We engineered Escherichia coli for improved ethylene glycol (EG) biosynthesis using two-stage dynamic metabolic control coupled with pathway optimization. An engineered Dahms pathway (xylB, xylC, xylD, yqhD) was induced upon phosphate depletion to enable EG biosynthesis during stationary phase. The performance of our initial strain, expressing a baseline pathway, was improved by the addition of "stoichiometric" valves enabling proteolytic degradation of xylose isomerase and soluble transhydrogenase, upon phosphate depletion. Reduction of these enzymes, which compete for substrates with EG biosynthesis, led to a 3-fold increase in specific productivity. In contrast, enabling "regulatory" dynamic metabolic valves targeting enoyl-ACP reductase and glucose-6-phosphate dehydrogenase, designed to enhance NADPH flux, did not improve production in this control. Following pathway optimization, regulatory strains exhibited enhanced production rates exceeding 0.5 g gCDW- 1 h- 1, a 14-fold improvement over the control, while stoichiometric valves conferred no benefit. Scale-up in fed-batch bioreactors achieved 140 g L- 1 EG in 70 h with a yield of 0.38 g g- 1 xylose (92% of theoretical), representing one of the highest reported titers in E. coli. These results demonstrate that optimal engineering strategies shift with pathway capacity. While elimination of competing fluxes benefits low-flux systems, high-flux pathways require enhanced cofactor regeneration.

Integrating molecular recognition, tracing, and interactome capture within a single nanoparticle could allow for probing, manipulation, and control of complex biological systems. Achieving such multifunctionality within a single well-defined nanostructure remains challenging. T7 bacteriophage-based biological nanoparticles provide a robust and genetically programmable scaffold; however, conventional T7 display systems typically accommodate only one exogenous peptide species per phage particle, limiting multifunctional and combinatorial applications. Here, we describe a mosaic phage platform that enables the simultaneous display of two peptides on separate T7 coat protein subunits. By combining genome-encoded and plasmid-driven expression of peptide-fused capsid proteins, the system provides a modular route for assembling multifunctional phage nanoparticles. We generated mosaic T7 phages co-presenting the neuropilin-1 (NRP-1)-binding peptide RPARPAR alongside either a HA-tag or a Twin-Strep (Tstrep) tag affinity handle. These engineered phages specifically target NRP-1-expressing cells and enable immunofluorescence visualization and pull-down of NRP-1 from complex lysates. Furthermore, a random peptide T7 phage library co-displaying the constant lung-homing peptide RPARPAR shows preferential pulmonary accumulation in vivo, demonstrating utility for combinatorial targeting and tissue-directed phage selection. Collectively, this dual-display approach expands phage display by enabling cell targeting, visualization, molecular interactome probing, and in vivo tissue homing within a single genetically encoded nanoparticle platform.

Efficient production of human serum albumin (HSA) fusion proteins in yeast remains constrained by the combined demands of folding, secretion, and cellular resource allocation. Rational optimization of endoplasmic reticulum folding and unfolded protein response pathways can improve production, but additional cellular constraints may persist in engineered strains. Here, we combined iterative atmospheric and room-temperature plasma (ARTP) mutagenesis, activity-coupled screening, and genome- and transcriptome-guided validation to improve production of an amylase-HSA fusion protein (AGSH) in Saccharomyces cerevisiae. Using extracellular α-amylase activity as the screening indicator, five rounds of ARTP mutagenesis in the folding-optimized strain AH28 generated mutants with enhanced AGSH production. The final mutant N5S14 reached 2.12-fold the AH28 level in AGSH-associated α-amylase activity. Physiological profiling showed that enhanced production was associated with altered growth-production balance and changes in glucose, ethanol, and glycerol profiles. Whole-genome sequencing identified 33 candidate mutation sites, and reverse engineering validated genes associated with improved AGSH production, including BUD27, NPR3, NPR2, and IML1. RNA-seq revealed distinct transcriptional states involving central carbon metabolism, protein folding, and stress-related processes. Transcriptome-guided validation identified PFY1 deletion and SSA3 overexpression as additional beneficial interventions. Overall, this strategy improved AGSH production and identified engineering targets beyond previously optimized folding-related modules.

