Tyrosine sulfation is a post-translational modification that has been reported to occur infrequently on recombinant monoclonal antibodies (mAbs). We recently demonstrated that tyrosine sulfation occurred on a bispecific antibody (bsAb) produced in Chinese hamster ovary (CHO) cells, using a multi-enzymatic approach in combination with intact mass and peptide-based mass spectrometry analysis supplemented with the use of synthetic peptides. Tyrosine sulfation needs to be controlled during the manufacturing process due to potential undesired effects, such as impact on potency and immunogenicity. Here, we report that tyrosine sulfation was not significantly inhibited by the addition of chemical inhibitors, such as sodium chlorate. Individual knockout and double knockout (DKO) of two key genes in the tyrosine sulfation pathway were carried out sequentially. Tyrosyl protein sulfotransferase 1/2 (TPST1/2) DKO by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing eliminated tyrosine sulfation while maintaining cell growth, antibody production, and overall product quality.
S-Adenosylmethionine (SAM) is a high-value biomolecule with critical applications in nutraceuticals, pharmaceuticals, and health supplements. However, the detailed metabolic mechanism by which sodium citrate promotes SAM production in Pichia pastoris has not yet been elucidated. Here, a comprehensive time-series transcriptomic analysis revealed that sodium citrate profoundly influences gene expression across multiple pathways. Sodium citrate supplementation redirects carbon flux toward oxidative energy metabolism by upregulating amino acid biosynthesis, translation, and glycolysis. Crucially, the transcriptional upregulation of argininosuccinate synthase (ARG1) and argininosuccinate lyase (ARG4) was identified as a key node driving fumarate-mediated TCA cycle anaplerosis and ATP supply. This foundational analysis led to the identification of ARG4 as a key metabolic engineering target. Overexpression of ARG4 significantly improved SAM production, achieving a 56.46% increase in shake flasks and 9.06 g/L SAM (a 22.10% improvement) in 500 mL fermenters compared to control strains. Integrated physiological and metabolic flux analysis (MFA) demonstrated that ARG4 overexpression redirects metabolic flow, channeling energy substrates toward SAM synthesis, effectively maintaining cellular respiratory metabolism and alleviating energy limitations in the late fermentation phase. This work establishes a novel strategy for enhancing product synthesis by restructuring energy allocation rather than merely increasing overall energy supply.
The recent spread of human monkeypox (mpox) beyond Africa has attracted global attention. Although mpox is a DNA virus with relatively lower pandemic potential, and smallpox vaccines and therapeutics may offer protection, the distinct clinical features observed in the current outbreak highlight the need for novel antiviral drugs. Here, we developed a drug repurposing workflow based on interspecies protein analysis and advanced virtual screening, integrating pre- and post-docking processes. After screening 6,074 approved or clinical-stage drugs against viral DNA topoisomerase, we expanded the analysis to viral DNA polymerase, increasing the compound library to 11,484 molecules, including approved or clinical-stage drugs and DNA polymerase inhibitors from ChEMBL. To assess dynamic stability, we performed molecular dynamics (MD) simulations and quantified protein-ligand contact occupancy, distinguishing stably bound candidates from transient docking poses. The MD and convolutional neural-network (GNINA) scoring analyses converged on vindesine for DNA topoisomerase and LY2090314 for DNA polymerase. Importantly, the workflow was applied to targets with distinct prior information, including a structurally characterized DNA polymerase supported by homologue inhibitor data and a less-characterized DNA topoisomerase requiring homology modelling. These results illustrate an adaptable repurposing pipeline for prioritizing therapeutic candidates against emerging infectious diseases, although experimental validation remains required.
The use of transposon-based technologies in cell line development has gained significant traction over the past decade. This study investigates the piggyBac transposon system combined with bicistronic vectors for monoclonal antibody expression, helping address the complex demands of multi-specific antibody formats that require multiple cistrons. We systematically evaluated bicistronic vector modifications within the piggyBac framework, including promoter sequences, reporter placements, and cassette configurations, and compared performance to piggyBac/single-gene vector co-transfection methods. Our findings demonstrate that double human CMV promoter configurations driving both heavy and light chain genes significantly enhanced pool productivity (2 to 2.5-fold) and reporter expression compared to separate promoter designs. Among tested cassette arrangements, the Light chain-GS-Heavy chain configuration yielded optimal productivity (1.5 to 6-fold) and superior heavy chain/light chain RNA transcript ratios (1.4 to 3.8-fold and 1.3 to 6.1-fold, respectively). While unfed batch conditions showed comparable productivities between optimized piggyBac/bicistronic and piggyBac/single-gene systems, the bicistronic approach exhibited superior productivity performance under fed-batch conditions (1.5-fold), with the resulting clones demonstrating significantly high productivity (top clone 9.6 g/L). This represents the first reported use of LC-selection marker-HC topology for monoclonal antibody expression, establishing a foundation for improved therapeutic biologic production through multicistronic vector systems combined with the piggyBac transposon strategy.
