Jinjin Chen

Dairy processing generates large volumes of lactose-rich permeate whose disposal can be costly. Lacticaseibacillus rhamnosus GG (LGG), a widely used probiotic strain, grows poorly on lactose, limiting the potential to valorize this stream. An endogenous Cas9-based genome-editing system successfully enabled adhE deletion and was subsequently used for strain engineering. Partial deletion of the lactose operon t1 terminator was followed by selection in MRS–lactose medium. Growth curves of 75 mutants were assessed; the ten with the highest maximum growth rates (MaxV) were sequenced and further adapted to lactose. Two were tested on glucose, lactose, and treated milk permeate. Subsequent pta deletion reduced acetate but delayed growth, while plasmid-borne ldhL1 overexpression provided no final lactate advantage. In 2 mL tube fermentations, LGGm produced 40.71 ± 2.09 g/L lactic acid within 24 h, versus 2.14 ± 0.18 g/L for wild-type LGG. The apparent yield was 1.02 g/g initial lactose, with approximately 98% L-lactate. These findings demonstrate the potential of engineered LGG for lactic acid production in MRS-supplemented dairy permeate at laboratory scale, providing a basis for further evaluation with reduced supplementation and larger fermentation volumes.

The global probiotic market is expanding rapidly, driven by growing demand for accessible strategies to support gut health, preventive care, and microbiome-based interventions. However, this commercial growth contrasts with the limited number of clinically validated, mechanism-driven products, highlighting a persistent gap between market expansion, scientific evidence, and therapeutic translation. Most current probiotics remain dominated by conventional genera, including Lactobacillus, Bifidobacterium, Bacillus, Saccharomyces, and Streptococcus, whereas live biotherapeutic products (LBPs) remain scarce. Synthetic biology is beginning to address this gap by transforming probiotics from empirically selected strains into programmable microbial platforms that sense disease-associated signals and produce defined therapeutic outputs. Escherichia coli Nissle 1917 (EcN) offers a valuable model chassis for engineered probiotics because of its long history of human use, safety record, genetic tractability, transient gut colonization, and scalable cultivation. As a rare Gram-negative probiotic, EcN naturally produces outer membrane vesicles that support host interaction, immunomodulation, and therapeutic cargo delivery. This review links probiotic market expansion with live biotherapeutic development and uses EcN to discuss emerging engineering strategies, therapeutic opportunities, and remaining translational barriers.

The gut microbiome plays a pivotal role in athletic health and performance by influencing metabolism, immunity, gastrointestinal integrity, and recovery. Advances in high-throughput sequencing and integrative multi-omics approaches have provided unprecedented insights into how microbial communities adapt to the physiological demands of training and competition. Key microbial taxa, including short-chain fatty acid producers, lactate utilizers, and carbohydrate fermenters, have been linked to enhanced endurance, reduced inflammation, and improved recovery, opening exciting opportunities for targeted probiotic interventions. While conventional probiotics remain valuable, next-generation engineered strains hold particular promise, supported by recent regulatory milestones such as FDA approvals of engineered probiotics and gene-edited livestock. In this narrative review, we synthesize recent multi-omics research to examine mechanistic links between the athlete gut microbiome and performance, evaluate current and emerging probiotic strategies, and highlight challenges related to personalization, standardization, and regulation. With longitudinal, performance-oriented trials and harmonized frameworks, microbiome-targeted interventions are poised to move beyond exploratory research toward evidence-based, precision tools that optimize athletic performance and recovery.

