Murideoxycholic acid (MDCA), as a significant secondary bile acid derived from the metabolism of α/β-muricholic acid in rodents, is an important component in maintaining the bile acid homeostasis. However, the biosynthesis of MDCA remains a challenging task. Here, we present the development of cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, employing semi-rational protein engineering technique. Following three rounds of mutagenesis, a triple variant (T260G/G328A/L82V) has been discovered that proficiently catalyzes the 6β-hydroxylation of lithocholic acid (LCA), thereby generating MDCA with an impressive 8.5-fold increase in yield compared to the template P450 BM3 mutant. The MDCA selectivity has been also promoted from 62.0% to 96.3%. This biocatalyst introduces a novel approach for the biosynthesis of MDCA from LCA. Furthermore, molecular docking and dynamics simulations have been employed to unravel the molecular mechanisms underlying the enhanced LCA conversion and MDCA selectivity.

L-asparaginases (EC 3.5.1.1) are amidohydrolase enzymes that predominantly catalyze conversion of L-asparagine to L-aspartic acid and ammonia. In addition, some exhibit secondary L-glutaminase activity. Escherichia coli and Erwinia chrysanthemi L-asparaginases are widely used in the pharmaceutical industry to produce therapeutically important compounds. In the therapeutic use of enzymes, bacterial L-asparaginases can trigger immune responses, leading to a high rate of adverse effects that diminish the effectiveness of the treatment. This situation has forced scientists to search for promising L-asparaginases from new sources. Yeast L-asparaginases could be useful in reducing toxicity and enhancing efficacy but they have been poorly studied to date. Here, we characterized the yeast Lachancea thermotolerans L-asparaginase (LtASNase) purified by affinity chromatography. It has a specific activity of 313.8 U/mg and a high k value (312.4 s). We demonstrated through a semi-rational design that the mutations of Lys99 show varying effects on catalytic activity, with the Lys99Ala mutant increasing specific activity 3.3-fold. Furthermore, the in vitro antileukemic activity of the non-formulated form of Lys99Ala LtASNase was evaluated against SUP-B15 and REH cell lines. The results demonstrated that LtASNase exhibits significant antileukemic potential, comparable to commercial type II bacterial enzymes. The understanding of the mutant L-asparaginases examined in this study will significantly contribute to the development of new and more effective yeast-derived asparaginases.

Amino acids, including asparagine, aspartate, glutamine, and glutamate, play important roles in purine and pyrimidine biosynthesis as well as serve as anaplerotic sources fueling the tricarboxylic acid (TCA) cycle for mitochondrial energy generation. Despite extensive studies on glutamine and glutamate in CHO cell cultures, the roles of asparagine and aspartate, especially in feed media, remain underexplored. In this study, we utilized a CHO genome scale model to first deeply characterize the intracellular metabolic states of CHO cells cultured in different combinations of basal and feed media to understand the traits of asparagine/aspartate-dependent and glutamate-dependent feeds. Subsequently, we identified the critical role of asparagine and aspartate in the feed media as anaplerotic sources and conducted in silico simulations to ascertain their optimal ratios to improve cell culture performance. Finally, based on the model simulations, we reformulated the feed media by tailoring the concentrations of asparagine and aspartate. Our experimental data reveal a CHO cell preference for asparagine compared with aspartate, and thus maintaining an optimal ratio of these amino acids is a key factor for achieving optimal CHO cell culture performance in biopharmaceutical production.

Boldenone (BD), a protein anabolic hormone, is commonly used to treat muscle damage, osteoporosis, and off-season muscle building in athletes. Traditional BD synthesis methods rely on chemical processes, which are costly and environmentally impactful. Therefore, developing a more sustainable and economical biosynthetic pathway is crucial for BD production. This study aimed to achieve efficient production of BD. Firstly, the catalytic performance of 17β-hydroxysteroid dehydrogenase and 3-ketosteroid-Δ-dehydrogenase was improved through enzyme engineering, and their expression in the new strain of Mycobacterium neoaurum was enhanced using metabolic engineering. These improvements significantly increased BD production to 4.05 g/L, with a significant decrease in by-product generation. To further increase the yield, a multi-enzyme fusion expression system was constructed, and a key cell wall gene kasB was knocked out, resulting in a spatial-time yield of BD reaching 1.02 g/(L·d). Subsequent optimization of the transformation system further increased the BD production to 5.56 g/L, with a spatiotemporal yield of 1.39 g/(L·d). The green biosynthetic route of phytosterol one-step conversion to BD developed in this study lays the foundation for industrial production.

