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Ulcerative colitis (UC) remains a significant therapeutic challenge due to its complex pathogenesis involving oxidative stress, immune dysregulation, and gut microbiota dysbiosis. Melanin, a natural biopolymer with robust anti‐inflammatory and antioxidant properties, presents a promising treatment avenue for UC. Probiotics, particularly Escherichia coli Nissle 1917 (EcN), have gained recognition for their role in restoring gut homeostasis. In this study, we genetically engineered EcN to overexpress tyrosinase (EcN‐T), facilitating the biosynthesis of melanin specifically for UC treatment. The engineered probiotics demonstrated superior therapeutic efficacy compared to either melanin or EcN administered alone, highlighting a synergistic effect. EcN‐T not only exhibited significant capabilities in scavenging reactive oxygen species and restoring gut microbiota but also possessed the characteristic of enhancing gut colonization time, thereby extending the dosing frequency. Moreover, EcN‐T showcased novel mechanisms, such as the restoration of the intestinal mucosal barrier and the elevation of short‐chain fatty acid levels. Additionally, EcN‐T inhibited M1 macrophage polarization through Hypoxia‐Inducible Factor 1‐alpha (HIF‐1α)dependent glycolytic reprogramming, underscoring its immunomodulatory potential. Collectively, these findings provide new insights into the therapeutic potential of EcN‐T for UC treatment, offering a novel strategy that enhances treatment efficacy while potentially reducing side effects associated with conventional therapies.

Engineered probiotics are a kind of new microorganisms produced by modifying original probiotics through gene editing. With the continuous development of tools and technology progresses, engineering renovation of probiotics are becoming more diverse and more feasible. In the past few years there have been some advances in the development of engineered probiotics that will benefit humankind. This review briefly introduces the theoretical basis of gene editing technology and focuses on some recent engineered probiotics researches, including inflammatory bowel disease, bacterial infection, tumor and metabolic diseases. It is hoped that it can provide help for the further development of genetically modified microorganisms, stimulate the potential of engineered probiotics to treat intractable diseases, and provide new ideas for the diagnosis of some diseases or some industrial production.

Inflammatory bowel disease (IBD) is a chronic condition affecting the intestines, marked by immune-mediated inflammation. This disease is known for its recurrent nature and the challenges it presents in treatment. Recently, probiotic have gained attention as a promising alternative to traditional small molecular drugs and monoclonal antibody chemotherapies for IBD. Probiotic, recognized as a \"living\" therapeutic agent, offers targeted treatment with minimal side effects and the flexibility for biological modifications, making them highly effective for IBD management. This comprehensive review presents the latest advancements in engineering probiotic-based materials, ranging from basic treatment mechanisms to the modification techniques used in IBD management. It delves deep into how probiotic produces therapeutic effects in the intestinal environment and discusses various strategies to enhance probiotic's efficacy, including genetic modifications and formulation improvements. Additionally, the review addresses the challenges, practical application conditions, and future research directions of probiotic-based therapies in IBD treatment, providing insights into their feasibility and potential clinical implications.

Most inflammatory bowel disease (IBD) patients are unable to maintain a lifelong remission. Developing a novel therapeutic strategy is urgently needed. In this study, we adopt a new strategy to attenuate colitis using the Escherichia coli Nissle 1917 probiotic strain to express a schistosome immunoregulatory protein (Sj16) in the gastrointestinal tract. The genetically engineered Nissle 1917 (EcN‐Sj16) highly expressed Sj16 in the gastrointestinal tracts of dextran sulfate sodium‐induced colitis mice and significantly attenuated the clinical activity of colitis mice. Mechanistically, EcN‐Sj16 increased the intestinal microbiota diversity and selectively promoted the growth of Ruminococcaceae and therefore enhanced the butyrate production. Butyrate induced the expression of retinoic acid, which further attenuated the clinical activity of colitis mice by increasing Treg cells and decreasing Th17. Strikingly, retinoic acid inhibitor inhibited the therapeutic effects of EcN‐Sj16 in colitis mice. These findings suggest that EcN‐Sj16 represents a novel engineered probiotic that may be used to treat IBD.

