Biobutanol produced in acetone-butanol-ethanol fermentation at batch mode cannot compete with chemically derived butanol because of the low reactor productivity. Continuous fermentation can dramatically enhance productivity and lower capital and operating costs but are rarely used in industrial fermentation because of increased risks in culture degeneration, cell washout, and contamination. In this study, cells of the asporogenous Clostridium acetobutylicum ATCC55025 were immobilized in a single-pass fibrous-bed bioreactor (FBB) for continuous production of butanol from glucose and butyrate at various dilution rates. Butyric acid in the feed medium helped maintaining cells in the solventogenic phase for stable continuous butanol production. At the dilution rate of 1.88 h -1, butanol was produced at 9.55 g/L with a yield of 0.24 g/g and productivity of 16.8 g/L∙h, which was the highest ever achieved for biobutanol fermentation and an 80-fold improvement over the conventional ABE fermentation. The extremely high productivity was attributed to the high density of viable cells (~100 g/L at >70% viability) immobilized in the fibrous matrix, which also enabled the cells to better tolerate butanol and butyric acid. The FBB was stable for continuous operation for an extended period of over one month.
Microorganisms build fatty acids with biocatalytic assembly lines, or fatty acid synthases (FASs), that can be repurposed to produce a broad set of fuels and chemicals. Despite their versatility, the product profiles of FAS-based pathways are challenging to adjust without experimental iteration, and off-target products are common. This study uses a detailed kinetic model of the E. coli FAS as a foundation to model nine oleochemical pathways. These models provide good fits to experimental data and help explain unexpected results from in vivo studies. An analysis of pathways for alkanes and fatty acid ethyl esters, for example, suggests that reductions in titer caused by enzyme overexpression can result from shifts in pools of metabolic intermediates that are incompatible with the substrate specificities of downstream enzymes. In general, different engineering objectives (i.e., production, unsaturated fraction, and average chain length) show experimentally consistent sensitivities to pathway enzymes, and model-based compositional analyses indicate simple shifts in enzyme concentrations can alter the product profiles of pathways with promiscuous components. The study concludes by integrating all models into a graphical user interface. The models supplied by this work provide a versatile kinetic framework for studying oleochemical pathways in different biochemical contexts.
7-Methylxanthine, a derivative of caffeine (1,3,7-trimethylxanthine), is a high-value compound that has multiple medical applications, particularly with respect to eye health. Here, we demonstrate the biocatalytic production of 7-methylxanthine from caffeine using Escherichia coli strain MBM019, which was constructed for production of paraxanthine (1,7-dimethylxanthine). The mutant N-demethylase NdmA4, which was previously shown to catalyze N 3-demethylation of caffeine to produce paraxanthine, also retains N 1-demethylation activity toward paraxanthine. This work demonstrates that whole cell biocatalysts containing NdmA4 are more active toward paraxanthine than caffeine. We used four serial resting cell assays, with spent cells exchanged for fresh cells between each round, to produce 2,120 μM 7-methylxanthine and 552 μM paraxanthine from 4,331 μM caffeine. The purified 7-methylxanthine and paraxanthine were then isolated via preparatory-scale HPLC, resulting in 177.3 mg 7-methylxanthine and 48.1 mg paraxanthine at high purity. This is the first reported strain genetically optimized for the biosynthetic production of 7-methylxanthine from caffeine.
Hairy root systems have proven to be a viable alternative for recombinant protein production. For recalcitrant proteins, maximizing the productivity of hairy root cultures is essential. The aim of this study was to optimize a Brassica rapa rapa hairy root process for secretion of α-L-iduronidase (IDUA), a biologic of medical value. The process was first optimized with hairy roots expressing eGFP. For the biomass optimization, the highest biomass yields were achieved in modified Gamborg B5 culture medium. For the secretion induction, the optimized secretion media was obtained with additives (1.5g/l PVP + 1mg/l 2,4-D + 20.5g/l KNO 3) resulting in 3.4 fold eGFP secretion when compared to the non-induced control. These optimized conditions were applied to the IDUA-expressing hairy root clone, confirming that the highest yields of secreted IDUA occurred when using the already defined additive combination. The functionality of the IDUA protein, secreted and intracellular, was confirmed with an enzymatic activity assay. A >150-fold increase of the IDUA activity was observed using an optimized secretion medium, compared with a non-induced medium. We have proven that our B. rapa rapa hairy root system can be harnessed to secrete recalcitrant proteins, illustrating the high potential of hairy roots in plant molecular farming.
