Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is one of the most widespread infectious diseases, with nearly 2 billion people infected globally. We present an innovative approach for the real-time detection of TB antigens Mpt64 and Ag85B using DNA aptamers in combination with a graphene oxide (GO)-assisted optical microfiber super-sensor. The high surface-to-volume ratio and superior properties of the GO layer significantly enhance the microfiber's fixation capabilities. To validate the clinical applicability of this sensing method, we employed the optical sensor to successfully detect Mpt64 and Ag85B in serum samples within 10 s, achieving limits of detection of 4.23 × 10⁻²⁰ M and 3.11 × 10⁻¹⁹ M, respectively. Due to the high conservation of Mpt64 and Ag85B in human and bovine MTB strains, our detection system can be used to identify MTB in both humans and bovine. These results demonstrate the sensor's high sensitivity for quantifying MTB particles, enabling rapid identification of infected individuals or bovine. Overall, the optical microfiber sensor system offers a promising platform for diagnosing MTB due to its straightforward detection scheme and potential for miniaturization.

The increasing demand for advanced biosystems necessitates innovative approaches to store and process genetic information. DNA, as a high-density storage medium, offers a promising solution for creating genetic memory systems that can provide state-dependent responses to various stimuli. To date, numerous studies have reported on genetic memory systems in living organisms. However, developing modular, orthogonal, and quantifiable in vitro genetic memory systems with scalable biological components remains a significant challenge. In this study, we present an in vitro genetic memory system utilizing three orthogonal serine integrases for DNA-based information storage and processing. By organizing the system into three standardized modules featuring two noncovalent chemical interactions (streptavidin-biotin and parS-ParB), we successfully designed and tested the orthogonality, scalability, and functionalization of these systems. Notably, we expanded the application to implement a cascade biotransformation process converting styrene to (S)-1-phenyl-1,2-ethanediol ((S)-PED) with remarkable efficiency, achieving up to double the transformation rate compared to free-floating purified enzymes. We anticipate that these constructions hold significant potential for advancing artificial memory systems in vitro and will provide a reliable framework for the development of programmable biochemical functions in synthetic biology.

Carotenoids, a class of lipid-soluble isoprenoid pigments, play essential roles in determining coloration and enhancing nutritional value across various food products. Sporobolomyces pararoseus has emerged as a promising microbial platform for industrial-scale biosynthesis of high-value carotenoids, particularly β-carotene, torulene, and torularhodin. The study evaluated the specific impacts of low and high temperatures on carotenoid production in S. pararoseus. Quantitative analysis demonstrated a statistically significant reduction in total carotenoid content across temperature treatments, with values decreasing from 1347.03 μg/gdw under optimal conditions (25°C) to 180.77 μg/gdw at low temperature (12°C) and 1100.13 μg/gdw at high temperature (33°C), representing 86.6% and 18.3% reductions, respectively. The observed reduction in total carotenoid content can be predominantly ascribed to the downregulation of key enzymatic pathways involved in both terpenoid and carotenoid biosynthesis. Conversely, torularhodin production and its relative proportion within the total carotenoid profile were significantly increased under high-temperature conditions. The increase in torularhodin levels may represent an emergency antioxidant response designed to counteract the heightened oxidative stress induced by high temperature. These findings deepen our understanding of how cultural temperatures influence carotenoid levels in S. pararoseus and offer valuable molecular insights for further enhancing its carotenoid synthesis through genetic modifications.

Electrical stimulation (ES) can effectively regulate cell behavior and promote bone tissue regeneration, and conductive biomaterials can further enhance this effect by enhancing the conduction of electrical signals between cells. In this study, poly(lactic-co-glycolic acid) (PLGA) and poly(l-lactide)-aniline pentamer triblock copolymer (PAP) were used as raw materials to prepare a conductive bionic scaffold (PLGA/PAP). Subsequently, bone morphogenetic protein 2 mimetic peptide containing a DOPA tag (DBMP2MP) was loaded on the scaffold surface. The prepared scaffold (DBMP2MP@PLGA/PAP) had a porosity of 79.17% and a porous structure similar to that of natural cancellous bone. After PAP was added, the mechanical strength and electrical conductivity of the scaffold were increased to 2.79 ± 0.1 kPa and 1.29 ± 0.023 × 10 s/cm. The addition of DBMP2MP significantly improved the hydrophilicity of the scaffold material, and the contact Angle of the scaffold material decreased from 102.45 ± 7.67° to 30.36 ± 5.25°. At the same time, DBMP2MP and scaffold surface bonding ability increased by two times compared with commercial BMP2. The polypeptide DBMP2MP can bind to the surface of scaffolds and exhibit long-lasting biological effects. In vitro cell experiments revealed that the DBMP2MP@PLGA/PAP scaffold could significantly promote the proliferation and adhesion of MC3T3-E1 cells and that the combination of DBMP2MP@PLGA/PAP with pulsed ES could further synergistically induce cell mineralization and osteogenic differentiation. The results of the rabbit radius defect experiments revealed that grafting the DBMP2MP@PLGA/PAP scaffold at the defect site significantly promoted the formation of new bone and collagen fibers. When the DBMP2MP@PLGA/PAP scaffold was combined with ES, the regeneration rate of bone tissue further improved, and the newborn collagen tissue is close to normal bone collagen. Therefore, this bionic scaffold with excellent electrical and biological activity shows considerable potential in the field of bone defect repair.