Immunotherapy has revolutionized cancer treatment by leveraging the patient's immune system, yet its efficacy is often hampered by the immunosuppressive tumor microenvironment (TME). Adenosine, a key player in this milieu, suppresses immune cell activity via cAMP signaling. Here, an innovative strategy to remodel the TME using a genetically engineered strain of Escherichia coli Nissle 1917 that expresses adenosine deaminase on its surface under hypoxic conditions is presented. This engineered probiotic targets tumors, converts immunosuppressive adenosine to inosine, and enhances anti‐tumor immune responses. In vivo, the engineered probiotic significantly improved immune cell infiltration and demonstrated synergistic effects with low‐dose doxorubicin in both subcutaneous and orthotopic mouse colorectal cancer model. Furthermore, the engineered probiotic modulated the TME, promoting a shift from M2‐like to M1‐like macrophages and increasing effector T cell populations. These findings highlight the potential of using engineered probiotics for metabolic modulation of the TME, offering a novel approach for enhancing cancer immunotherapy. This study presents engineered probiotics that convert immunosuppressive adenosine into inosine in tumors, alleviating immunosuppression and enhancing antitumor immunity. Demonstrating efficacy in murine ectopic and orthotopic tumor models, this approach offers a promising probiotic‐based cancer immunotherapy strategy, highlighting the potential of microbial therapeutics in oncology.

Slow transit constipation is an intractable constipation with unknown aetiology and uncertain pathogenesis. The gut microbiota maintains a symbiotic relationship with the host and has an impact on host metabolism. Previous studies have reported that some gut microbes have the ability to produce 5-hydroxytryptamine (5-HT), an important neurotransmitter. However, there are scarce data exploiting the effects of gut microbiota-derived 5-HT in constipation-related disease. We genetically engineered the probiotic Escherichia coli Nissle 1917 (EcN-5-HT) for synthesizing 5-HT in situ. The ability of EcN-5-HT to secrete 5-HT in vitro and in vivo was confirmed. Then, we examined the effects of EcN-5-HT on intestinal motility in a loperamide-induced constipation mouse model. After two weeks of EcN-5-HT oral gavage, the constipation-related symptoms were relieved and gastrointestinal motility were enhanced. Meanwhile, administration of EcN-5-HT alleviated the constipation related depressive-like behaviors. We also observed improved microbiota composition during EcN-5-HT treatment. This work suggests that gut microbiota-derived 5-HT might promise a potential therapeutic strategy for constipation and related behavioral disorders.

Helicobacter pylori ( H. pylori ) infection has emerged as a serious risk factor for global human health. Current antibiotic-based clinical eradication therapies still have many challenges, including inadequate drug permeability across the gastric mucus barrier, H. pylori biofilm-induced antibiotic resistance and recurrent infections, as well as gut dysbacteriosis. Herein, we report a probiotic-nanozyme hybrid system (LP@FeTA/CuPt), which is formed by coupling polyphenol-coated Lactobacillus plantarum (LP@FeTA) and CuPt nanozymes. LP@FeTA effectively penetrates the gastric mucus barrier via autokinetic movement, and facilitates the targeted delivery of CuPt nanozymes within H. pylori biofilms by “eating” extracellular polymeric substances (EPS). CuPt nanozymes respond to the acidic and H 2 O 2 -rich biofilm microenvironment to generate reactive oxygen species (ROS) and release Cu ions, thereby achieving multi-target eradication of biofilms by interfering with H. pylori flagellar self-assembly, urease activity, outer membrane function and energy metabolism. Importantly, LP@FeTA/CuPt can be degraded by transferrin at the infection site, allowing the released CuPt nanozymes to reactivate the antibacterial immune functions of macrophage, thus eliminating biofilm-escaped bacterioplankton to prevent recurrent infections. Notably, LP@FeTA/CuPt also modulates gut microbiota homeostasis. This study provides a promising non-antibiotic therapeutic strategy for H. pylori infection.

