Half of patients with renal cell carcinoma (RCC) will develop metastases. The disease is likely to be curable at early stages but incurable when metastatic. New and non-invasive biomarkers are needed for the diagnosis of RCC. Extracellular vesicles (EVs) are considered promising new biomarker targets for the diagnosis of various diseases. Our study aimed to develop an EV-based assay for the detection of RCC using a highly sensitive nanoparticle-aided time-resolved fluorescence immunoassay (NP-TRFIA). To confirm that the tetraspanins were located on EVs, we used size exclusion chromatography to separate EV- and PE (protein-enriched)-fractions from RCC4 and 786-O RCC cancer cell lines and HEK293. EV- and PE-fractions were quantified using NP-TRFIA assays established for tetraspanins CD9, CD63, CD81, and CD151. Tetraspanin biomarkers were further measured from RCC cell culture medium as well as serum samples of RCC (n=14), benign (n=17), and healthy (n=9) individuals. Among the tetraspanins, CD63 showed 3-5-fold higher expression on EVs derived from RCC4 and 786-O cell lines compared to those from the HEK293. A sandwich CD63-CD63 assay demonstrated significant discrimination of RCC patients from benign (p=0.0003), and healthy (p= 0.005) individuals, respectively. Similarly, the CD81-CD81 assay also enabled significant separation of RCC patients compared to benign (p=0.014), and healthy (p= 0.003) controls, respectively. This result suggests that RCC cell lines and serum of RCC patients show higher amounts of CD63- and CD81-enriched EVs compared to controls. Detection of these tetraspanin-enriched EVs using our NP-TRFIA approach may play a vital role in the detection of RCC.
Scalable single use adherent cell-based biomanufacturing platforms are part of solutions to realize the full potential of cell and gene therapies. Here, we reported the development of an innovative fixed bed bioreactor platform for the scale-up of adherent cell culture. The bioreactor platform is centered on a packed bed of woven polyethylene terephthalate mesh discs that are vertically stacked and sandwiched between two fluid guide plates. Computational fluid dynamics modeling was used to direct the design and development of bioreactor series, targeting uniform flow with minimal shear stress. Residence time distribution measurements revealed that a pulse injected dye tracer solution passed through the bioreactors with great uniformity and narrow distribution of residence time, mimicking plug flow. Periodic media sampling with an offline analyzer showed that there was minimal gradient of four important metabolites (glucose, glutamine, lactate, and ammonia) across the bioreactor throughout cell growth. The bioreactor platform was further validated in automated cell harvesting with ~96% efficiency and ~98% viability, as well as linear scalability, in terms of both operational parameters and performance, for cell culture and adeno-associated virus vector production. Finally, mathematic models based on oxygen uptake rates were developed and proven effective to model cell growth curves and estimate biomass in real-time. This study shows that this innovative fixed bed bioreactor platform enables linearly scalable adherent cell-based biomanufacturing with high productivity.
Bioconversion of Rebaudioside D faces high-cost obstacles. Herein, a novel glycosyltransferase StUGT converting Rebaudioside A to Rebaudioside D was screened and characterized, which exhibits stronger affinity and substrate specificity for Rebaudioside A than previously reported enzymes. A whole-cell catalytic system was thus developed using the StUGT strain. The production of Rebaudioside D was enhanced significantly by enhancing cell permeability, and the highest production of 6.12 g/L (yield=98.08%) by cell catalyst was obtained by statistical-based optimization. A new cascade process utilizing this recombinant strain and E. coli expressing sucrose synthase was further established to reduce cost through replacing expensive UDPG with sucrose. A StUGT-GsSUS1 system exhibited high catalytic capability, and 5.27 g/L Rebaudioside D was achieved finally without UDPG addition by systematic optimization. This is the best performance reported in cell-cascaded biosynthesis, which paves a new cost-effective strategy for sustainable synthesis of scarce premium sweeteners from biomass.
