Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

Engineering a complex, multiple enzyme-mediated synthesis of natural plant pigments in the silkworm, Bombyx mori.

Plants produce various pigments that not only appear as attractive colors but also provide valuable resources in applications in daily life and scientific research. Biosynthesis pathways for these natural plant pigments are well studied, and most have multiple enzymes that vary among plant species. However, adapting these pathways to animals remains a challenge. Here, we describe successful biosynthesis of betalains, water-soluble pigments found only in a single plant order, Caryophyllales, in transgenic silkworms by coexpressing three betalain synthesis genes, cytochrome P450 enzyme CYP76AD1, DOPA 4,5-dioxygenase, and betanidin 5-O-glucosyltransferase. Betalains can be synthesized in various tissues under the control of the ubiquitous IE1 promoter but accumulate mainly in the hemolymph with yields as high as 274 µg/ml. Additionally, transformed larvae and pupae show a strong red color easily distinguishable from wild-type animals. In experiments in which expression is controlled by the promoter of silk gland-specific gene, fibroin heavy-chain, betalains are found predominantly in the silk glands and can be secreted into cocoons through spinning. Betalains in transformed cocoons are easily recovered from cocoon shells in water with average yields reaching 14.4 µg/mg. These data provide evidence that insects can synthesize natural plant pigments through a complex, multiple enzyme-mediated synthesis pathway. Such pigments also can serve as dominant visible markers in insect transgenesis applications. This study provides an approach to producing valuable plant-derived compounds by using genetically engineered silkworms as a bioreactor.

Engineered Living Material Bioreactors with Tunable Mechanical Properties using Vat Photopolymerization.

3D-printed engineered living materials (ELM) are promising bioproduction platforms for agriculture, biotechnology, sustainable energy, and green technology applications. However, the design of these platforms faces several challenges, such as the processability of these materials into complex form factors and control over their mechanical properties. Herein, ELM are presented as 3D-printed bioreactors with arbitrary shape geometries and tunable mechanical properties (moduli and toughness). Poly(ethylene glycol) diacrylate (PEGDA) is used as the precursor to create polymer networks that encapsulate the microorganisms during the vat photopolymerization process. A major limitation of PEGDA networks is their propensity to swell and fracture when submerged in water. The authors overcame this issue by adding glycerol to the resin formulation to 3D print mechanically tough ELM hydrogels. While polymer concentration affects the modulus and reduces bioproduction, ELM bioreactors still maintain their metabolic activity regardless of polymer concentration. These ELM bioreactors have the potential to be used in different applications for sustainable architecture, food production, and biomedical devices that require different mechanical properties from soft to stiff.

Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems.

Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self-assembling bacterial microcompartment (BMC) shell proteins and liquid-liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid-liquid interfaces between either 1) the dextran-rich droplets and PEG-rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two-phase system, or 2) the polypeptide-rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically-driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three-phase system wherein coacervate droplets are contained within dextran-rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three-phase system by changing the polyelectrolyte charge ratio. The tens-of-micron scale BMC shell protein-coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

Bacterial growth-mediated systems remodelling of Nicotiana benthamiana defines unique signatures of target protein production in molecular pharming.

The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.

Development of a highly cytotoxic, clinical-grade virus-specific T cell product for adoptive T cell therapy.

At present, recipients of allogeneic hematopoietic stem-cells are still suffering from recurrent infections after transplantation. Infusion of virus-specific T cells (VST) post-transplant reportedly fights several viruses without increasing the risk of de novo graft-versus-host disease. This study targeted cytomegalovirus (CMV) for the development of an innovative approach for generating a very specific VST product following Good Manufacturing Practices (GMP) guidelines. We used a sterile disposable compartment named the Leukoreduction System Chamber (LRS-chamber) from the apheresis platelet donation kit as the starting material, which has demonstrated high levels of T cells. Using a combination of IL-2 and IL-7 we could improve expansion of CMV-specific T cells. Moreover, by developing and establishing a new product protocol, we were able to stimulate VST proliferation and favors T cell effector memory profile. The expanded VST were enriched in a closed automated system, creating a highly pure anti-CMV product, which was pre-clinically tested for specificity in vitro and for persistence, biodistribution, and toxicity in vivo using NOD scid mice. Our results demonstrated very specific VST, able to secrete high amounts of interferon only in the presence of cells infected by the human CMV strain (AD169), and innocuous to cells partially HLA compatible without viral infection.