Using transformation-associated recombination (TAR) technology, a lovastatin biosynthetic gene cluster was assembled in Saccharomyces cerevisiae BY4741, in which pathway-related enzyme genes were controlled by strong fungal promoters and terminators. Using Aspergillus niger SH-2 as the starting strain, a copy of Monacolin J (MJ) and a copy of lovastatin expression cassette were integrated into its genome, MJ heterologous expression strains and lovastatin heterologous expression strains were successively constructed. To facilitate the synthesis of MJ and lovastatin, ATP-dependent citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) were overexpressed in the MJ- and lovastatin-engineered strains. For fermentation optimization, various carbon sources were tested to determine the best one. Furthermore, the roles of glutamic acid, methionine, and linoleic acid in the production of MJ and lovastatin were investigated. Finally, in a 500 mL shake flask with 100 mL of liquid medium, using hydrolyzed starch as the carbon source and adding 10 mM glutamate, a MJ titer of 1606 mg/L and a lovastatin of 1345 mg/L were obtained, and after overexpressing ACL and ACC enzymes in the MJ strain, a MJ titer of 1786 mg/L were obtained. During fed-batch fermentation in a 2-L bioreactor, the lovastatin yield reached 2915 mg/L.

Dengue virus (DENV) and Zika virus (ZIKV) are public health threats, causing substantial morbidity and mortality worldwide. The co-circulation of DENV and ZIKV complicates prevention and control efforts, highlighting the importance of studying cross-reactive antibodies, vaccines, and immune modulators. This study explores the cross-reactivity and neutralizing capacity of antibodies against DENV elicited by immunization with a ZIKV protein extract. Female isogenic BALB/c mice were immunized with ZIKV protein extract derived from C6/36 cell cultures. Splenic B lymphocytes from immunized mice were fused with myeloma cells using polyethylene glycol (PEG) to generate hybridomas. The resulting antibodies were assessed using ELISA and plaque reduction neutralization tests (PRNT) against ZIKV and DENV including their structural and nonstructural proteins. Our findings showed that immunization induced a diverse antibody response, including antibodies recognizing epitopes unique to ZIKV as well as epitopes shared with DENV. Notably, neutralizing antibodies targeting conserved epitopes, particularly those shared with DENV-1, were also identified against other DENV serotypes. These results suggest the presence of cross-reactive neutralizing antibody responses between ZIKV and DENV, potentially exhibiting serotype-specific reactivity. Collectively, this study provides experimental evidence of flavivirus cross-reactivity and contributes to a more refined understanding of humoral responses in settings where these viruses co-circulate.

Santalene is the core aromatic component of natural sandalwood essential oil and the key precursor of santalol, which is a high-value sesquiterpenoid with unique woody aroma and bioactivities. Microbial production emerges as a promising alternative supply route other than inefficient plant extraction. Herein, we systematically engineer a peroxisomal factory for α-santalene overproduction in Ogataea polymorpha by optimizing mevalonate pathway in peroxisomes and enhancing the supply of reducing power NADPH and precursor acetyl-CoA. The engineered strain produced 556.2 mg/L and 3.4 g/L α-santalene in shake flask batch and fed-batch fermentations, respectively. This work suggests O. polymorpha is a promising host for peroxisome engineering and offers a sustainable bio-manufacturing route for santalol derivatives.

Lactiplantibacillus plantarum has emerged as a promising synthetic biology chassis owing to its food-grade safety, probiotic properties, and adaptability to the gastrointestinal tract. Its engineering potential has expanded rapidly in live biotherapeutics, functional foods, biosensors, and animal health. However, its broader application is still limited by the lack of standardized genetic parts, versatile engineering tools, and industrially compatible chassis. This review summarizes recent advances in the synthetic biology toolbox of L. plantarum, including expression elements, genome engineering strategies, and representative applications. Particular emphasis is placed on the development of genetic components, conventional and CRISPR-based engineering technologies, and their applications in medicine, food biotechnology, biosensing, and animal nutrition. Current challenges and future opportunities, including standardized genetic parts, precision genome engineering, AI-assisted strain design, and high-performance chassis development, are also discussed. This review provides a comprehensive framework for developing next-generation programmable probiotic cell factories based on L. plantarum.

