage was improved in a hyperuricemia mouse model. YES301 has shown comparable efficacy to traditional uric acid lowering drugs such as allopurinol, but with fewer side effects and higher biosafety, highlighting the potential of engineered probiotics in reducing intestinal purine levels to manage hyperuricemia (Zou et al., 2024).

Firstly, many bacteria contain natural toxins with potential anti-cancer properties. To better utilize these bacterial toxins for cancer treatment, scientists have recombined the natural toxin genes to express and purify them through engineered bacteria. This not only enhances the targeting of cancer cells but also reduces damage to normal tissues. For instance, researchers have mutated the P. aeruginosa exotoxin A (PE) and fused it with anti-CD25 to form a new recombinant toxin (CD25-PE38KDELKQK). The recombinant toxin, expressed and purified by E. coli, significantly reduced the natural toxin’s toxicity to the pulmonary vessels in the lungs of mice with lung cancer (Wang et al., 2008). Researchers have also linked the B subunit of the natural bacterial toxin Shiga-like toxin (STXB) with the DT389 fragment of the natural diphtheria toxin (DT538) to form a fusion toxic protein (DT389-STXB). STXB is responsible for recognizing the cancer cell surface receptor GB3, while the DT389 toxin is responsible for eliminating cancer cells, showing high affinity for breast cancer cells (Mohseni et al., 2021).

Engineering bacteria can activate immune cells or guide them to recognize and kill cancer cells by expressing specific immune-stimulating molecules, such as cytokines or chemokines. This approach enhances the immune memory effect, preventing the recurrence of cancer.

Interleukin-2 (IL-2) can activate immune cells infiltrating tumor tissues, including T cells, NK cells, and ILC cells (Raeber et al., 2022). By electroporation of plasmids carrying the human IL-2 gene into the attenuated Salmonella typhimurium strain x455O, a drug delivery system x455O (pIL2) is constructed. Gavage of this engineered bacteria in MC-38 adenocarcinoma mice not only reduced liver metastasis of MC-38 adenocarcinoma but also did not observe toxicity of IL-2 or Salmonella (Saltzman et al., 1996). Subsequently, researchers successively attempted to use Clostridium butyricum secreting rat interleukin-2 (rIL2) (Barbé et al., 2005), and Bacillus cereus secreting mouse interleukin-2 (mIL2) as engineered bacteria for tumor treatment (Kubiak et al., 2021). Recently, scientists have progressively optimized the bioactivity of IL-2 using EcN, and mice with colon cancer models treated with intravenous injection of this engineered bacterial strain showed a moderate reduction in tumor growth rate and a significant increase in IL-2 levels in the tumor (Tumas et al., 2023).

Components of the interleukin-18 (IL-18) signaling pathway are upregulated on tumor-infiltrating lymphocytes, indicating that IL-18 can enhance the immune system’s ability to combat tumors (Zhou et al., 2020). Researchers synthesized IL-18 using an engineered attenuated Salmonella typhimurium strain (VNP20009), and after intravenous injection of this engineered bacteria into a mouse model, the growth of colon cancer and breast cancer cells was significantly inhibited, as well as lung metastasis in immunocompetent mice (Loeffler et al., 2008).

L-arginine in tumor tissue can enhance the antitumor capability of immune checkpoint inhibitors. To increase the production of L-arginine, scientists deleted the arginine repressor gene (ArgR) of EcN and mutated the N-acetylglutamate synthase gene (ArgAfbr). The engineered bacteria, when co-injected with anti-PD-L1, significantly reduced tumor growth rate (Canale et al., 2021).

Researchers used EcN with the lysis gene (ΦX174E) to express nanobodies targeting immune checkpoints PD-L1nb and CTLA-4nb. After intratumoral injection of this engineered bacteria, the tumor partially or completely regressed in the mouse model, and no visible liver metastasis was observed (Gurbatri et al., 2020).

Another study utilized engineered Salmonella (△ppGpp) to secrete the Del-1 protein (a potent inhibitor of neutrophil recruitment, antagonizing the antigen-1 associated with lymphocyte function on vascular endothelial cells). After tail vein injection of this engineered bacteria into a mouse model, the number of neutrophils in the tumor decreased, the number of M1-type macrophages increased, and tumor elimination was observed in some samples (Tian et al., 2022).

Engineering bacteria can serve as drug delivery systems, directly delivering anticancer drugs to tumor cells. This approach can increase the local concentration of drugs and reduce systemic side effects. Researchers have used E. coli DH5α that can specifically target tumor tissues to express beta-glucuronidase (βG), which converts inactive prodrugs into anticancer active drugs at the tumor site, significantly inhibiting tumor growth (Cheng et al., 2008; Afkhami-Poostchi et al., 2020).