Degradation of Listeria monocytogenes biofilm by phages belonging to the genus Pecentumvirus.

Listeria monocytogenes is a pathogenic foodborne bacterium that is a significant cause of mortality associated with foodborne illness and causes many food recalls attributed to a bacteriological cause. Their ability to form biofilms contributes to the persistence of Listeria spp. in food processing environments. When growing as biofilms, L. monocytogenes are more resistant to sanitizers used in the food industry, such as benzalkonium chloride (BAC), as well as to physical stresses like desiccation and starvation. Lytic phages of Listeria are antagonistic to a broad range of Listeria spp. and may, therefore, have utility in reducing the occurrence of Listeria-associated food recalls by preventing food contamination. We screened nine closely related Listeria phages, including the commercially available Listex P100, for host range and ability to degrade microtiter plate biofilms of L. monocytogenes ATCC 19111 (serovar 1/2a). One phage, CKA15, was selected and shown to rapidly adsorb to its host under conditions relevant to applying the phage in dairy processing environments. Under simulated dairy processing conditions (SDPC), CKA15 caused a 2-log reduction in Lm19111 biofilm bacteria. This work supports the biosanitation potential of phage CKA15 and provides a basis for further investigation of phage-bacteria interactions in biofilms grown under SDPC. IMPORTANCE: Listeria monocytogenes is a pathogenic bacterium that is especially dangerous for children, the elderly, pregnant women, and immune-compromised people. Because of this, the food industry takes its presence in their plants seriously. Food recalls due to L. monocytogenes are common with a high associated economic cost. In food-processing plants, Listeria spp. typically reside in biofilms, which are structures produced by bacteria that shield them from environmental stressors and are often attached to surfaces. The significance of our work is that we show a bacteriophage-a virus-infecting bacteria-can reduce Listeria counts by two orders of magnitude when the bacterial biofilms were grown under simulated dairy processing conditions. This work provides insights into how phages may be tested and used to develop biosanitizers that are effective but are not harmful to the environment or human health.

Mechanical loading rescues mechanoresponsiveness in a human osteoarthritis explant model despite Wnt activation.

OBJECTIVES: Optimizing rehabilitation strategies for osteoarthritis necessitates a comprehensive understanding of chondrocytes' mechanoresponse in both health and disease, especially in the context of the interplay between loading and key pathways involved in osteoarthritis (OA) development, like canonical Wnt signaling. This study aims to elucidate the role of Wnt signaling in the mechanoresponsiveness of healthy and osteoarthritic human cartilage. METHODS: We used an ex-vivo model involving short-term physiological mechanical loading of human cartilage explants. First, the loading protocol for subsequent experiments was determined. Next, loading was applied to non-OA-explants with or without Wnt activation with CHIR99021. Molecular read-outs of anabolic, pericellular matrix and matrix remodeling markers were used to assess the effect of Wnt on cartilage mechanoresponse. Finally, the same set-up was used to study the effect of loading in cartilage from patients with established OA. RESULTS: Our results confirm that physiological loading maintains expression of anabolic genes in non-OA cartilage, and indicate a deleterious effect of Wnt activation in the chondrocyte mechanoresponsiveness. This suggests that loading-induced regulation of chondrocyte markers occurs downstream of canonical Wnt signaling. Interestingly, our study highlighted contrasting mechanoresponsiveness in the model of Wnt activation and the established OA samples, with established OA cartilage maintaining its mechanoresponsiveness, and mechanical loading rescuing the chondrogenic phenotype. CONCLUSION: This study provides insights into the mechanoresponsiveness of human cartilage in both non-OA and OA conditions. These findings hold the potential to contribute to the development of strategies that optimize the effect of dynamic compression by correcting OA pathological cell signaling.

HSP70-A key regulator in chondrocyte homeostasis under naturally coupled hydrostatic pressure-thermal stimuli.