Microbial cell factory development requires iterative optimization of enzymes, pathway fluxes, and cell traits to achieve efficient and robust production of target chemicals. However, the complexity of intracellular metabolic networks often limits predictable identification and control of engineering targets. Genetically encoded metabolite-responsive RNA biosensors based on aptamer recognition and riboswitch- or ribozyme-mediated regulation have emerged as versatile tools for linking intracellular metabolite levels to measurable or selectable outputs. Their rapid signal transduction, compact genetic architecture, and low metabolic burden make them attractive for high-throughput screening and dynamic regulation. This Review adopts a hierarchical engineering framework that examines RNA biosensor applications across progressively broader levels, from enzyme engineering to pathway-level regulation and host-level cellular trait engineering. We further discuss their molecular organization and technological evolution, including ligand recognition, regulatory coupling, sensor tuning, and computational approaches. Finally, challenges associated with sensor robustness, host dependency, scalability, and translation into production-relevant phenotypes are discussed together with opportunities in cell-free characterization and data-driven design. This hierarchical perspective highlights RNA biosensors as engineering interfaces connecting intracellular sensing with interventions across multiple scales of microbial cell factory engineering.

Enhancing nitrogen assimilation and coordinating carbon-energy metabolism in the autotrophic chassis Cupriavidus necator remain key challenges for maximizing single-cell protein (SCP) production from CO2. In this study, multilevel engineering was performed on C. necator. First, glnA was overexpressed to enhance nitrogen assimilation, which increased crude protein content to 72.75% in the best-performing strain, GS13. Preliminary physiological characterization suggested that this increase correlated with enhanced heme level. Second, glcR was disrupted to alter carbon allocation toward amino acid precursor metabolism, and epr-1 was deleted to inhibit extracellular protein degradation during the secretion process. The double-knockout strain Δepr-1ΔglcR achieved a high crude protein content of 70.13%. However, when the GS13 module was introduced into this background, OD600 increased to 13.12 while crude protein content decreased to 68.05%, indicating a non-additive interaction between the engineered modules. Overall, our study provided improved strains and a modular framework for future SCP optimization and mechanistic studies of carbon-nitrogen coordination in C. necator.

Calpain is a family of cysteine proteases whose role in sea cucumber autolysis was previously unreported. In this study, specific inhibition of calpain notably alleviated autolysis in the respiratory tree and intestine, significantly reduced body wall hardness loss. During an 8 h autolysis period, calpain activity in the body wall continuously declined, whereas activity in the respiratory tree and intestine peaked at 2 h. The contents of calpain 5 & 9 were highest in the respiratory tree and remained relatively stable, whereas these two isoforms showed distinct temporal patterns in the intestine and body wall. Immunohistochemistry revealed high calpain expression in the muscle layers and at the mucosal fold tips of the intestine and respiratory tree, but sparse distribution in the body wall connective tissue. After 4 h of autolysis, disintegration of the intestinal serosal layer, disorganization of the respiratory tree cellular structure, and destruction of the epidermis and endodermis of the body wall were observed. Co-immunoprecipitation coupled with mass spectrometry identified 40 calpain-interacting proteins, and fluorescence colocalization confirmed direct interaction with tubulin. This first report establishes calpain involvement in sea cucumber autolysis, clarifying its dynamic activity, tissue distribution, and interacting proteins, and suggests calpain may act by degrading tubulin.