A promising approach to treat colorectal cancer (CRC) involves combining chemotherapy, epigenetics, and gene therapy to combat drug resistance. Multifunctional nanocarriers have emerged as a valuable tool for targeted CRC therapy. By delivering multiple treatments directly to cancer cells, these nanocarriers offer the potential for improved outcomes and reduced side effects. PAMAM-based dendrimers were functionalized with a unique combination of folic acid, 5-FU, SAHA, and plasmid DNA pCIneoGFP for targeted delivery to CRC cells. Biophysical characterizations of therapeutic loaded dendrimers and their complexes with pCIneoGFP were performed by: dynamic light scattering, fluorescence spectroscopy, and gel electrophoresis. Further, cellular analyses of dendriplexes demonstrated high transfection efficiency and anticancer activity on HCT 116 and HT-29 cell lines. We have successfully developed a multifunctional nanocarrier platform based on PAMAM dendrimers, offering a promising tool for targeted combination therapy of CRC.

Saccharomyces boulardii, as a probiotic yeast, has shown great potential in regulating gut health and treating gastrointestinal diseases. Due to its unique antimicrobial and immune-regulating functions, it has become a significant subject of research in the field of probiotics. In this study, we aim to enhance the antioxidant properties of S. boulardii by producing l-ergothioneine (EGT). We first constructed a double knockout of ura3 and trp1 gene in S. boulardii to facilitate plasmid-based expressions. To further enable effective genome editing of S. boulardii, we implemented the PiggyBac system to transpose the heterologous gene expression cassettes into the chromosomes of S. boulardii. By using enhanced green fluorescent protein (EGFP) as the reporter gene, we achieved random chromosomal integration of EGFP expression cassette. By using PiggyBac transposon system, a great variety of EGT-producing strains was obtained, which is not possible for the conventional single target genome editing, and one best isolated top producer reached 17.50 mg/L EGT after 120 h cultivation. In summary, we have applied the PiggyBac transposon system to S. boulardii for the first time for genetic engineering. The engineered probiotic yeast S. boulardii has been endowed with new antioxidant properties and produces EGT. It has potential applications in developing novel therapeutics and dietary supplements for the prevention and treatment of gastrointestinal disorders.

Existing low pH viral inactivation methods for continuous downstream processing of biologics typically rely on predictive models to estimate the necessary pH adjustments. However, these methods are of limited use during the process development stage due to the dynamic nature of capture chromatography, where batch variations can alter the eluted protein titer. This study introduces an inline viral inactivation system (IVIS) that utilizes real-time adaptive control and inline sensor readings to precisely regulate the pH manipulation for inline acidification and continuous viral inactivation. The IVIS, which includes a coiled flow inversion reactor (CFIR), is integrated with a multicolumn capture chromatography system to demonstrate a fully continuous process from protein capture chromatography to inline pH manipulation. The system achieved precise inline pH manipulation within ±0.15 and a narrow residence time distribution of 13.5 min with a relative width of 0.7. The introduction of real-time inline pH manipulation with the IVIS signifies a notable advancement in managing critical process parameters (CPPs) and ensuring consistent product quality across varied production environments for continuous downstream bioprocessing.

Following the recent COVID-19 pandemic, mRNA manufacturing processes are being actively developed and optimized to produce the next generation of mRNA vaccines and therapeutics. Herein, the performance of the tangential flow filtration (TFF) was evaluated for high-recovery, and high-purity separation of mRNA from unreacted nucleoside triphosphates (NTPs) from the in vitro transcription (IVT) reaction mixture. For the first time, the fouling model was successfully validated with TFF experimental data to describe the adsorption of mRNA on filtration membrane. The fouling model enables monitoring of the mRNA purification processes, designing an appropriate strategy for filter clean-up, replacing the column at the right time and reducing the process cost. Recovery greater than 70% mRNA without degradation was obtained by implementing a capacity load of ∼19 g/m, <2.5 psi transmembrane pressure (TMP) and feed flux of 300 LMH. This approach also enables the purification of multiple mRNA drug substance sequences for the treatment of a wide range of different diseases.

Over the past decade, engineered producer cell lines have led 10-fold increases in antibody yield, based on an improved understanding of the cellular machinery influencing cell health and protein production. With prospects for further production improvements, increased antibody production would enable a significant cost reduction for life-saving therapies. In this study, we strategized methods to increase cell viability and the resulting cell culture duration to improve production lifetimes. By overexpressing the cell surface adenosine A receptor (AR), the Akt pathway was activated, resulting in improved cellular proliferation. Alternatively, by inducing autophagy through temperature downshift, we were able to significantly enhance cellular-specific productivity, with up to a three-fold increase in total antibody production as well as three-fold higher cell-specific productivity. Interestingly, the expression levels of the autophagy pathway protein Beclin-1 appeared to correlate best with the total antibody production, of autophagy-related proteins examined. Thus, during cell clonal development Beclin-1 levels may serve as a marker to screen for conditions that optimize antibody titer.

Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.

In light-based 3D-bioprinting, gelatin methacrylate (GelMA) is one of the most widely used materials, as it supports cell attachment, and shows good biocompatibility and degradability in vivo. However, as an animal-derived material, it also causes safety concerns when used in medical applications. Gelatin is a partial hydrolysate of collagen, containing high amounts of hydroxyproline. This causes the material to form a thermally induced gel at ambient temperatures, a behavior also observed in GelMA. This temperature-dependent gelation requires precise temperature control during the bioprinting process to prevent the gelation of the material. To avoid safety concerns associated with animal-derived materials and reduce potential issues caused by thermal gelation, a recombinant human alpha-1 collagen I fragment was expressed in Komagataella phaffii without hydroxylation. The resulting protein was successfully modified with methacryloyl groups and underwent rapid photopolymerization upon ultraviolet light exposure. The developed material exhibited slightly slower polymerization and lower storage modulus compared to GelMA, while it showed higher stretchability. However, unlike the latter, the material did not undergo physical gelation at ambient temperatures, but only when cooled down to below 10°C, a characteristic that has not been described for comparable materials so far. This gelation was not caused by the formation of triple-helical structures, as shown by the absence of the characteristic peak at 220 nm in CD spectra. Moreover, the developed recombinant material facilitated cell adherence with high cell viability after crosslinking via light to a 3D structure. Furthermore, desired geometries could be easily printed on a stereolithographic bioprinter.

Oligoclonal antibodies, which are carefully defined mixtures of monoclonal antibodies, are valuable for the treatment of complex diseases, such as infectionss and cancer. In addition to these areas of medicine, they could be utilized for the treatment of snakebite envenoming, where recombinantly produced monoclonal human antibodies could overcome many of the drawbacks accompanying traditional antivenoms. However, producing multiple individual batches of monoclonal antibodies in an industrial setting is associated with significant costs. Therefore, it is attractive to produce oligoclonal antibodies by mixing multiple antibody-producing cell lines in a single batch to have only one upstream and downstream process. In this study, we selected four antibodies that target different toxins found in the venoms of various elapid snake species, such as mambas and cobras, and generated stable antibody-producing cell lines. Upon co-cultivation, we found the cell line ratios to be stable over 7 days. The purified oligoclonal antibody cocktail contained the anticipated antibody concentrations and bound to the target toxins as expected. These results thus provide a proof of concept for the strategy of mixing multiple cell lines in a single batch to manufacture tailored antivenoms recombinantly, which could be utilized for the treatment of snakebite envenoming and in other fields where oligoclonal antibody mixtures could find utility.

The performance of industrial strains has gradually improved with the rapid development of synthetic biotechnology. The production efficiency of traditional batch and fed-batch culture is limited and product quality varies since both are dynamic processes, whereas multi-stage continuous culture can maximise the production efficiency of specific fermentation processes and achieve consistent product quality. However, each single-stage fermentation under multi-stage continuous fermentation requires accurate steady-state control, and a model with adequate accuracy is required for designing and controlling a multi-stage continuous fermentation process. At present, there are few reports on kinetic models for the control of multi-stage continuous fermentation. In this work, we constructed a hybrid model for Saccharomyces cerevisiae multi-stage continuous culture, taking both oxygen limitation and Crabtree effect. The accuracy of the model was ∼80%, the advantages and limitations of the model are discussed and a potential improvement strategy is proposed.

Glucose sensors are essential tools for monitoring blood glucose concentration in diabetic patients. In recent years, with the increasing number of individuals suffering from diabetes, blood glucose monitoring has become extremely necessary, which expedites the iteration and upgrade of glucose sensors greatly. Currently, two main types of glucose sensors are available for blood glucose testing: enzyme-based glucose sensor (EBGS) and enzyme-free glucose sensor (EFGS). For EBGS, several progresses have been made to comprehensively improve detection performance, ranging from enhancing enzyme activity, thermostability, and electron transfer properties, to introducing new materials with superior properties. For EFGS, more and more new metallic materials and their oxides are being applied to further optimize its blood glucose monitoring. Here the latest progress of electrochemical glucose sensors, their manufacturing methods, electrode materials, electrochemical parameters, and applications were summarized, the development glucose sensors with various noninvasive sampling modes were also compared.