Phytosterols usually have to be esterified to various phytosterol esters to avoid their disadvantages of unsatisfactory solubility and low bioavailability. The enzymatic synthesis of phytosterol esters in solvent-free system has advantages in terms of environmental friendliness, sustainability, and selectivity. However, the limitation of the low stability and recyclability of the lipase in the solvent-free system, which often requires a relatively high temperature to induce the viscosity, also increased the industrial production cost. In this context, a low-cost material, namely diatomite, was employed as the support in the immobilization of Candida rugosa lipase (CRL) due to its multiple modification sites. The Fe3O4 was also then introduced to this system for quick and simple separation via the magnetic field. Moreover, to further enhance the immobilization efficiency of diatomite, a modification strategy which involved the octadecyl and sulfonyl group for regulating the hydrophobicity and interaction between the support and lipase was successfully developed. The optimization of the ratio of the modifiers suggested that the -SO3H/C18 (1:1.5) performed best with an enzyme loading and enzyme activity of 84.8 mg·g-1 and 54 U·g-1, respectively. Compared with free CRL, the thermal and storage stability of CRL@OSMD was significantly improved, which lays the foundation for the catalytic synthesis of phytosterol esters in solvent-free systems. Fortunately, a yield of 95.0% was achieved after optimizing the reaction conditions, and a yield of 70.0% can still be maintained after 6 cycles.
Schizochytrium sp. is a heterotrophic microorganism capable of accumulating polyunsaturated fatty acids, and has achieved industrial production of docosahexaenoic acid (DHA). It also has the potential for eicosapentaenoic acid (EPA) production. In this study, it was found that the cell growth, lipid synthesis and fatty acid composition of Schizochytrium sp. were significantly affected by the level of cobalamin in the medium, especially with regards to the content of EPA in the fatty acids. The content of EPA in the fatty acids increased 17.91 times, reaching 12.0%, but cell growth and lipid synthesis were significantly inhibited under cobalamin deficiency. The response mechanism for this phenomenon was revealed through combined lipidomic and transcriptomic analysis. Although cell growth was inhibited under cobalamin deficiency, the genes encoding key enzymes in central carbon metabolism were still up-regulated to provide precursors (Acetyl-CoA) and reducing power (NADPH) for the synthesis and accumulation of fatty acids. Moreover, the main lipid subclasses observed during cobalamin deficiency were glycerolipids (including glycerophospholipids), with EPA primarily distributed in them. The genes involved in the biosynthesis of these lipid subclasses were significantly up-regulated, such as the key enzymes in the Kennedy pathway for the synthesis of triglycerides. Thus, this study provided insights into the specific response of Schizochytrium sp. to cobalamin deficiency and identified a subset of new genes that can be engineered for modification.
The use of a combination of several antibacterial agents for therapy holds great promise in reducing the dosage and side effects of these agents, improving their efficiency, and inducing potential synergistic therapeutic effects. Herein, R12-AgNPs were produced with the supernatant of an ionizing radiation-tolerant bacterium Deinococcus wulumuqiensis R12 by one-step under room temperature. The biosynthesized AgNPs presented fascinating antibacterial activity and peroxidase-like properties, which endowed it with the capability to catalyse the decomposition of H2O2 to generate hydroxyl radical. After combination of R12-AgNPs and H2O2, an excellent synergistic bacteriostatic activity was observed for both E. coli and S. aureus, especially at low concentrations. In addition, in vitro cytotoxicity tests showed R12-AgNPs had good biocompatibility. Thus, this work presents a novel antibacterial agent that exhibits favorable synergistic antibacterial activity and low toxicity, without the use of antibiotics or a complicated synthesis process.
Antibody mimetics is a novel antibody engineering approach after the development of polyclonal, monoclonal antibodies, and genetically engineered antibody fragments. Inspired by the structure and function of natural antibodies, antibody mimetics offer many advantages over conventional antibodies and can be constructed by protein-directed evolution, peptide design and synthesis, or fusion of complementarity-determining regions through intervening framework regions. A series of parent protein/peptide structures and technical roadmaps have been established to induce better recognising properties, superior affinity, stability, penetrability, and cost-effectiveness of the designed mimetics. This article aims to summarise the evolution of antibody mimetics engineering, illustrate the highlights and hotpots in this research field using scientometric analysis, and give an anticipatory analysis on this increasing research topic.