Plastics are indispensable to modern life, but their widespread use has created an environmental crisis due to inefficient waste management. Mixed plastic waste, comprising diverse polymers, presents significant recycling challenges due to the high costs of sorting and processing, leading to ecosystem accumulation and harmful by-product generation. This study addresses this issue by engineering a synthetic bacterial consortium (SBC) designed to degrade mixed plastic monomers. The consortium pairs Escherichia coli Nissle 1917, which uses ethylene glycol (EG), a monomer derived from polyethylene terephthalate (PET), as a carbon source, with Pseudomonas putida KT2440, which metabolizes hexamethylenediamine (HD), a monomer from nylon-6,6, as a nitrogen source. Adaptive evolution of the SBC revealed a novel metabolic interaction where P. putida developed the ability to degrade both EG and HD, while E. coli played a critical role in degrading glycolate, mitigating its by-product toxicity. The evolved cross-feeding pattern enhanced biomass production, metabolic efficiency, and community stability compared to monocultures. The consortium’s performance was validated through flux balance analysis (FBA), high-performance liquid chromatography (HPLC), and growth assays. These findings highlight the potential of cross-feeding SBCs in addressing complex plastic waste, offering a promising avenue for sustainable bioremediation and advancing future polymer degradation strategies.

Bacterial membrane vesicles (BMVs) are emerging as powerful natural nanoparticles with transformative potential in medicine and industry. Despite their promise, scaling up BMV production and ensuring stable isolation and storage remain formidable challenges that limit their broader application. Inspired by eukaryotic mechanisms of membrane curvature, we engineered Escherichia coli DH5α to serve as a high-efficiency BMV factory. By fusing the ethanolamine utilization microcompartment shell protein EutS with the outer membrane via the ompA signal peptide, we induced dramatic membrane curvatures that drove enhanced vesiculation. Simultaneously, overexpression of fatty acyl reductase led to the production of amphiphilic fatty alcohols, further amplifying the BMV yield. Dynamic modulation of peptidoglycan hydrolase (PGase) expression facilitated efficient BMV release, resulting in a striking 149.11-fold increase in vesicle production. Notably, the high-yield BMVs from our engineered strain, without the need for purification, significantly bolstered innate immune responses and demonstrated therapeutic efficacy in treating inflammatory bowel disease (IBD). This study presents a strategy to overcome BMV production barriers, showcasing the therapeutic potential of engineered bacteria and BMVs for IBD treatment, while highlighting their potential applications in diverse biomedical fields.

Probiotics, living microorganisms with demonstrated health benefits when administered in sufficient quantities, have a rich history as dietary supplements to benefit human health. Recently, understanding of their mechanisms in the gastrointestinal tract has prompted exploration of probiotics in treating human diseases. However, the effective and precise delivery of probiotics remains a significant challenge in therapeutic applications. Here, we review the mechanisms of action of probiotics in human health and the most advanced strategies for efficient probiotic delivery. We also discuss the potential applications of engineered probiotics in disease treatment. This review contributes insights into the evolving landscape of probiotic research for therapeutic applications.

Probiotics, living microorganisms with demonstrated health benefits when administered in sufficient quantities, have a rich history as dietary supplements to benefit human health. Recently, understanding of their mechanisms in the gastrointestinal tract has prompted exploration of probiotics in treating human diseases. However, the effective and precise delivery of probiotics remains a significant challenge in therapeutic applications. Here, we review the mechanisms of action of probiotics in human health and the most advanced strategies for efficient probiotic delivery. We also discuss the potential applications of engineered probiotics in disease treatment. This review contributes insights into the evolving landscape of probiotic research for therapeutic applications.

Biomining processes utilize microorganisms, such as Acidithiobacillus, to extract valuable metals by producing sulfuric acid and ferric ions that dissolve sulfidic minerals. However, excessive production of these compounds can result in metal structure corrosion and groundwater contamination. Synthetic biology offers a promising solution to improve Acidithiobacillus strains for sustainable, eco-friendly, and cost-effective biomining, but genetic engineering of these slow-growing microorganisms is challenging with current inefficient and time-consuming methods. To address this, we established a CRISPR-dCas9 system for gene knockdown in A. ferridurans JAGS, successfully downregulating the transcriptional levels of two genes involved in sulfur oxidation. More importantly, we constructed an all-in-one CRISPR-Cas9 system for fast and efficient genome editing in A. ferridurans JAGS, achieving seamless gene deletion (HdrB3), promoter substitution (Prus to Ptac), and exogenous gene insertion (GFP). Additionally, we created a HdrB-Rus double-edited strain and performed biomining experiments to extract Ni from pyrrhotite tailings. The engineered strain demonstrated a similar Ni recovery rate to wild-type A. ferridurans JAGS but with significantly lower production of iron ions and sulfuric acid in leachate. These high-efficient CRISPR systems provide a powerful tool for studying gene functions and creating useful recombinants for synthetic biology-assisted biomining applications in the future.