OBJECTIVE: During physical activities, chondrocytes experience coupled stimulation of hydrostatic pressure (HP) and a transient increase in temperature (T), with the latter varying within a physiological range from 32.5 °C to 38.7 °C. Previous short-term in vitro studies have demonstrated that the combined hydrostatic pressure-thermal (HP-T) stimuli more significantly enhance chondroinduction and chondroprotection of chondrocytes than isolated applications. Interestingly, this combined benefit is associated with a corresponding increase in HSP70 levels when HP and T are combined. The current study therefore explored the indispensable role of HSP70 in mediating the combined effects of HP-T stimuli on chondrocytes. DESIGN: In this mid-long-term study of in vitro engineered cartilage constructs, we assessed chondrocyte responses to HP-T stimuli using customized bioreactor in standard and HSP70-inhibited cultures. RESULTS: Surprisingly, under HSP70-inhibited conditions, the usually beneficial HP-T stimuli, especially its thermal component, exerted detrimental effects on chondrocyte homeostasis, showing a distinct and unfavorable shift in gene and protein expression patterns compared to non-HSP70-inhibited settings. Such effects were corroborated through mechanical testing and confirmed using a secondary cell source. A proteomic-based mechanistic analysis revealed a disruption in the balance between biosynthesis and fundamental cellular structural components in HSP70-inhibited conditions under HP-T stimuli. CONCLUSIONS: Our results highlight the critical role of sufficient HSP70 induction in mediating the beneficial effects of coupled HP-T stimulation on chondrocytes. These findings help pave the way for new therapeutic approaches to enhance physiotherapy outcomes and potentially shed light on the elusive mechanisms underlying the onset of cartilage degeneration, a long-standing enigma in orthopedics.

A continuous flow bioreactor system for high-throughput hyperpolarized metabolic flux analysis.

Hyperpolarized carbon-13 labeled compounds are increasingly being used in medical MR imaging (MRI) and MR imaging (MRI) and spectroscopy (MRS) research, due to its ability to monitor tissue and cell metabolism in real-time. Although radiological biomarkers are increasingly being considered as clinical indicators, biopsies are still considered the gold standard for a large variety of indications. Bioreactor systems can play an important role in biopsy examinations because of their ability to provide a physiochemical environment that is conducive for therapeutic response monitoring ex vivo. We demonstrate here a proof-of-concept bioreactor and microcoil receive array setup that allows for ex vivo preservation and metabolic NMR spectroscopy on up to three biopsy samples simultaneously, creating an easy-to-use and robust way to simultaneously run multisample carbon-13 hyperpolarization experiments. Experiments using hyperpolarized [1-13C]pyruvate on ML-1 leukemic cells in the bioreactor setup were performed and the kinetic pyruvate-to-lactate rate constants ( k PL ) extracted. The coefficient of variation of the experimentally found k PL s for five repeated experiments was C V = 35 % . With this statistical power, treatment effects of 30%-40% change in lactate production could be easily differentiable with only a few hyperpolarization dissolutions on this setup. Furthermore, longitudinal experiments showed preservation of ML-1 cells in the bioreactor setup for at least 6 h. Rat brain tissue slices were also seen to be preserved within the bioreactor for at least 1 h. This validation serves as the basis for further optimization and upscaling of the setup, which undoubtedly has huge potential in high-throughput studies with various biomarkers and tissue types.

Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost.

BACKGROUND AIMS: Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. METHODS: Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. RESULTS: The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. CONCLUSIONS: These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.

Dual Enzyme-Encapsulated Materials for Biological Cascade Chemistry and Synergistic Tumor Starvation.

Framework and polymeric nanoreactors (NRs) have distinct advantages in improving chemical reaction efficiency in the tumor microenvironment (TME). Nanoreactor-loaded oxidoreductase enzyme is activated by tumor acidity to produce H2O2 by increasing tumor oxidative stress. High levels of H2O2 induce self-destruction of the vesicles by releasing quinone methide to deplete glutathione and suppress the antioxidant potential of cancer cells. Therefore, the synergistic effect of the enzyme-loaded nanoreactors results in efficient tumor ablation via suppressing cancer-cell metabolism. The main driving force would be to take advantage of the distinct metabolic properties of cancer cells along with the high peroxidase-like activity of metalloenzyme/metalloprotein. A cascade strategy of dual enzymes such as glucose oxidase (GOx) and nitroreductase (NTR) wherein the former acts as an O2-consuming agent such as overexpression of NTR and further amplified NTR-catalyzed release for antitumor therapy. The design of cascade bioreductive hypoxia-responsive drug delivery via GOx regulates NTR upregulation and NTR-responsive nanoparticles. Herein, we discuss tumor hypoxia, reactive oxygen species (ROS) formation, and the effectiveness of these therapies. Nanoclusters in cascaded enzymes along with chemo-radiotherapy with synergistic therapy are illustrated. Finally, we outline the role of the nanoreactor strategy of cascading enzymes along with self-synergistic tumor therapy.

