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.
mRNA vaccines have emerged as a promising platform in vaccinology, enabling rapid and flexible antigen design. By delivering mRNA encoding target antigens, they allow in situ protein expression and induction of adaptive immune responses. Advances in sequence optimization, structural engineering, and delivery systems—particularly lipid nanoparticles (LNPs)—have improved mRNA stability, translational efficiency, and immunogenic performance in preclinical and clinical studies. Beyond infectious diseases, mRNA-based approaches are being explored in cancer immunotherapy, autoimmune disease modulation, and personalized medicine. Emerging platforms, including self-amplifying RNA and circular RNA, expand their potential applications. Despite these advances, challenges remain, such as the durability of immune responses, inter-individual variability, delivery specificity, and cold-chain requirements. Ongoing efforts are focused on improving these aspects through optimized delivery systems, novel administration routes, and the integration of artificial intelligence for sequence and formulation design. Notably, this review provides updated coverage of sequence design, manufacturing processes, delivery mechanisms, clinical applications, challenges, and future directions, with particular emphasis on recent advances in bacterial and parasitic mRNA vaccines and the AI-empowered closed-loop design of mRNA vaccines. Overall, mRNA technology represents a rapidly evolving and versatile platform with significant potential for future therapeutic development.
Sourcing protein-based biomaterials directly from nature presents persistent challenges in scalability, processing, and batch-to-batch variability. Recombinant engineering has focused largely on spider silks, and hagfish intermediate filament (HIF) proteins remain a comparatively unexplored alternative, with small non-repetitive subunits that have been produced full-length at high yield. This review examines the biology of naturally produced HIF protein threads from the Pacific hagfish, Eptatretus stoutii, advances in recombinant expression and purification, and their translation into functional biomaterials. We first describe the tripartite domain architecture and biochemical classification of the threonine-rich α- and γ-keratin-like subunits, and then the defensive biomechanics of HIF threads in hagfish slime, where osmolyte-regulated skeins rapidly uncoil to clog predator gills. We evaluate isolation methods from native exudate alongside thread mechanics, including a strain-induced α-to-β secondary structure transition that could account for the material’s toughness. Moving to engineered platforms, we analyze heterologous production of recombinant HIF (rHIF) in Escherichia coli and Pichia pastoris and compare purification strategies including centrifugation, size-exclusion chromatography, tangential flow filtration, and selective precipitation. We then survey rHIF biomaterial development across dry fibers, hydrogel fibers, and standalone thin films, with a focus on biomedical applications, specifically high-fidelity in vitro modeling of Bruch’s membrane.
Minimal gene set design, reducing a system to its smallest functional part set, is a core synthetic biology strategy. Bacteriophages are attractive but underused chassis, yet wild-type phages are difficult to engineer. We present a comparative genomics-guided computational framework for synthetic minimal phage gene set design, demonstrated in silico on Streptococcus mutans, the primary agent of dental caries. Three S. mutans phages (M102AD, φAPCM01, SMHBZ8) were compared by synteny analysis, and their 123 proteins were annotated with InterProScan, EggNOG-mapper, and HHpred. Conserved essential genes were selected by cross-phage conservation and annotation evidence, assembled as standardized transcriptional units, codon-optimized for E. coli K-12 with a modern folding-aware optimizer, and cloned in silico into the broad host range vector pBBR1MCS-2. Seventeen genes were assembled into a 16,403 bp synthetic insert; the final 21,559 bp construct raised the mean codon adaptation index (CAI) from 0.58 to 0.96, brought GC content into the optimal range, and eliminated rare codons, while removing all internal cloning sites. A 5′ mRNA-folding screen confirmed accessible ribosome binding sites for all 17 genes; a sensitivity analysis confirmed that the gene set was robust to the selection thresholds; and AlphaFold-Multimer modeling of representative inter-module interfaces is reported. Multi-host CAI analysis indicates strong codon usage compatibility with Enterobacteriaceae hosts. We emphasize that the construct is a computationally designed multi-gene expression plasmid, not a self-replicating or packageable phage genome, and that all results are in silico predictions requiring experimental validation. Gibson Assembly, not restriction cloning, is recommended. The design logic is transferable to other phage–host systems.
Quorum sensing (QS) has emerged as a central mechanism by which bacteria coordinate gene expression across populations. As synthetic biology has advanced, QS has been developed into a widely exploited design layer for programming collective behaviors in living systems. At the same time, the diversity of QS chemistries and architectures discovered in natural communities suggests that microbial communication contains a much larger and more versatile design space than the classical LuxI/LuxR QS paradigms alone. These developments open the door to future applications in programmable therapies, bioprocess control, and microbiome engineering that harness QS as an internal decision-making and coordination module. In this article, we review the historical development and canonical architectures of natural QS systems, including acyl-homoserine lactones, autoinducing peptides, and AI-2 pathways. We then outline design principles that govern QS-based communication and show how they guide the construction of synthetic gene circuits. Finally, we survey engineered uses of QS, highlighting applications in synchronized gene expression, spatial pattern formation, stabilized consortia, and emerging bacterial therapeutics, and discuss how expanding QS repertoires and design frameworks could enable the next generation of programmable population-aware synthetic microbes.