The use of self-healing mineralized hydrogels in 3D printing has demonstrated significant advantages, including enhanced printing accuracy and the ability to maintain high shape fidelity throughout the printing process. After conducting an initial optimization study, we incorporated our self-healing mineralized hydrogel into semi-solid extrusion-based 3D printing to print diclofenac-loaded oral films. The dependence of the print speed on the nature of the material was established by varying the print speed. The process of optimizing the print speed was conducted using a blank hydrogel, which involved analyzing specific parameters, such as printing accuracy and the percentage of pore area under sizing. The results demonstrated that 2 mm/sec print speed showed a higher printing accuracy of 98.13% and pore area under-sizing value of 41.31%. Interestingly, the viscosity of the hydrogel increased from 5.30 to 133 PaS upon addition of the drug. The percentage pore area under sizing also decreased from 41.31% to 11.48% as the drug loading was increased from 0% to 3% w/w. The in vitro drug release study demonstrated that the 3% w/w diclofenac sodium-loaded oral films printed at 2 mm/sec exhibited a faster release profile. Furthermore, considerable bioavailability of diclofenac sodium (DS) was achieved from the 3D-printed oral films during the in vivo study. These results can be effectively used to develop a drug delivery system that can release medications accurately and consistently, either in a targeted area or systemically.

Protein developability is an important, yet often overlooked, aspect of protein discovery campaigns that is a key driver of utility. Recent advances have improved developability screening capacity, making it an increasingly viable option in early-stage discovery. Here, we engineered one component of developability, stability, of two affibody proteins-one that targets death receptor 5 and another that targets tumor necrosis factor receptor 1-previously evolved to bind receptor and non-competitively inhibit signaling via conformational modulation. Starting from an error-prone PCR library of each affibody, variants were screened via yeast surface display binder selections, including depletion of non-specific binders, followed by developability assessment using the on-yeast protease and yeast display level assays. Multiplex deep sequencing identified variants for further evaluation. Purified variants exhibited elevated stability-8°C to 14°C increase in T-with maintained 1-2 nM affinity for the TNFR1 affibody and 30-fold improvement in the DR5 affibody affinity to 0.8 nM.

3-hydroxybutyrate (3-HB), an essential endogenous metabolite, shows significant therapeutic potential in several disease contexts. However, its clinical application has been hampered by limitations, such as adverse effects on the gut microbiota. This study introduces a genetically engineered strain of Lactobacillus rhamnosus GG (LGGK) that integrates the benefits of 3-HB production with the probiotic properties of LGG. Using a murine colon cancer cachexia (CAC) model, LGGK supplementation significantly improved survival, reduced tumor progression, and alleviated muscle wasting. LGGK restored gut microbial diversity, increased the abundance of beneficial bacteria, and increased the production of short-chain fatty acids while reducing harmful microbial populations. In addition, LGGK supplementation demonstrated anti-inflammatory effects, effectively reducing elevated pro-inflammatory cytokines in serum and skeletal muscle. These findings highlight LGGK as a dual-action therapeutic approach that utilizes the metabolic benefits of 3-HB and the gut-modulating properties of LGG. This innovation offers a promising strategy for the treatment of CAC and potentially other metabolic and inflammatory disorders, and highlights the potential of engineered probiotics in advanced therapeutic applications.