Listeriosis is a foodborne infection caused by Listeria monocytogene s that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, Listeria is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of L. monocytogene s, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. Saccharomyces boulardii is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered Saccharomyces cerevisiae through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against L. monocytogene s, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast S. boulardii and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce L. monocytogenes levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for Listeria . Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic S. boulardii. Key points CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared, both allowing the expression and activity of endolysin Ply511 against L. monocytogenes. Endolysin Ply511 retained its activity against L. monocytogenes in simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI). Using fecal samples from human donors, the anti-Listeria effect was reduced potentially due to the lower metabolic activity of S. boulardii and the higher competition with the intestinal microbiome. Graphical Abstract

In humans, nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease due to the increased prevalence of obesity. While treatment of NAFLD is often geared toward lifestyle changes, such as diet and exercise, the use of dietary supplements such as probiotics is underinvestigated. Here, we report that probiotic Lactobacillus reuteri reduces fatty liver in a mouse model of diet-induced obesity. This phenotype was further enhanced upon delivery of recombinant interleukin-22 by engineered Lactobacillus reuteri . These observations pave the road to a better understanding of probiotic mechanisms driving the reduction of diet-induced steatosis and to development of next-generation probiotics for use in the clinic. Ultimately, these studies may lead to rational selection of (engineered) probiotics to ameliorate fatty liver disease. The incidence of metabolic syndrome continues to rise globally. In mice, intravenous administration of interleukin-22 (IL-22) ameliorates various disease phenotypes associated with diet-induced metabolic syndrome. In patients, oral treatment is favored over intravenous treatment, but methodologies to deliver IL-22 via the oral route are nonexistent. The goal of this study was to assess to what extent engineered Lactobacillus reuteri secreting IL-22 could ameliorate nonalcoholic fatty liver disease. We used a mouse model of diet-induced obesity and assessed various markers of metabolic syndrome following treatment with L. reuteri and a recombinant derivative. Mice that received an 8-week treatment of wild-type probiotic gained less weight and had a smaller fat pad than the control group, but these phenotypes were not further enhanced by recombinant L. reuteri . However, L. reuteri secreting IL-22 significantly reduced liver weight and triglycerides at levels that exceeded those of the probiotic wild-type treatment group. Our findings are interesting in light of the observed phenotypes associated with reduced nonalcoholic liver disease, in humans the most prevalent chronic liver disease, following treatment of a next-generation probiotic that is administered orally. Once biological and environmental containment strategies are in place, therapeutic applications of recombinant Lactobacillus reuteri are on the horizon. IMPORTANCE In humans, nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease due to the increased prevalence of obesity. While treatment of NAFLD is often geared toward lifestyle changes, such as diet and exercise, the use of dietary supplements such as probiotics is underinvestigated. Here, we report that probiotic Lactobacillus reuteri reduces fatty liver in a mouse model of diet-induced obesity. This phenotype was further enhanced upon delivery of recombinant interleukin-22 by engineered Lactobacillus reuteri . These observations pave the road to a better understanding of probiotic mechanisms driving the reduction of diet-induced steatosis and to development of next-generation probiotics for use in the clinic. Ultimately, these studies may lead to rational selection of (engineered) probiotics to ameliorate fatty liver disease.

Recent progress in synthetic biology has empowered engineered probiotics to sense tumor‐specific physicochemical signals, thereby facilitating targeted in situ drug delivery. Here, an engineered probiotic consortium capable of integrating multiple tumor microenvironment (TME) signals and orchestrating multi‐therapeutic payloads release through an orthogonal quorum‐sensing system is designed. The probiotic consortium can respond to three characteristic TME parameters, pH, hypoxia, and high‐lactate levels, in order to achieve controlled release of lactate depletion enzyme (LdhA) for metabolic environment improvement and the programmed death ligand 1 (PD‐L1) nanobody for immune checkpoint inhibition. Using the humanized PD‐1 mouse model bearing hPD‐L1 MC38 tumor and the humanized peripheral blood mononuclear cells (PBMC) mouse model bearing HT‐29 tumor, it is demonstrated that this self‐regulating microbial consortium achieves sustained oscillations and significantly suppresses tumor progression. Mechanistic studies reveal that the antitumor efficacy activates CD8+, CD4+, and IFN‐γ+ T cells, coupled with diminished immunosuppressive Foxp3+ regulatory T cell infiltration. This work advances the development of engineered live biotherapeutic products for cancer therapy and provides a modular platform for microbial consortium‐based precision medicine. This work develops an engineered probiotic consortium sensing pH, hypoxia, and lactate in the tumor microenvironment. It releases a lactate‐depleting enzyme (LdhA) and a PD‐L1 nanobody. In humanized mouse models (MC38 and HT‐29 tumors), it achieves durable oscillations, suppresses tumors, activates CD8+, CD4+, IFN‐γ+ T cells, and reduces Foxp3+ regulatory T cells, highlighting that engineered microbial consortia can be potential therapies.