Cannabis sativa (Cannabis) is a high-value medicinal crop whose functional genomics and metabolic engineering remain constrained by limited genetic tools. The species is regarded as recalcitrant to stable transformation, and existing transient expression systems exhibit low efficiency or are restricted to hemp cultivars. Here, we report two Agrobacterium-mediated agroinfiltration protocols enabling rapid, robust, and reproducible transient gene expression in drug-type, high-THC Cannabis tissues. Using the RUBY betalain reporter system, we demonstrate transformation efficiencies of up to 100% in mature leaf disks and 81%-100% in whole seedlings. By independently comparing Agrobacterium strains and vector configurations, we demonstrate that transformation efficiency and transgene expression are influenced by distinct experimental variables, with EHA105 providing the highest transformation efficiency and p35S-based vectors producing the strongest reporter expression. Complementing these genetic tools, we developed a UPLC-PDA method enabling simultaneous detection and quantification of cannabinoids and cannflavins. While not intended to replace mass spectrometry-based analyses, the method provides an accessible and cost-effective platform for comparative metabolite profiling during functional genomics and metabolic engineering studies. Together, these advances establish an integrated workflow linking high-efficiency transient gene expression with streamlined biochemical analysis, providing a practical foundation for gene function studies, pathway interrogation, and biotechnology-driven trait engineering in Cannabis.

Recombinant adeno-associated virus (rAAV) vectors are essential for gene therapy, but their production generates empty and partial capsids as product-related impurities that can compromise efficacy and safety. While empty capsids have been partially characterized, the isolation and study of partial capsids remain hindered by a lack of robust separation methods. Here, we present an optimized iodixanol density gradient ultracentrifugation strategy enabling high-resolution separation and enrichment of empty, partial, and full AAV capsids. Key innovations include expanding the 40% iodixanol layer to create a broader intermediate-density zone and implementing controlled bottom-puncture fractionation. Applied to a rAAV2 preparation, this method enriched partial capsids from 18.9% in the starting material to approximately 94.9% in the target fraction (determined by AUC integration under the equal response assumption). For a rAAV2 variant with a low baseline of partial capsids (1.3%), over 20-fold enrichment was achieved, reaching 30.1% purity as determined by AUC integration under the equal response assumption. This approach provides partial-capsid-enriched fractions, enabling detailed characterization of partial capsids, supporting critical quality attribute assessment, and facilitating the development of safer, more effective rAAV-based therapeutics.

Aromatic noncanonical amino acids (aromatic ncAAs) are valuable building blocks for pharmaceuticals, agrochemicals, food additives, and specialty chemicals, yet their sustainable and stereoselective synthesis remains challenging. Ammonia-lyases (ALs) have emerged as powerful biocatalysts for the one-step asymmetric hydroamination of arylacrylic acids, and recent advances in protein engineering have substantially enhanced their catalytic performance including efficiency, selectivity, promiscuity, and productivity. This review integrates catalytic mechanisms with the deployment of engineering strategies to illustrate how aromatic amino acid ammonia-lyases (AALs) have evolved into versatile biocatalysts for aromatic ncAAs synthesis. Particular attention is devoted to the engineering of AALs (including phenylalanine, tyrosine, histidine ammonia lyase) and (methyl)aspartate ammonia lyases, highlighting progress in substrate scope expansion, catalytic promiscuity, selectivity modulation, and hybrid catalytic systems. Current limitations and emerging opportunities are further assessed to delineate future directions for next-generation ALs engineering. The engineering strategies and current states of ALs summarized herein provide a conceptual basis for the efficient development of ALs for sustainable aromatic ncAAs biomanufacturing.

Filamentous microorganisms play a central role in industrial biotechnology, where pellet morphology affects mass transfer, rheology, and process performance. However, time-resolved monitoring of filamentous biomass and pellet size dynamics in stirred-tank reactors remains challenging due to offline sampling limitations. We present a noninvasive in situ imaging approach to observe biomass-related light scattering and projected pellet size. A custom-built camera and ring-light setup with MATLAB-based image acquisition and processing was used to quantify image luminosity as a proxy for biomass content and to determine projected object area (POA) and the number of detected objects per image above a POA threshold of 0.5 mm[2]. To characterize systematic biases, polyoxymethylene spheres of 1-4 mm diameter served as a model system. Luminosity scaled with object concentration, whereas object detection and apparent size estimation were increasingly affected by object size and concentration. In a bench-scale cultivation of malic acid-producing Aspergillus oryzae, image luminosity followed biomass content, while in situ POA analysis revealed increasing projected pellet size coinciding with Zn[2+] limitation. These observations differed from ex situ sampling, indicating that image-based in situ monitoring provides time-resolved information on biomass formation and pellet size dynamics that may not be captured by offline sampling alone.