Evaluating the Gibbs-Donnan and volume exclusion effects during protein ultrafiltration and diafiltration (UF/DF) is crucial in biopharmaceutical process development to precisely control the concentration of the drug substance in the final formulation. Understanding the interactions between formulation excipients and proteins under these conditions requires a domain-specific knowledge of molecular-level phenomena. This study developed gradient boosted tree models to predict the Gibbs-Donnan and volume exclusion effects for amino acids and therapeutic monoclonal antibodies using simple molecular descriptors. The models' predictions were interpreted by information gain and Shapley additive explanation (SHAP) values to understand the modes of action of the antibodies and excipients and to validate the models. The results translated feature effects in machine learning to real-world molecular interactions, which were cross-referenced with existing scientific literature for verification. The models were validated in pilot-scale manufacturing runs of two antibody products requiring high levels of concentration. By only requiring a molecule's biophysicochemical descriptors and process conditions, the proposed models provide an in silico alternative to conventional UF/DF experiments to accelerate process development and boost process understanding of the underlying molecular mechanisms through rational interpretation of the models.

Pichia pastoris possesses the unique ability to utilize methanol as its sole carbon source, which makes it a proper host for producing various high-value-added products via metabolic engineering. Nevertheless, cell death has been observed during the fermentation of modified P. pastoris, with limited literature elucidating the underlying causes and mechanisms. Understanding the death mechanisms during methanol-based fermentation is crucial for optimizing fermentation strategies, enhancing the accumulation of target products, and reducing production costs. Here, we first sought to eliminate the potential causes of cell death during fermentation, such as inadequate inorganic salts and toxic by-product accumulation. The elimination of these potential causes was achieved efficiently utilizing the high-throughput fermentation equipment. Subsequently, we established a correlation between yeast cell death and the duration of the methanol metabolism period by monitoring the growth of the yeast at different fermentation stages. A critical revelation from this work came from analyzing the yeast's transcriptomic data at various stages of methanol metabolism. It was observed that a significant characteristic of yeast cell death during fermentation was the marked down-regulation of transcript levels of key enzymes involved in the methanol assimilation pathway and genes related to their biosynthesis process. The findings of this work are crucial for better understanding the causes and mechanisms of cell death for engineered P. pastoris during methanol-utilized fermentation.

A new method has been developed to improve the detection of norovirus (NoV) in complex fecal samples using nanocatalyst-based immunoassays. The method involves using multifunctional trimetallic nanoparticles, known as Ag@FeO@Au NPs. These nanoparticles consist of a core of silver (Ag) and a shell of iron oxide (FeO) decorated with isolated gold nanoparticles (Au NPs). The nanoparticles have enhanced catalytic activity, making them an ideal nanocatalyst for reducing 4-nitrophenol (4-NP, yellow) to 4-aminophenol (4-AP, colorless). The developed Ag@FeO@Au NPs-based immunoassay achieved a limit of detection (LOD) of 1.9 pg/mL for norovirus-like particles (NoV-LP) and 6.97 RNA copy number/mL for fecal NoV. In fecal sample analysis for NoV, a heat treatment at 65°C was necessary to prevent degradation of the target protein, ensuring sensitive detection. This work successfully combined multifunctional nanocatalysts for advanced immunoassays, which could contribute to developing nano-biosensing platforms.

Currently, the cells, which are urgently required for large-scale application in biomedical-related fields, harvested by traditional trypsin digestion are usually subject to repeated digestion, leading to a reduction of cell activity. In this study, poly (N-isopropylacrylamide) (PNIPAAm) was grafted onto the lignocellulose hollow fiber membranes (HFMs) with cerium ammonium nitrate (CAN) as the initiator to prepare thermosensitive HFMs, which was combined with a rotation system of culture (RSOC) to achieve dynamic culture and non-destructive harvesting of cells from the HFMs. The results of ATR-FTIR, elemental analysis, and SEM confirmed the successful preparation of PNIPAAm-grafted-HFMs, which also showed good biocompatibility to apply for cell culture carriers. In cooling detachment, the HFMs-0.01 group could completely detach the cells within 1 h with a cell separation efficiency of more than 90%. The laminin (LN) and fibronectin (FN) harvested by cooling detachment of P8 generation PC12 cells reached 0.0531 ± 0.0032 and 2.5045 ± 0.0001 pg/cell, respectively, which were significantly higher than that by trypsin digestion. In addition, the cells on the thermosensitive HFMs proliferated fastest in RSOC at 30 rpm with higher glucose consumption and lactate metabolism than in static conditions. Moreover, the cells that had dynamic detachment at 20 rpm had the highest cell density and activity. Therefore, the thermosensitive HFMs could be applied as cell culture carriers in RSOC for cell culturing at 30 rpm and harvesting at 20 rpm, which would provide considerable potential for large-scale cell culture in vitro.