Bacterial spores can also serve as carriers for anticancer drugs because obligate anaerobic bacterial spores can only germinate in the hypoxic necrotic areas present in solid tumors, while remaining dormant in other parts of the body (Barbé et al., 2006; Kubiak and Minton, 2015). However, the original bacterial spores cannot eliminate cancer cells and require genetic modification or combination with other anticancer strategies. Scientists have covalently attached the chemotherapeutic drug gemcitabine (MGEM) to Clostridium butyricum spores (SPORE), and after oral administration, the drug (SPORE-MGEM) migrates from the upper gastrointestinal tract to pancreatic tumors, with a threefold increase in drug accumulation within the tumor compared to MGEM alone. In a mouse model of pancreatic cancer, SPORE-MGEM inhibited tumor growth without significant side effects (Han et al., 2022).

The primary mode of transmission for HIV/AIDS is sexual contact, with the cervicovaginal mucosa being the main route of HIV infection in women. Scientists have engineered Lactobacillus jensenii, isolated from vaginal mucosa, to express a bispecific CD4 (2D CD4) molecule on its surface that can bind to HIV proteins, which can impede heterosexual transmission of HIV (Chang et al., 2003; Liu et al., 2008). Subsequently, a study used engineered Lactobacillus acidophilus (ATCC 4356) to express human CD4, with the expressed CD4 protein anchored on the outer wall of the engineered bacteria to adsorb HIV-1 virus. In a humanized mouse model, the engineered bacteria, when stimulated rectally, prevented HIV-1 infection, but the vaginal effect was suboptimal (Wei et al., 2019). Similarly, the probiotic EcN was designed to secrete HIV-gp41-lysin peptides, thereby preventing HIV fusion and subsequent invasion. This engineered bacterial strain can colonize persistently and stably in the colon and cecum, releasing anti-HIV peptides in mice for several months (Rao et al., 2005).

Cholera is a life-threatening gastrointestinal infectious disease caused by the infection of toxigenic Vibrio cholerae (Kanungo et al., 2022). Engineered bacterial interventions have been used for the diagnosis and treatment of cholera. Scientists have constructed an intelligent engineered bacterium using Lactococcus lactis, whose recombinant transmembrane receptor module HR4M can specifically detect the quorum-sensing signal CAI-1 of Vibrio cholerae in the intestine and trigger the expression of the reporter gene β-lactamase. The expressed product is easily detectable in fecal samples, thereby assisting in the diagnosis of cholera (Mao et al., 2018). Holowko et al. (2016) developed an intelligent diagnostic engineered bacterium based on the quorum-sensing mechanism of Vibrio cholerae and CRISPRi technology for detecting Vibrio cholerae. It uses E. coli as a vector, and when Vibrio cholerae is at a high density, the transmembrane receptor module CqsS-LuxU-LuxO reacts with the signal molecule CAI-1, and the CRISPRi-based transmission control system triggers the expression of the diagnostic factor GFP, which has been successfully used for detecting Vibrio cholerae. Similarly, Jayaraman et al. (2017) replaced the GFP from the previous study with the lytic protein YebF-Art-085, and introduced the expression of the killing protein Art-085. The lysed engineered bacteria release the killing protein, thereby eliminating Vibrio cholerae. In vitro experiments have shown that this intelligent engineered bacterium can effectively inhibit the growth of Vibrio cholerae cells.

P.aeruginosa primarily colonizes the respiratory and gastrointestinal tracts (Ungor and Apidianakis, 2024). It is one of the main causes of hospital-acquired infections due to its resistance to many antibiotics and antimicrobial agents (Jurado-Martín et al., 2021). Utilizing engineered bacteria to effectively sense and kill P. aeruginosa provides a novel antibacterial strategy for controlling infections caused by this bacterium. Researchers have designed a smart E. coli that can detect acyl-homoserine lactone 3OC12HSL produced by P. aeruginosa using the intracellular receptor protein lasR. Upon detection, the bacteriocin Pyocin S5 is produced, which can be released by the E7 lysis protein after lysing E. coli, and then kills P. aeruginosa in the infected environment (Saeidi et al., 2011). Building on the aforementioned research, another study changed the release mode of the smart engineered bacteria from lysis to secretion. When lasR binds to the P. aeruginosa infection signal 3OC12HSL, the pathogen-specific toxin CoPy, which carries the signal peptide Flgm, is expressed and can be released through the flagellar secretion system. This design can obviously kill P. aeruginosa more effectively and for a longer duration (Gupta et al., 2013). Subsequently, a similar study used the probiotic strain EcN as a host and added the expression of the biofilm-degrading enzyme DspB to the bactericidal protein Pyocin S5. This engineered bacterium showed both preventive and therapeutic activity in Caenorhabditis elegans and mouse models of P. aeruginosa intestinal infection (Hwang et al., 2017). Recently, Gao et al. (2023) constructed a chimeric pyocin (ChPy) to specifically kill P. aeruginosa and designed a near-infrared (NIR) light-responsive strain to produce and deliver this drug. This smart engineered bacterial strain can continuously produce ChPy without light and release it to kill P. aeruginosa upon NIR light-induced bacterial lysis.