Cancer is one of the major health-related issues affecting the population worldwide and subsequently accounts for the second-largest death. Genetic and epigenetic modifications in oncogenes or tumor suppressor genes affect the regulatory systems that lead to the initiation and progression of cancer. Conventional methods, including chemotherapy/radiotherapy/appropriate combinational therapy and surgery, are being widely used for theranostics of cancer patients. Surgery is useful in treating localized tumors, but it is ineffective in treating metastatic tumors, which spread to other organs and result in a high recurrence rate and death. Also, the therapeutic application of free drugs is related to substantial issues such as poor absorption, solubility, bioavailability, high degradation rate, short shelf-life, and low therapeutic index. Therefore, these issues can be sorted out using nano lipid-based carriers (NLBCs) as promising drug delivery carriers. Still, at most, they fail to achieve site targeted drug delivery and detection. This can be achieved by selecting a specific ligand/antibody for its cognate receptor molecule expressed on the cancer cell surface. In this review, we have mainly discussed the various types of ligands used to decorate NLBCs. A list of the ligands used to design nanocarriers to target malignant cells specifically has been extensively undertaken, and the approved ligand decorated lipid-based nanomedicines with their clinical status has been explained in tabulated form to provide a wider scope to the readers regarding ligand coupled NLBCs.
The plant-sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2-pyrone synthase gene from Gerbera hybrida ( GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl-CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12- ACL1-ACC1 using fed-batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 g/L or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for production of TAL and other acetyl-CoA-derived polyketides from low-cost carbon sources.
In high-performance industrial fermentation processes, stirring and aeration may account for significant production costs. Compared to the widely applied Rushton impellers, axial-pumping impellers are known to yield a lower power draw and at the same time improve mixing. However, their lower gas dispersion capability requires stronger agitation, compromising these benefits. Diverse advanced impeller forms have been developed to cope with this challenge. We apply alternating radial and axial impellers and demonstrate strong gas dispersion and energy-efficient mixing for the first time in a large-scale (160 m 3) bioreactor, based on experimental and CFD simulation data. For equal operating conditions (stirrer speed, aeration rate), this setup yielded similar gas hold-ups and better mixing times (-35 %) compared to a classical Rushton-only configuration. Hence, applying a radial impeller on an upper level for improving gas dispersion maintains the benefits of axial impellers in terms of reducing energy demand (up to -50 %). We conclude that this effect is significant only at large-scale, when bubbles substantially expand due to the release of the hydrostatic pressure and have time to coalesce. The work thus extends current knowledge on mixing and aeration of large-scale reactors using classical impeller types.
The osteopontin released from mesenchymal stem cells (MSC) undergoing lineage differentiation can negatively influence the expansion of hematopoietic stem cells (HSCs) in co-culture systems developed for expanding HSCs. Therefore, minimising the amount of osteopontin in the co-culture system is important for the successful ex vivo expansion of HSCs. Towards this goal, a bioengineered 3D-microfibrous matrix that can maintain MSCs in less osteopontin releasing condition has been developed and its influence on the expansion of HSCs has been studied. The newly developed 3D-matrix significantly decreased the release of osteopontin, depending on the MSC culture conditions used during the priming period before HSC seeding. The culture system with the lowest amount of osteopontin facilitated more than 40-fold increase in HSC number in 1 weeks’ time period. Interestingly, the viability of expanded cells and the CD34+ pure population of HSCs found to be the highest in the low osteopontin containing system. Therefore, bioengineered microfibrous 3D-matrices seeded with MSCs, primed under suitable culture conditions can be an improved ex vivo expansion system for HSC culture.
Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately removed to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taken into consideration of residual HCP profile in the product, the dose, dosage form, and administration route etc. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of a Merck investigational stimulator of interferon genes protein (STING) agonist, MK-1454, which is a cyclic dinucleotide synthesized using E. coli cell lysate overexpressing cyclic GMP-AMP synthase (cGAS) as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 API.
The Ocean covers two-third of our planet and has great biological heterogeneity. Marine organisms like algae, vertebrates, invertebrates, and microbes are known to provide many natural products with biological activities as well potent sources of biomaterials for therapeutic, biomedical, biosensors, and climate stabilization. Over the years, the field of biosensors have gained huge attention due to their extraordinary ability in providing early diseases diagnosis and treatment as well as environmental pollutants. This review focuses on various biomaterials (Carbohydrtae polymers, proteins, polyacids etc) of marine origin such as Alginate, Chitin, Chitosan, Fucoidan, Carrageenan, Chondroitin Sulfate (CS), Hyaluronic acid (HA), Collagen, marine pigments, marine nanoparticles, Hydroxyapatite (HAp), Biosilica, lectins, and marine whole cell. Further, it mentions the source of such marine biomaterials and their promising evolution for the development of biosensors that are potent to be employed in the biomedical, environmental science and agricultural sciences domains.