Nanoparticles (NPs) have emerged as a promising solution for many biomedical applications. Although not all particles have antimicrobial or regenerative properties, certain NPs show promise in enhancing wound healing by promoting tissue regeneration, reducing inflammation, and preventing infection. Integrating various NPs can further enhance these effects. Herein, the zinc oxide (ZnO)-MXene-Ag nanocomposite was prepared, and the conjugation of its three components was confirmed through scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) mapping analysis. In vitro analysis using the agar well diffusion technique demonstrated that ZnO-MXene-Ag nanocomposite exhibited high antimicrobial efficacy, significantly inhibiting Escherichia coli, Salmonella, and Candida albicans, and showing enhanced potency when combined with tetracycline, resulting in a 2.6-fold increase against Staphylococcus and a 2.4-fold increase against Pseudomonas. The efficacy of nanocomposite-loaded carboxymethyl cellulose (CMC) gel on wound healing was investigated using varying concentrations (0, 1, 5, and 10 mg/mL). Wound healing was monitored over 21 days, with results indicating that wounds treated with 1 mg/mL ZnO-MXene-Ag gel exhibited superior healing compared to the control group (0 mg/mL), with significant improvements noted from Day 3 onward. Conversely, higher concentrations (10 mg/mL) resulted in reduced healing efficiency, particularly notable on Day 15. In conclusion, the ZnO-MXene-Ag nanocomposite-loaded CMC gel is a promising agent for enhanced wound healing and antimicrobial applications. These findings highlight the importance of optimizing NP concentration to maximize therapeutic benefits while minimizing potential cytotoxicity.

Halomonas elongata thrives in hypersaline environments producing polyhydroxyalkanoates (PHAs) and osmoprotectants such as ectoine. Despite its biotechnological importance, several aspects of the dynamics of its metabolism remain elusive. Here, we construct and validate a genome-scale metabolic network model for H. elongata 153B. Then, we investigate the flux distribution dynamics during optimal growth, ectoine, and PHA biosynthesis using statistical methods, and a pipeline based on shadow prices. Lastly, we use optimization algorithms to uncover novel engineering targets to increase PHA production. The resulting model (iEB1239) includes 1534 metabolites, 2314 reactions, and 1239 genes. iEB1239 can reproduce growth on several carbon sources and predict growth on previously unreported ones. It also reproduces biochemical phenotypes related to Oad and Ppc gene functions in ectoine biosynthesis. A flux distribution analysis during optimal ectoine and PHA biosynthesis shows decreased energy production through oxidative phosphorylation. Furthermore, our analysis unveils a diverse spectrum of metabolic alterations that extend beyond mere flux changes to encompass heightened precursor production for ectoine and PHA synthesis. Crucially, these findings capture other metabolic changes linked to adaptation in hypersaline environments. Bottlenecks in the glycolysis and fatty acid metabolism pathways are identified, in addition to PhaC, which has been shown to increase PHA production when overexpressed. Overall, our pipeline demonstrates the potential of genome-scale metabolic models in combination with statistical approaches to obtain insights into the metabolism of H. elongata. Our platform can be exploited for researching environmental adaptation, and for designing and optimizing metabolic engineering strategies for bioproduct synthesis.

Targeted integration (TI) Chinese hamster ovary (CHO) platforms are commonly used for protein expression. However, the impact of epigenetic modifications on protein expression in TI cell lines remains elusive since almost all the epigenetic studies focus on random integration (RI) of the gene of interest and only within the promoter region. To address the impact of epigenetic modifications on TI CHO cells, we utilized a standard mAb-1 to identify and characterize TI clones with the same transgene copy numbers but different levels of transgene transcription and titer. Surprisingly, while CMV promoters were not methylated and histone acetylation/methylation was present, these epigenetic markers did not trend with mRNA transcription and protein expression in our TI model. Instead, ATAC-seq data analysis revealed that differences in chromatin accessibility within the TI site could be a major factor impacting these observed differences. However, neither chromatin accessibility nor histone acetylation/methylation profiles in early cultures were predictive of high-expressing clones early during the CLD process. Finally, modulation of the histone profiles (H3K27ac and H3K4me3) at the CMV promoters within the TI integration site using dCas9 fusion proteins was not effective in further increasing mAb titers which could have been likely due to interference of the dCas9 fusion proteins with transcription from the CMV promoters. Overall, our data suggests increasing chromatin accessibility at the TI site is the most effective way to increase mRNA transcription and hence, productivity in TI cell lines.