Both Gram-positive and Gram-negative bacteria release nanosized extracellular vesicles called membrane vesicles (MVs, 20-400 nm), which have great potential in various biomedical applications due to their abilities to deliver effector molecules and induce therapeutic responses. To fully utilize bacterial MVs for therapeutic purposes, regulated and enhanced production of MVs would be highly advantageous. In this study, we developed a universal method to enhance MV yields in both G+/G- bacteria through an autonomous controlled peptidoglycan hydrolase (PGase) expression system. A significant increase (9.37-fold) of MV concentration was observed in engineered E. coli Nissle 1917 compared to the wild-type. With the help of this autonomous system, for the first time we experimentally confirmed horizontal gene transfer and nutrient acquisition in a cocultured bacterial consortium. Furthermore, the engineered probiotic E. coli strains with high yield of MVs showed higher activation of the innate immune responses in human embryonic kidney 293T (HEK293T) and human colorectal carcinoma cells (HCT116), thereby demonstrating the great potential of engineering probiotics in immunology and further living therapeutics in humans.

Genus Acidithiobacillus includes a group of Gram-negative Fe/S-oxidizing acidophilic chemolithotrophic bacteria that are extensively studied and used for biomining processes. Synthetic biology approaches are key means to study and improve their biomining performance. However, efficient genetic manipulations in Acidithiobacillus are still major bottlenecks. In this study, we report a simple and efficient pAFi system (CRISPR-dCas9) and a scarless pAF system (CRISPR-Cas9) for genetic manipulations in A. ferridurans JAGS. The pAFi system harboring both dCas9 and sgRNA was constructed based on pBBR1MCS-2 to knockdown HdrA and TusA genes, separately, of which the transcription levels were significantly downregulated by 48% and 93%, separately. The pAF system carrying pCas9-sgRNA-homology arms was constructed based on pJRD215 to delete HdrB3 gene and overexpress Rus gene. Our results demonstrated that the pAF system is a fast and efficient genome editing method with an average rate of 15-20% per transconjugant in one recombination event, compared to 10 ^-3 and then 10 ^-2 in two recombination events by traditional markerless engineering strategy. Moreover, with these two systems, we successfully regulated iron and sulfur metabolisms in A. ferridurans JAGS: the deletion of HdrB3 reduced 48% of sulfate production, and substitution overexpression of Rus promoter showed 8.82-fold of mRNA level and enhanced iron oxidation rate. With these high-efficient genetic tools for A. ferridurans , we will be able to study gene functions and create useful recombinants for biomining applications. Moreover, these systems could be extended to other Acidithiobacillus strains and promote the development of synthetic biology-assisted biomining. Highlights: Two shuttle vectors were constructed for Acidithiobacillus ferridurans All-in-one pAFi (CRISPR-dCas9) and pAF (CRISPR-Cas9) systems were built up for gene knockdown and genome editing, separately The transcription levels of HdrA and TusA were reduced 48% and 93% using pAFi system and thus suppressed sulfur oxidation HdrB3 deletion and Rus overexpression were achieved using pAF system and showed significant effects on sulfur and iron oxidation respectively Our pAF system facilitated genome editing in Acidithiobacillus ferridurans with high efficiency (15-20%) in less than 4 weeks