Biodiesels constitute a growing class of fuel in the world which is increasingly inclined towards more ecological and sustainable energy. Despite their advantages, biodiesels are of limited cold flow properties and of larger NOx emissions, which are mostly attributed to the chemical composition of their oil feedstocks. This study presents a novel approach to produce Genetically Engineered Biodiesel from genetically manipulated oleaginous yeast oils for improving biodiesel properties and performances. Using full-factorial central composite design, the best chemical composition of an optimal biodiesel was predicted. Then, simple and combined MFE1, PEX10 and POX2 mutants of the oleaginous yeast Yarrowia lipolytica were constructed and showed interesting lipid profiles whose biodiesel is predicted to have better cold flow properties. These mutants showed also higher lipid titers by 2-3 folds compared to the parent strain. This study provides a genetic engineering strategy for tailor design of biodiesel properties and performance.
9α-Hydroxyandroster-4-ene-3,17-dione (9-OH-AD) is a representative steroid drug intermediate that can be prepared by phytosterols (PS) biotransformation with mycobacteria in a resting cell-cyclodextrin system. In this study, over-expression of 17β-hydroxysteroid dehydrogenase (Hsd4A) was testified to enhance the side-chain degradation of PS and to reduce the incomplete degradation by-products. Meanwhile, the complete degradation product 4-androstene-3,17-dione (AD) was increased due to lack of 3-Ketosteroid 9α-Hydroxylase (KshA1) activities. To increase the production and purity of 9-OH-AD, the metabolic pathway of the side-chain degradation of PS and 9-position hydroxylation was modulated by balancing the over-expression of Hsd4A and KshA1 in mycobacteria and reducing the bioconversion rate via lowering the ratio of PS and cyclodextrin. The production and purity of 9-OH-AD in broth were improved from 22.18 g/L and 77.13% to 28.27 g/L and 87.84%, with a molar yield of 78.32%.
Background: To obtain high yields of recombinant insulin and advancing therapeutic avenues for diabetes patients, the development of innovative designer insulin analogs have critical importance. The modified insulin analog presents a cost-effective remedy by being produced as inclusion bodies (IBs) within Escherichia coli BL21 (DE3) Rosetta-2 strain. This approach to production not only offers reduced production time but also yields high recovery rates. The prime aim of this investigation was to optimize the composition of the cultivation media, thereby accomplishing higher cell density fermentation of the proinsulin. Result: Various factors, including carbon and nitrogen sources, salts, metal ions, and pH, were systematically investigated through experimental screening using the BioLector multiwell bright plate. Additionally, computational analysis employing the Plackett-Burman Design within the Design Expert software was utilized to assess their effectiveness in terms of insulin concentration as a surrogate measure of insulin yield. Among the tested variables, glucose, glycerol, MgSO4, and lower Luria-Bertani mix concentration have a significant influence on insulin production, as determined by the screening experiment. Subsequently, the Central Composite Design approach was operated to further evaluate and optimize the precise levels of these influential variables. This systematic methodology achieved an optimized cultivation media formulation, resulting in a remarkable enhancement of insulin production, with levels reaching up to 13 mg/ml when applied in BioLector fermentation. Conclusion: The formulated cultivation media exhibited suitability for promoting high cell density fermentation of the modified insulin, thereby facilitating the attainment of optimal yields for the proinsulin expression.
We designed and constructed a green, sustainable, and nongenetically modified organism (non-GMO) bioprocess to efficiently coproduce D-tagatose, bioethanol, and microbial protein from whey powder. First, a one-pot biosynthesis process involving lactose hydrolysis and D-galactose redox reactions for D-tagatose production was established in vitro via a three-enzyme cascade. Second, a nicotinamide adenine dinucleotide phosphate-dependent galactitol dehydrogenase mutant, D36A/I37R, based on the nicotinamide adenine dinucleotide-dependent polyol dehydrogenase from Paracoccus denitrificans was created through rational design and screening. Moreover, an NADPH recycling module was created in the oxidoreductive pathway, and the tagatose yield increased by 3.35-fold compared with that achieved through the pathway without the cofactor cycle. The reaction process was accelerated using an enzyme assembly with a glycine–serine linker, and the tagatose production rate was 9.28-fold higher than the initial yield. Finally, Saccharomyces cerevisiae was introduced into the reaction solution, and 266.5 g of D-tagatose, 371.3 g of bioethanol, and 215.4 g of dry yeast (including 38% protein) were obtained from 1 kg of whey powder (including 810 g lactose). This study provides a promising non-GMO process for functional food (D-tagatose) production. Moreover, this process fully utilized whey powder, demonstrating good atom economy.