The separation of empty and full adeno-associated viruses (AAVs) is particularly critical because of the similarity between the two. Anion-exchange chromatography represents a scalable tool, leveraging their slight difference in isoelectric point. However, traditional resin chromatography can only deliver poor resolution, mainly due to the restricted accessibility of the capisds to the bead pores. To cover this gap, in this work, we designed and optimized the separation of empty and full AAVs using anion-exchange membrane chromatography. The role played by flowrate, gradient slope and wash step parameters on the chromatographic resolution was first investigated with two consecutive designs of experiments (DoE). The response surfaces indicated that high process flowrates, that is 15 membrane volumes (MVs)/min, with a gradient from 115 mM to 250 mM sodium acetate in 350 MVs maximize the chromatographic resolution. Then, preparative experiments showed that fast loading is possible, with residence time as short as 24 s, without product breakthrough. Moreover, membrane loading up to 1.2E17 viral particles (vp)/Lmembrane could be reached, providing one order of magnitude higher productivity with respect to standard column operations. With these results, we demonstrated the great potential of anion-exchange membrane chromatography in the polishing of AAVs, overcoming traditional limitations of packed-bed operations.
Capsanthin is a high-value tetraterpenoid widely utilized in the food, cosmetic, and pharmaceutical industries. However, its traditional production via plant extraction and chemical synthesis faces severe sustainability and cost constraints. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) offers a superior chassis due to its robust mevalonate pathway and abundant lipid droplets for pigment sequestration. In this study, we systematically engineered Y. lipolytica for the de novo biosynthesis of capsanthin. By heterologously co-expressing of Capsicum annuum capsanthin/capsorubin synthase (CaCCS) and Arabidopsis thaliana zeaxanthin epoxidase (AtZEP), we achieved the first reported biosynthesis of capsanthin in Y. lipolytica. Combinatorial screening revealed that full-length sequences of CaCCS and AtZEP yielded optimal production of 6.92 mg/L. To further enhance the productivity of capsanthin, we evaluated spatial compartmentalization strategies. The results demonstrated that targeting the biosynthetic enzymes to the peroxisome via Ser-Lys-Leu (SKL) tags successfully increased the titer to 8.8 mg/L by leveraging its specialized lipophilic and redox microenvironment. Finally, a 5-L fed-batch fermentation was conducted, achieving a maximum capsanthin titer of 48.15 mg/L. This work provides a sustainable and scalable alternative to plant extraction and chemical synthesis for capsanthin production.
Retinol, a derivative of vitamin A with potent antioxidant and therapeutic properties, is in high market demand. In response to the low productivity of conventional methods, metabolic engineering has been explored for microbial retinol production. However, systematic engineering strategies for high-level retinol synthesis in Komagataella phaffii remain limited. In this study, the methylotrophic yeast K. phaffii was developed as an engineered chassis for efficient de novo retinol biosynthesis. Based on a previously constructed β-carotene-producing strain, β-carotene-15,15'-dioxygenase (Blh) and retinol dehydrogenase (RDH12) were screened and introduced to establish the synthetic pathway of retinol. To increase precursor supply, key genes in the β-carotene biosynthetic pathway were overexpressed. The mevalonate (MVA) pathway was further optimized, and central carbon metabolism was reprogrammed to enhance metabolic flux toward retinol. Transport engineering was also performed to improve retinol secretion. Several candidate transporters were overexpressed, and the protein encoded by chr1-4_0619 in K. phaffii was identified as an endogenous retinol transporter. The final engineered strain produced 3.38 g/L retinol with BHT supplementation in fed-batch fermentation using a 1.5 L bioreactor. This work represents de novo microbial synthesis of retinol from a one-carbon feedstock, demonstrating the formidable potential of K. phaffii as a sustainable chassis for retinol production.