Increasing demand for recombinant adeno-associated virus (rAAV)-based gene therapies necessitates increased manufacturing production. Transient transfection of mammalian cells remains the most commonly used method to produce clinical-grade rAAVs due to its ease of implementation. However, transient transfection processes are often characterized by suboptimal yields and low fractions of full-to-total capsids, both of which contribute to the high cost of goods of many rAAV-based gene therapies. Our previously developed mechanistic model for rAAV2/5 production indicated that the inadequate capsid filling is due to a temporal misalignment between viral DNA replication and capsid synthesis within the cells and the repression of later phase capsid formation by Rep proteins. We experimentally validated this prediction and showed that performing multiple, time-separated doses of plasmid increases the production of rAAV. In this study, we use the insights generated by our mechanistic model to develop an intensified process for rAAV production that combines perfusion with high cell density re-transfection. We demonstrate that performing multiple, time-separated doses at high cell density boosts both cell-specific and volumetric productivity and improves plasmid utilization when compared to a single bolus at standard operating conditions. Our results establish a new paradigm for continuously manufacturing rAAV via transient transfection that improves productivity and reduces manufacturing costs.

Miniature CRISPR/Cas systems possess delivery advantages for gene therapy. The type V-F Cas12f1 from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been engineered by several studies as compact genome editing tools through protein and single guide RNA (sgRNA) engineering. However, a comparative evaluation of gene editing and activation efficiencies mediated by different AsCas12f1 variants and sgRNA scaffolds is lacking. This study tested combinations of four AsCas12f1 protein variants and six sgRNA scaffolds for their gene editing and transcription activation efficiencies. The protein variant AsCas12f1-HKRA performed the best in gene editing and activation when paired with sgRNA-en_v2.1 scaffold. Furthermore, we validated a super miniature gene activator by fusing a small activation domain to AsCas12f1-HKRA. Our findings recommend using AsCas12f1-HKRA and sgRNA-en_v2.1 for gene editing and activation applications.

Escherichia coli accumulates acetate as a byproduct in fast growth aerobic conditions when using glucose as carbon source. This phenomenon, known as overflow metabolism, has negative impacts on cell growth and protein expression, also causes carbon loss during biosynthesis in most microbial production scenarios. In this study, we regarded the "waste" metabolite as a useful metabolism indicator, constructed an overflow biosensor to monitor the change of acetate concentration and converted the signal into various regulation outputs. Phloroglucinol is a phenolic compound with several derivatives that exhibit various pharmacological activities. By applying the bifunctional dynamic regulation system on the phloroglucinol production, we released the cellular redox pressure in real-time and reduced the waste of carbon flux on overflow metabolism. Finally, carbon flux was redirected more favorably towards the desired product, resulting in a boosted phloroglucinol titer of 1.30 g/L, increased by 2.04-fold. Overall, this study explored the use of a central byproduct-responsive biosensor system on improving cellular metabolic status, providing a general approach for enhancing bioproduction.

Alternative fermentation feedstocks such as ethanol can be produced from CO via electrocatalytic processes that coproduce O. In this study, industrial-scale fermentation of ethanol with pure O for single cell protein (SCP) production was studied using a modeling approach. This approach considered (i) microbial kinetics, (ii) gas-liquid transfer, and (iii) an exploration of potential operational constraints. The technical feasibility for producing up to 58 kt/y of SCP in a 600 m bubble column operating in continuous mode was assessed and attributed mainly to a high O transfer rate of 1.1 mol/(kg h) through the use of pure O. However, most of the pure O fed to the fermenter remains unconsumed due to the large gas flows needed to maximize mass transfer. In addition, biomass production may be hampered by high dissolved CO concentrations and by large heat production. The model estimates a microbial biomass concentration of 114 g/kg, with a yield on ethanol of 0.61 g/g (> 95% ). Although the large predicted O transfer capacity seems technically feasible, it needs further experimental validation. The model structure allows the analysis of alternative substrates in the same way as identifying the best carbon feedstock.

Gastrodin, the principal bioactive component of the renowned herb Gastrodia elata, is extensively utilized in medicinal drugs and nutraceuticals. This study seeks to enhance microbial production of gastrodin through high-throughput screening (HTS) and transcriptomics-guided optimization. Initially, atmospheric pressure and room temperature plasma (ARTP)-mediated mutagenesis were employed to develop a library of mutant strains. Furthermore, a transcription factor-based biosensor and a high-throughput solid-phase extraction mass spectrometry (HP-SPE-MS) were evaluated to establish an HTS method for gastrodin. Consequently, mutant strain MT8 was isolated, producing 9.8 g/L of gastrodin in YPD medium, which represents a 55.6% increase compared to the control strain. Next, key genes identified via transcriptomics were overexpressed in strain MT8, with the overexpression of gene YALI2E01737g, a gene involved in the synthesis of aromatic amino acids, significantly enhancing gastrodin production to reach 10.1 g/L. In addition, fermentation process optimization further improved gastrodin titer up to 13.1 g/L in shaking flasks. This study demonstrated the utility of HTS techniques to enhance gastrodin production and paved the way for its future industrial application.