Aquaporins (AQPs) are intrinsic membrane proteins responsible for facilitating water transport across biological membranes. AQPs found in plant membrane vesicles (MV) have been related to the functionality and stability of the vesicles. In this study, we focused on AQPs obtained from Brassica oleracea var. L. italica (broccoli) by the great potential for different biotechnological applications. To gain further insight into the role of AQPs in MV and advance the biotechnological applications of AQPs, we describe the heterologous overexpression of two broccoli AQPs (BoPIP1;2 and BoPIP2;2) in Pichia pastoris, resulting in the purification of both AQPs with high yield (0.14 and 0.99 mg per gram cells for BoPIP1;2 and BoPIP2;2, respectively). We reconstituted purified AQPs in liposomes to study their functionality, showing no changes in size compared to liposomes. BoPIP2;2 facilitated water transport, which was preserved for seven days at 4oC and 25ºC but not at 37oC, whereas BoPIP1;2 did not enhance water transport across the proteoliposome membrane. Additionally, BoPIP2;2 was incorporated into liposomes to encapsulate a resveratrol extract in proteoliposome vesicles, resulting in increased entrapment efficiency compared to conventional liposomes. Molecular docking identified potential binding sites for resveratrol in PIP2s, highlighting the role of AQPs in the improved entrapment efficiency of resveratrol. Moreover, a modelling study was conducted, demonstrating interactions between a plant AQP and human integrin, which may be a benefit to increase contact and internalization by the human target cells. Thus, our results suggest that AQPs-based alternative encapsulation systems can be used in specifically target biotechnological applications.
Organ-on-a-chip technology has shown great potential in disease modeling and drug evaluation. However, traditional organ-on-a-chip devices are mostly pump-dependent with low throughput, which makes it difficult to leverage their advantages. In this study, we have developed a generic, pump-free organ-on-a-chip platform consisting of a 32-unit chip and an adjustable rocker, facilitating high-throughput dynamic cell culture with straightforward operation. By utilizing the rocker to induce periodic fluid forces, we can achieve fluidic conditions similar to those obtained with traditional pump-based systems. Through constructing a gut-on-a-chip model, we observed remarkable enhancements in the expression of barrier-associated proteins and the spatial distribution of differentiated intestinal cells compared to static culture. Furthermore, RNA sequencing analysis unveiled enriched pathways associated with cell proliferation, lipid transport and drug metabolism, indicating the ability of the platform to mimic critical physiological processes. Additionally, we tested seven drugs which represent a range of high, medium, and low in vivo permeability using this model and found a strong correlation between their Papp values and human Fa, indicating reliable and predictive simulation outcomes for drug absorption. Our findings highlight the potential of this pump-free organ-on-a-chip platform as a valuable tool for advancing drug development and enabling personalized medicine.
Biopharmaceuticals, including therapeutic antibodies, are rapidly growing products in the pharmaceutical market. Mammalian cells, such as Chinese hamster ovary (CHO) cells, are widely used as production hosts because recombinant antibodies require complex three-dimensional structures modified with sugar chains. Recombinant protein production using mammalian cells is generally performed in conjunction with cell growth. In this study, we developed a technology that controls cell growth and recombinant protein production to induce recombinant protein production with arbitrary timing. Expression of green fluorescent protein (GFP) gene and a single-chain antibody fused with the Fc-region of the human IgG1 (scFv-Fc) gene can be induced and mediated by the estrogen receptor-based artificial transcription factor Gal4-ERT2-VP16 and corresponding inducer drugs. We generated CHO cells using an artificial gene expression system. The addition of various concentrations of inducer drugs to the culture medium allowed control of proliferation and transgene expression of the engineered CHO cells. Use of 4-hydroxytamoxifen, an antagonist of estrogen, as an inducing agent yielded high gene expression at a concentration more than 10-fold lower than that of β-estradiol. When scFv-Fc was continuously produced under inducing conditions, stable production was possible for more than 2 weeks while maintaining high specific productivity (57 pg cell-1 day-1). This artificial gene expression control system that utilizes the estrogen response of estrogen receptors can be an effective method for inducible production of biopharmaceuticals.