Expression of a single-chain monellin (MNEI) mutant with enhanced stability in transgenic mice milk.

Monellin is a sweet protein that may be used as a safe and healthy sweetener. However, due to its low stability, the application of monellin is currently very limited. Here, we describe a wild-type, a double-sites mutant (E2N/E23A) and a triple-sites mutant (N14A/E23Q/S76Y) of single-chain monellin (MNEI) expressed in transgenic mice milk. Based on enzyme-linked immunoassay (ELISA), Western blot, and sweetness intensity testing, their sweetness and stability were compared. After boiling for 2 min at different pH conditions (2.5, 5.1, 6.8, and 8.2), N14A/E23Q/S76Y-MNEI showed significantly higher sweetness and stability than the wild-type and E2N/E23A-MNEI. These results suggest that N14A/E23Q/S76Y-MNEI shows remarkable potential as a sweetener in the future.

Recent advances in upstream process development for production of recombinant adeno-associated virus.

Recombinant adeno-associated virus (rAAV) is rapidly emerging as the preferred delivery vehicle for gene therapies, with promising advantages in safety and efficacy. Key challenges in systemic in-vivo rAAV gene therapy applications are the gap in production capabilities versus potential market demand and complex production process. This review summarizes current available information on rAAV upstream manufacturing processes and proposed optimizations for production. The advancements in rAAV production media were reviewed with proposals to speed up the cell culture process development. Furthermore, major methods for genetic element delivery to host cells were summarized with their advantages, limitations, and future directions for optimization. In addition, culture vessel selection criteria were listed based on production cell system, scale, and development stage. Process control at the production step was also outlined with an in-depth understanding of production kinetics and quality control.

Bioprocess development for cord blood mesenchymal stromal cells on microcarriers in Vertical-Wheel bioreactors.

Equine mesenchymal stromal cells (MSCs) have been found to be beneficial for the treatment of many ailments, including orthopedic injuries, due to their superior differentiation potential and immunomodulating properties. Cell therapies require large cell numbers, which are not efficiently generated using conventional static expansion methods. Expansion of equine cord blood-derived MSCs (eCB-MSCs) in bioreactors, using microcarriers as an attachment surface, has the potential to generate large numbers of cells with increased reproducibility and homogeneity compared with static T-flask expansion. This study investigated the development of an expansion process using Vertical-Wheel (VW) bioreactors, a single-use bioreactor technology that incorporates a wheel instead of an impeller. Initially, microcarriers were screened at small scale to assess eCB-MSC attachment and growth and then in bioreactors to assess cell expansion and harvesting. The effect of different donors, serial passaging, and batch versus fed batch were all examined in 0.1 L VW bioreactors. The use of VW bioreactors with an appropriate microcarrier was shown to be able to produce cell densities of up to 1E6 cells/mL, while maintaining cell phenotype and functionality, thus demonstrating great potential for the use of these bioreactors to produce large cell numbers for cell therapies.

Reinforcement learning based temperature control of a fermentation bioreactor for ethanol production.

Ethanol production is a significant industrial bioprocess for energy. The primary objective of this study is to control the process reactor temperature to get the desired product, that is, ethanol. Advanced model-based control systems face challenges due to model-process mismatch, but Reinforcement Learning (RL) is a class of machine learning which can help by allowing agents to learn policies directly from the environment. Hence a RL algorithm called twin delayed deep deterministic policy gradient (TD3) is employed. The control of reactor temperature is categorized into two categories namely unconstrained and constrained control approaches. The TD3 with various reward functions are tested on a nonlinear bioreactor model. The results are compared with existing popular RL algorithm, namely, deep deterministic policy gradient (DDPG) algorithm with a performance measure such as mean squared error (MSE). In the unconstrained control of the bioreactor, the TD3 based controller designed with the integral absolute error (IAE) reward yields a lower MSE of 0.22, whereas the DDPG produces an MSE of 0.29. Similarly, in the case of constrained controller, TD3 based controller designed with the IAE reward yields a lower MSE of 0.38, whereas DDPG produces an MSE of 0.48. In addition, the TD3 trained agent successfully rejects the disturbances, namely, input flow rate and inlet temperature in addition to a setpoint change with better performance metrics.

Modeling large-scale bioreactors with diffusion equations. Part I: Predicting axial dispersion coefficient and mixing times.