The evolution of SARS-CoV-2 necessitates next-generation vaccine strategies targeting conserved viral elements to ensure broad and durable protection. In this study, we designed and evaluated novel mRNA-based vaccines encoding conserved domains of SARS-CoV-2, including the spike S2 subunit, NSP3 Macrodomain I (Mac I), and papain-like protease (PLpro). These mRNAs were synthesized, cloned, and encapsulated into lipid nanoparticles (LNPs) with 70%-80% efficiency and favorable physicochemical properties. In vitro characterization showed efficient antigen expression with 60%-80% cell survival. Immunization of BALB/c mice induced strong humoral responses, particularly against S2 and Mac I, marked by IgG titers, class switching, and durable antibody presence up to 84 days post-booster. PLpro elicited minimal antibody responses, likely due to limited stability or intrinsic low immunogenicity. Combinatorial LNPs encoding multiple antigens maintained robust expression and elicited comparable immune responses despite dose splitting. Anti-S2 antibodies effectively inhibited spike protein uptake in vitro. Importantly, the selected antigenic regions exhibited high sequence conservation across major SARS-CoV-2 variants, underscoring their broad-spectrum vaccine potential. These findings support further development of domain-targeted, multi-antigen mRNA vaccines to enhance cross-variant protection and T-cell-mediated immunity.
Polyphenols are bioactive compounds used in food, beverages, cosmetics, and medicine. Despite their expanding use, the mechanisms of polyphenol toxicity in microbial polyphenol producers and the adaptive strategies that confer tolerance, remain poorly characterized. Here, we applied adaptive laboratory evolution to evolve tolerance to three structurally distinct polyphenols, curcumin, naringenin, and resveratrol in Yarrowia lipolytica. Whole-genome resequencing of tolerant strains revealed recurrent loss-of-function mutations in the morphogenetic transcription factor MHY1 (YALI1_B28150g) and large segmental duplications on chromosome E, indicating multiple evolutionary solutions. Notably, resveratrol-evolved strains showed strong tolerance in the absence of MHY1 mutations, instead relying on chromosome-scale copy-number variation, highlighting compound-specific adaptive routes. Functional reconstruction demonstrated that MHY1 inactivation is sufficient to confer high-level tolerance, as both gene deletion (ΔMHY1) and independent point mutations (F240L, C153*) reproduce the tolerant phenotype across polyphenols. Transcriptomic analysis showed that MHY1 inactivation is associated with a distinct regulatory state characterized by reduced transcriptional representation of stress-associated pathways and relative stabilization of core metabolic functions. Together, these results identify MHY1 loss-of-function as a dominant and transferable regulatory mechanism for polyphenol tolerance, while also revealing alternative genome-level adaptations selected under severe chemical stress.
The rapid expansion of the global seafood processing industry has generated substantial quantities of shrimp shells and other chitin-rich waste. Recombinant chitinases provide a promising strategy for converting this biomass into high-value chitin oligosaccharides. In this study, we systematically optimized the secretory expression of the GH18 exochitinase CtChi70 in Komagataella phaffii (formerly Pichia pastoris) and examined its applicability for the enzymatic conversion of shrimp shell-derived chitin into N,N'-diacetyl-chitobiose ((GlcNAc)2). Through signal peptide optimization, gene copy number amplification, and HAC1 overexpression, extracellular chitinase activity in shake-flask cultures reached 181 U/L. Scale-up using high-cell-density fed-batch fermentation in a 7-L bioreactor further increased activity to 2151 U/L. A mild chitin extraction process was established using citric acid demineralization and low-concentration NaOH deproteinization, yielding a suitable substrate for enzymatic hydrolysis. The resulting chitin was successfully converted into high-purity (GlcNAc)2 within 24 h. Collectively, this work establishes an efficient expression strategy for CtChi70 production and demonstrates an integrated, environmentally sustainable approach for shrimp shell valorization.
Periodontitis is a chronic inflammatory disease characterized by irreversible destruction of alveolar bone, periodontal ligament attachment, and supporting tooth structures. Emerging evidence suggests that periodontal tissue breakdown and regenerative failure are primarily driven by dysregulation of the local immune microenvironment rather than by direct bacterial insult. Therefore, conventional therapies focused primarily on microbial control are insufficient to restore immune homeostasis and functional regeneration of the cementum-periodontal ligament-alveolar bone complex. Effective periodontal regeneration requires coordinated infection management, immune modulation, oxidative stress clearance, and inflammatory microenvironment reprogramming to relieve the suppression of regenerative cells, particularly periodontal ligament stem cells. This review summarizes key regulatory networks of the periodontal immune microenvironment, with emphasis on macrophage polarization, neutrophil heterogeneity, and the imbalance between Th17 cells and regulatory T cells. We further discuss recent immune-engineering strategies for restoring periodontal homeostasis, including surface-modified biomaterials, ion-delivery systems, stem cell-derived extracellular vesicles, gene-editing technologies, and smart responsive therapeutic scaffolds. Advances in single-cell and spatial transcriptomics have revealed previously unrecognized functional subpopulations, such as NLRP3+ macrophages and SAA1+ fibroblasts, offering new opportunities for precision immunomodulation. Collectively, this review provides an integrated and translational framework for leveraging immune microenvironment-based interventions to achieve functional and predictable periodontal regeneration.