Differentiating endothelial cells (ECs) from human pluripotent stem cells (hPSCs) typically takes 2 weeks and requires parameter optimization. Overexpression of cell type-specific transcription factors in hPSCs has shown efficient differentiation into various cell types. ETV2, a crucial transcription factor for endothelial fate, can be overexpressed in hPSCs to induce rapid and facile EC differentiation (iETV2-ECs). We developed a two-stage strategy which involves differentiating inducible ETV2-overexpressing hPSCs in a basal induction medium during stage I and expanding them in an endothelial medium during stage II. By optimizing seeding density and medium composition, we achieved 99% pure CD31+ CD144+ iETV2-ECs without cell sorting in 5 days. iETV2-ECs demonstrated in vitro angiogenesis potential, LDL uptake, and cytokine response. Transcriptomic comparisons revealed similar gene expression profiles between iETV2-ECs and traditionally differentiated ECs. Additionally, iETV2-ECs responded to Wnt signaling agonist and TGFβ inhibitor to acquire brain EC phenotypes, making them a scalable EC source for applications including blood-brain barrier modeling.

Production of therapeutic proteins, antibodies, and virus-like particles (VLP) using baculovirus expression systems (BEVS) has been explored for decades. However, we have realized an urgent need for accelerated production of recombinant proteins and VLPs to address critical situations in recent scenarios. In contrast to BEVSs, the virus-free method is significantly shorter as it bypasses the time-consuming process of infectivity monitoring and virus amplification. Moreover, in the virus-free method, complex steps of protein separation can be eliminated to ease downstream processing. Hence, we present a detailed review of the recent techniques for expressing recombinant proteins, therapeutics, and VLP in insect cells using virus-free methods. First, we focus on the specific methodologies used to optimize virus-free transfection. Here, we provide insight into the interplay between crucial factors, including concentration of transfection reagent, seeding density, and medium temperature. Secondly, we provide a structured review of the novel transfection reagents used for transient and stable transfection. Thirdly, we performed an assessment of the cell lines and plasmids used for virus-free expression and their evaluation based on corresponding protein yield. Finally, we provide the recent advancement in scaling up the transfection process from the shaker flask to the bioreactor level to achieve better yield. Various virus-free expression methodologies presented in this article are essential for evaluating the transfection processes toward improving protein yield. The readers can also use the information to design experiments and optimize process parameters for bioreactor operation.

The widespread consumption of PET worldwide has necessitated the search for environment-friendly methods for PET degradation and recycling. Among these methods, biodegradation stands out as a promising approach for recycling PET. The discovery of duo enzyme system PETase and MHETase in 2016, along with their engineered variants, has demonstrated significant potential in breaking down PET. Previous studies have also demonstrated that the activity of the enzyme PETase increases when it is immobilized on nanoparticles. To achieve highly efficient and complete PET depolymerization, we immobilized both FAST-PETase and MHETase at a specific ratio on magnetic nanoparticles. This immobilization resulted in a 2.5-fold increase in product release compared with free enzymes. Additionally, we achieved reusability and enhanced stability of the enzyme bioconjugates.

d-Glucaric acid is a value-added dicarboxylic acid that can be utilized in the chemical, food, and pharmaceutical industries. Due to the complex process and environmental pollution associated with the chemical production of d-glucaric acid, bioconversion for its synthesis has garnered increasing attention in recent years. In this study, a novel cell factory was developed for the efficient production of d-glucaric acid using d-xylose and methanol. Mdh, Hps, Phi, Miox, Ino1, Suhb, and Udh were first co-expressed in E. coli JM109 to construct the d-glucaric acid synthesis pathway. The deletion of FrmRAB, RpiA, PfkA, and PfkB was then performed to block or weaken the endogenous competitive pathways. Next, adaptive evolution was carried out to improve cell growth and substrate utilization. With the purpose of further increasing the product titer, the NusA tag and myo-inositol biosensor were introduced into engineered E. coli to enhance Miox expression. After medium optimization and fermentation process control, 3.0 g/L of d-glucaric acid was finally obtained in the fed-batch fermentation using modified Terrific Broth medium.