Corynebacterium glutamicum is a useful microbe to produce succinic acid, a bio-based platform chemical, under anaerobic condition. The knock-out mutant of lactate dehydrogenase 1 gene (ΔldhA-6) and co-expression of succinic acid transporter (Psod:sucE- ΔldhA) were generated by using CRISPR-Cpf1 genome editing system. HAPC (hydrogen peroxide and acetic acid) pretreatment is a highly efficient method for enzymatic hydrolysis of softwood and the hydrolysate was used for production of succinic acid. In the 15% hydrolysate (Pinus densiflora), the best condition for ΔldhA mutant to produce succinic acid from the hydrolysate was confirmed to ferment 4% hydrolysate, resulted in 14.82 g L-1 succinic acid production for 6 h, which reached to 2.47 g L-1 h-1 productivity. No production of acetic acid and lactic acid was detected during the fermentation. The co-expression transformant, [Psod:sucE- ΔldhA], produced 17.70 g L-1 succinic acid in 6 h, presenting a productivity of 2.95 g L-1 h-1 on the 4% hydrolysate. In the fed-batch system, 39.67 g L-1 succinic acid was produced for 48 h. The yield of succinic acid from reducing sugars in the hydrolysate is approximately 56.71%, while the yield of succinic acid from glucose alone as the main substrate is approximately 84.4%. These results indicated that the production of succinic acid from softwood has potential applications in alternative biochemical processes, and minimizing the loss of sugars during enzymatic hydrolysis and fermentation can lead to more economic benefits in succinic acid production from lignocellulosic biomass.
Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. We have assessed the α-casein (CSN1S1) gene as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but much lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression.
The stiffness of the tumor microenvironment (TME) is dynamic and drives metabolic reprogramming in cancer cells as a consequence of tumor progression. To demonstrate the possibility to modulate the mechano-metabolomic profile of breast cancers by tuning the mechanical property and dimensionality of extracellular matrices (ECMs), we cultured triple-negative MDA-MB-231 and luminal MCF-7 cells on 2D and in 3D hydrogels based on tyramine functionalized hyaluronic acid (HTA). Using high-throughput metabolomics analyses, we established that we can differentially regulate breast cancer mechano-metabolome. The stiff hydrogels resulted in upregulated lipid and amino acid metabolism along with increasing malignancy and chemoresistancy. Reprogramming in glucose metabolism is primarily observed in cells seeded on 2D hydrogels, whereas modifications in amino acid metabolism is predominant in cells embedded in 3D stiff hydrogels. These findings suggest that matrix stiffness and dimensions have decisive roles in reprogramming breast cancer metabolome, which is the hallmark of breast cancer development and progression.
Recently, nanomedicine have been successfully applied in the cancer therapy. However, how to precisely control the drug release from nanomedicine in tumor tissue and overcome the hypoxic microenvironment of tumor tissue is still an important challenge in the development of nanomedicine. In this work, a new type of drug-loaded nanoparticles P(AAm-co-AN)-AuNRs@CeO2-DOX (PA-DOX) was prepared by combining high-efficiency photothermal reagents, critical up-conversion temperature polymer layer and anti-cancer drug doxorubicin (DOX) for the treatment of liver cancer. In this system, CeO2 can decompose hydrogen peroxide to H2O2 and O2 alleviate the anaerobic microenvironment of liver cancer cells. As a photothermal reagent, AuNRs@CeO2 can convert near-infrared light into heat energy to achieve local heat to kill cancer cells and ablate solid tumors. In addition, the elevated temperature would enable the polymer layer to undergo a phase transition to release more DOX to achieve a controlled release mechanism, which will open up a new horizon for clinical cancer treatment