Single gene encoded phage lysis proteins offer a promising strategy for bacterial ghost production, yet their host-dependent regulatory mechanisms remain poorly understood. Here, we investigated the lysis protein E from phage ID52 (ID52-E), which exhibits stronger lytic activity than φX174 E. By screening ID52-E-resistant mutants, we identified a four-base insertion in secB as the genetic alteration associated with lysis resistance in BL21, and CRISPR-Cas9-mediated secB disruption confirmed that SecB is required for ID52-E-mediated lysis. Proteomic analysis revealed altered protein expression in resistant mutants despite preserved bacterial morphology. Co-immunoprecipitation and biolayer interferometry supported an apparent interaction between SecB and ID52-E, with an apparent KD of 3.541 × 10- 8 M under the tested 1:1 fitting model. Molecular docking, molecular dynamics simulations, mutagenesis, lysis assays, and binding measurements further implicated SecB Ala145 as a key interface residue. Together, these findings identify SecB as a host factor that facilitates ID52-E-mediated bacterial lysis and provide mechanistic insight for improving bacterial ghost production.
Sclareol is a highly valued bicyclic diterpene widely used as a precursor for the fragrance ambroxide and has also attracted interest as a bioactive natural product. Traditional production relies heavily on plant extraction, primarily from Salvia sclarea, an approach constrained by environmental variability, long cultivation cycles, and costly downstream purification. To overcome these supply bottlenecks, synthetic biology-enabled biomanufacturing has emerged as a sustainable and scalable alternative. Recent advances in synthetic biology and metabolic engineering have enabled the elucidation and reconstruction of the sclareol biosynthetic pathway in heterologous hosts. This review summarizes current knowledge of sclareol biosynthesis, highlights representative engineering strategies for its production in microbial cell factories, while briefly introducing emerging photosynthetic and plant-based platforms as complementary green production systems. Finally, we outline the current challenges and future perspectives for the industrial biomanufacturing of sclareol and other high-value terpenoids.
Chitosan, a derivative of the abundant biopolymer chitin, holds significant biotechnological potential but is limited by its poor solubility. Its oligomeric forms, chitooligosaccharides (COSs), exhibit superior bioactivity and solubility, driving demand for efficient enzymatic production methods. This study reports the biochemical and functional characterization of a novel chitosanase, SlCsn46A, identified from Streptomyces lydicus M01 and classified into the glycoside hydrolase family 46 (GH46). The enzyme was successfully heterologously expressed in Escherichia coli and purified. SlCsn46A demonstrated high catalytic efficiency, with an optimal activity at 60°C and pH 6.0 and a maximum specific activity of 957.80 U·mg- 1. It exhibited broad pH stability and significantly enhanced activity in the presence of Mn2 + and Tween 80. Kinetic analysis revealed a low Michaelis constant (Km = 0.61 mg·mL- 1), indicating strong substrate affinity. Product analysis confirmed its endo-type action mode, specifically hydrolyzing chitosan to yield chitobiose [(GlcN)2] and chitotriose [(GlcN)3] as the predominant end products. These properties collectively establish SlCsn46A as an efficient and specific biocatalyst, demonstrating great potential for the targeted and efficient synthesis of low-degree-of-polymerization COS for applications in the food, agricultural, and biomedical industries.
Trichoderma reesei (T. reesei), a filamentous fungus, has emerged as a pivotal organism in the realm of cellulase production, garnering significant attention from researchers due to its exceptional cellulolytic properties and the potential for genetic manipulation to enhance enzyme yields. Cellulases, a group of enzymes that catalyze the hydrolysis of cellulose into glucose, hold immense importance in various biotechnological applications, including biofuel production, waste management, and the textile industry. As a model organism, T. reesei offers a unique platform for studying the mechanisms underlying cellulase production, which encompasses genetic regulation, enzymatic pathways, and the influence of environmental factors such as substrate type, light, and metal ions. Multi-omics analyses have elucidated novel aspects of cellulase production mechanisms, and concurrent advances in genetic engineering provide new strategies for optimizing enzyme synthesis in T. reesei. Despite the progress made, several unresolved questions remain regarding the regulatory mechanisms of cellulase genes, highlighting the need for future research to explore these gaps and further enhance our understanding of cellulase production. This literature review aims to synthesize current knowledge on the mechanisms of cellulase production in T. reesei, discuss recent advancements, and outline future research opportunities that could lead to significant breakthroughs in this field.