Bioreactor scale-up is complicated by dynamic interactions between mixing, reaction, mass transfer, and biological phenomena, the effects of which are usually predicted with simple correlations or case-specific simulations. This two-part study investigated whether axial diffusion equations could be used to calculate mixing times and to model and characterize large-scale stirred bioreactors in a general and predictive manner without fitting the dispersion coefficient. In this first part, a resistances-in-series model analogous to basic heat transfer theory was developed to estimate the dispersion coefficient such that only available hydrodynamic numbers and literature data were needed in calculations. For model validation, over 800 previously published experimentally determined mixing times were predicted with the transient axial diffusion equation. The collected data covered reactor sizes up to 160 m3 , single- and multi-impeller configurations with diverse impeller types, aerated and non-aerated operation in turbulent and transition flow regimes, and various mixing time quantification methods. The model performed excellently for typical multi-impeller configurations as long as flooding conditions were avoided. Mixing times for single-impeller and few nonstandard bioreactors were not predicted equally well. The transient diffusion equation together with the developed transfer resistance analogy proved to be a convenient and predictive model of mixing in typical large-scale bioreactors.

Modeling large-scale bioreactors with diffusion equations. Part II: Characterizing substrate, oxygen, temperature, carbon dioxide, and pH profiles.

Large-scale fermentation processes involve complex dynamic interactions between mixing, reaction, mass transfer, and the suspended biomass. Empirical correlations or case-specific computational simulations are usually used to predict and estimate the performance of large-scale bioreactors based on data acquired at bench scale. In this two-part-study, one-dimensional axial diffusion equations were studied as a general and predictive model of large-scale bioreactors. This second part focused on typical fed-batch operations where substrate gradients are known to occur, and characterized the profiles of substrate, pH, oxygen, carbon dioxide, and temperature. The physically grounded steady-state axial diffusion equations with first- and zeroth-order kinetics yielded analytical solutions to the relevant variables. The results were compared with large-scale Escherichia coli and Saccharomyces cerevisiae experiments and simulations from the literature, and good agreement was found in substrate profiles. The analytical profiles obtained for dissolved oxygen, temperature, pH, and CO 2 ${\text{CO}}_{2}$ were also consistent with the available data. Distribution functions for the substrate were defined, and efficiency factors for biomass growth and oxygen uptake rate were derived. In conclusion, this study demonstrated that axial diffusion equations can be used to model the effects of mixing and reaction on the relevant variables of typical large-scale fed-batch fermentations.

Secretion of the cytoplasmic and high molecular weight ß-galactosidase of Paenibacillus wynnii with Bacillus subtilis.

BACKGROUND: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. RESULTS: In this study, the cytoplasmic and 120 kDa ß-galactosidase of Paenibacillus wynnii (ß-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the ß-gal-Pw gene led to an increase in extracellular ß-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular ß-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular ß-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. CONCLUSION: For the first time, the ß-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

Metabolic Profiles and Microbial Synergy Mechanism of Anammox Biomass Enrichment and Membrane Fouling Alleviation in the Anammox Dynamic Membrane Bioreactor.

The anammox dynamic membrane bioreactor (DMBR) is promising in applications with enhanced anammox biomass enrichment and fouling alleviation. However, the metabolic mechanism underlying the functional features of anammox sludge and the biofilm membrane is still obscure. We investigated the metabolic networks of anammox sludge and membrane biofilm in the DMBR. The cooperation between anammox and dissimilatory nitrate reduction to ammonium processes favored the robust anammox process in the DMBR. The rapid bacterial growth occurred in the DMBR sludge with 1.33 times higher biomass yield compared to the MBR sludge, linked to the higher activities of lipid metabolism, nucleotide metabolism, and B vitamin-related metabolism of the DMBR sludge. The metabolism of the DMBR biofilm microbial community benefited the fouling alleviation that the abundant fermentative bacteria and their cooperation with the anammox sludge microbial community promoted organics degradation. The intensified degradation of foulants by the DMBR biofilm community was further evidenced by the active carbohydrate metabolism and the upregulated vitamin B intermediates in the biofilms of the DMBR. Our findings provide insights into key metabolic mechanisms for enhanced biomass enrichment and fouling control of the anammox DMBR, guiding manipulations and applications for overcoming anammox biomass loss in the treatment of wastewater under detrimental environmental conditions.