Liver fibrosis is considered as a wound healing process in the presence of chronic hepatic injury. A hydrogel (CPDP) based on chitosan-phenol that undergoes fast gelling and owns self-healing and injectable properties was investigated for the effect on regression of liver fibrosis. For the purpose, we established both in vitro and in vivo liver fibrosis models and implanted CPDP hydrogel into the injured liver. The CPDP hydrogel not only provided a suitable microenvironment for hepatocyte spheroids, but also demonstrated a potential for the hepatocyte spheroid-embedded system to mimic the liver tissue in vitro. Furthermore, the urea synthesis of injured hepatocytes cultured on hepatocyte spheroid-embedded CPDP hydrogel was 1.12 times higher than that on hepatocyte spheroid-embedded collagen hydrogel after 7 days of culture, indicating that CPDP hydrogel effectively rescued hepatic function in the injured hepatocytes. Moreover, the hepatic injury was alleviated with improved hepatic function in the liver fibrosis model in vivo. A reduction of approximately 28% in serum AST/ALT ratios and a 70% decrease in the fibrotic area suggested the regression of liver fibrosis after 2 weeks of CPDP hydrogel administration. These findings suggest that CPDP hydrogel holds promise for applications in liver tissue engineering.

Cancer-associated fibroblasts are increasingly recognized to have a high impact on prostate tumor growth and drug resistance. Here, we bioengineered organotypic prostate cancer 3D in vitro models to better understand tumor-stroma interplay, the metabolomic profile underlying such interactions, and their impact on standard-of-care therapeutics performance. The assembly of robust and uniform spheroids was evaluated and compared in monotypic PC-3 and heterotypic microtumors comprised of either a healthy or malignant stroma and prostate cancer cells. Our findings demonstrate that the precise inclusion of prostate cancer stromal elements is crucial to generating robust PC-3 prostate cancer spheroids with reproducible morphology and size. The inclusion of cancer-associated fibroblasts promoted the establishment of more compact microtumors exhibiting characteristic expression of major proteins. Exometabolomic profile analysis also highlighted the impact of stromal cells on tumor models metabolism. The optimized heterotypic spheroids were additionally exploited for screening standard-of-care therapeutics, exhibiting a higher resistance when compared to their monotypic counterparts. Our findings demonstrate that including stromal elements in PC-3 prostate cancer models is crucial for their use as increasingly organotypic testing platforms, being relevant for screening candidate anti-cancer therapeutics and for the discovery of potential combinations with emerging anti-stroma therapies.

Bayesian optimization is a stochastic, global black-box optimization algorithm. By combining Machine Learning with decision-making, the algorithm can optimally utilize information gained during experimentation to plan further experiments-while balancing exploration and exploitation. Although Design of Experiments has traditionally been the preferred method for optimizing bioprocesses, AI-driven tools have recently drawn increasing attention to Bayesian optimization within bioprocess engineering. This review presents the principles and methodologies of Bayesian optimization and focuses on its application to various stages of bioprocess engineering in upstream and downstream processing.

Industrial-scale microbial fermentation processes often face limitations in mixing and mass transfer, leading to the formation of environmental gradients within the bioreactor. These gradients expose microbes to heterogeneous conditions over time and space. In this study, we evaluated the effects of combined substrate and dissolved oxygen (DO) gradients on the metabolic response of Penicillium chrysogenum at an industrial scale. Three representative heterogeneous environments were simulated in scale-down systems: (1) feed inlet (high glucose, low oxygen (HGLO): C > 20 mM, DO < 0.012 mM), (2) aeration inlet (high oxygen, low glucose (HOLG): C < 0.8 mM, DO > 0.2 mM), and (3) global environment (periodic 360 s fluctuation cycle with 45 s of HGLO and 75 s of HOLG conditions). Results showed that prolonged exposure to feed inlet conditions led to a complete loss of penicillin production capacity, accompanied by significant excretion of intracellular metabolites, and this effect was largely irreversible. While, cells randomly walking under the top impeller zone did not lose production capacity but showed signs of premature degeneration due to increased energy demand. When exposed to the global environment, cells finely tuned their metabolism in a periodical manner, with nearly a 50% loss of penicillin productivity. In summary, substrate gradients alone did not cause irreversible effects, but large substrate gradients contributed to reduced productivity. Oxygen gradients, however, not only reduced production but also caused irreversible cellular damage. These findings provide valuable insights for developing scale-up criteria and strain engineering strategies aimed at improving large-scale culture performance.