The immunomodulatory and regenerative effects of mesenchymal stem cell (MSC) extracellular vesicles (EVs) have spurred the development of strategies to manufacture these EVs as therapeutics. Clinical-grade EV manufacturing requires defined serum-free media (SFM) to reduce heterogeneity and improve batch reproducibility, yet standard protocols for collecting EVs from SFM are lacking. A primary concern is the co-isolation of SFM-derived proteins with EVs during separation, which can interfere with the characterization of the collected EVs, and their subsequent application. In this study, we evaluated how removing proteins from SFM affects EV yield and purity, and examined how collection timing and cell confluence influence EV yield and proteomic profile. A defined SFM (PPRF-msc6) was compared to (i) an ultracentrifuged medium (PPRF-msc6 after overnight ultracentrifugation), and (ii) a starvation medium (PPRF-msc6 without albumin and fetuin). MSC growth and viability were reduced in starvation medium, but were not adversely impacted when using ultracentrifuged medium. Compared to unmodified PPRF-msc6, ultracentrifuged medium improved EV purity but lowered EV yield. MSC confluence impacted the proteomic profile of EV fractions, demonstrating the importance of determining when EVs are collected during the culture period. Defining protocols for MSC-EV collection contributes to standardization within the rapidly growing EV field, and informs the development of clinically relevant bioprocesses.
817 DNAzyme has emerged as a potent catalytic nucleic acid tool for gene expression silencing, offering distinct advantages including programmable target recognition, enzymatic turnover capability, and high biostability. Despite its therapeutic potential, clinical applications of this metalloenzyme have been constrained by suboptimal catalytic performance under physiological conditions, which is primarily attributed to insufficient intracellular Mg 2+ concentrations (typically <1 mM). To address this critical limitation, we have developed an atomic probing approach through systematic nucleotide modifications at key catalytic residues, which has successfully reduced the Mg 2+ dependency by 50%. The optimized DNAzyme has largely enhanced the RNA cleavage (by 1.7 fold) at physiological Mg 2+ (0.5 mM), offering significantly higher gene silencing in 293T cells, compared to the wild‐type. By enabling efficient gene silencing in native biological environments, this novel advancement in metalloenzyme engineering and DNAzyme catalysis has established a chemical approach to enhance the DNAzyme activity under physiological Mg 2+ conditions, which is a critical prerequisite for future therapeutic applications and biotech developments.
Echinocandin B (ECB), a fungal non-ribosomal lipopeptide, serves as the exclusive natural precursor of the front-line antifungal anidulafungin. Despite its clinical importance, ECB production remains suboptimal due to incomplete understanding of its biosynthesis mechanism. The global regulator LaeA has been implicated in secondary metabolite production, yet its specific role in ECB biosynthesis remains unexplored. To address this, we successfully constructed laeA deletion and overexpression strains, and demonstrated that LaeA functionally couples morphological development with ECB productivity. Transcriptomic analysis revealed LaeA directly activated the sterigmatocystin cluster via pathway-specific regulator AflR, but indirectly influenced ECB through iron-heme cofactor synchronization rather than direct gene cluster activation. LaeA overexpression upregulated siderophore iron transporters and heme biosynthetic genes, with supplementation of Fe2 + or 5-ALA further boosting titers to 2487 ± 123 and 2697 ± 16 mg/L, respectively. To overcome P450s catalytic constraints, we screened out a novel cytochrome P450 reductase CPR2 as the optimal redox partner. Co-expression of CPR2 with bacterial hemoglobin VHb achieved synergistic enhancement, improving the ECB titer to 3170 ± 41 mg/L. This study provided a practical strategy for improving ECB production and offering insights into the versatile regulatory modes of global secondary metabolite regulators.
Female infertility, which affects millions of couples globally, has been a highly focused research field owing to the importance of reproduction in humans. Emerging bioengineering technologies, including tissue engineering, microfluidic chips, imaging techniques, personalized medicine, gene editing tools, and artificial intelligence, have the potential to revolutionize the existing assisted reproductive technology. These technologies have enabled creating artificial biomimetic systems for the culture of oocytes and embryos; changed the way they develop; and enhanced their competence evaluation in an automatic manner. However, the implementation and potential integration of these technologies have been a long-entrenched challenge due to the lack of standardized protocols and precise control over reproductive cycles. This review article summarizes recent advances in these innovative approaches, with an emphasis on tissue engineering and microfluidic technology. Their convergence is discussed as a potential pathway toward more integrated, precise, and personalized reproductive systems for next-generation assisted reproductive technology. In this context, key challenges related to ethics, standardization, cross-technology integration, and clinical translation are further discussed.