A novel fermentation process was developed in which renewable electricity is indirectly used as a fermentation substrate, synergistically decreasing both the consumption of sugar as a first generation carbon source and emission of the greenhouse gas CO2. To achieve this, a glucose-based process is co-fed with formic acid, which can be generated by capturing CO2 from fermentation offgas followed by electrochemical reduction with renewable electricity. This ‘closed carbon loop’ concept is demonstrated by a case study in which co-feeding formic acid is shown to significantly increase the yield of biomass on glucose of the industrially relevant yeast species Yarrowia lipolytica. First, the optimal feed ratio of formic acid to glucose is established using chemostat cultivations. Subsequently, guided by a dynamic fermentation process model, a fed-batch protocol is developed and demonstrated on laboratory scale. Finally, the developed fed-batch process is proven to be scalable to pilot scale. An extension of this proven concept to also recycle the O2 that is co-generated with the formic acid to the fermentation process for intensification purposes, and a potential further application of the concept to anaerobic fermentations are discussed.
The deformation and detachment of bacterial biofilm are related to the structural and mechanical properties of the biofilm itself. Extracellular polymeric substances (EPS) play an important role on keeping the mechanical stability of biofilms. The understanding of biofilm mechanics and detachment can help to reveal biofilm survival mechanisms under fluid shear and provide insight about what flows might be needed to remove biofilm in a cleaning cycle or for a ship to remove biofilms. However, how the EPS may affect biofilm mechanics and its deformation in flow conditions remains elusive. To address this, a coupled computational fluid dynamic – discrete element method (CFD-DEM) model was developed. The mechanisms of biofilm detachment, such as erosion and sloughing have been revealed by imposing hydrodynamic fluid flow at different velocities and loading rates. The model, which also allows adjustment of the proportion of different functional group of microorganisms in the biofilm, enables the study of the contribution of EPS towards biofilm resistance to fluid shear stress. Furthermore, the stress-strain curves during biofilm deformation have been captured by loading and unloading fluid shear stress to study the viscoelastic properties of the biofilm.
Three-dimensional (3D) bioprinting shows great potential for autologous vascular grafts due to its simplicity, accuracy, and flexibility. 6mm diameter vascular grafts are used in clinic. However, producing small-diameter vascular grafts are still an enormous challenge. Normally, sacrificial hydrogels are used as temporary lumen support to mold tubular structure which will affect the structure’s stability. In this study, we develop a new bioprinting approach to fabricating small-diameter vessel using two-step crosslinking process. ¼ lumen wall of bioprinted gelatin mechacrylate (GelMA) flat structure is exposed to ultraviolet (UV) light briefly for having certain strength, while ¾ lumen wall shows as concave structure remained uncrosslinked. Pre-crosslinked flat structure is merged towards the uncrosslinked concave structure. Two individual structures will be combined tightly into an intact tubular structure by receiving more UV exposure time. Complicated tubular structures are constructed by these method. Notably, the GelMA-based bioink loaded with smooth muscle cells (SMCs) are bioprinted as the outer layer and human umbilical vein endothelial cells (HUVECs) are seeded onto the inner surface. A bionic vascular vessel with dual layers is fabricated successfully and keeps good viability, and functionality. This study may provide a novel idea for fabricating biomimetic vascular network or other more complicated organs.
Transition of rapid, ready-to-use, and low-cost nucleic acid-based detection technologies from laboratories to points of sample collection has drastically accelerated. However, most of these approaches are still incapable of diagnosis starting from sampling, through nucleic acid isolation and detection in the field. Here, we developed a simple, portable, low-cost, colorimetric, and remotely controllable platform for reliable, high-throughput, and rapid diagnosis using loop mediated isothermal amplification (LAMP) assays. It consists of a thermally isolated cup, low-cost electronic components, a polydimethylsiloxane sample well, and a fast prototyped case that covers electronic components. The steady-state temperature error of the system is less than 1%. We performed LAMP, Colony-LAMP, and Colony PCR reactions using the yaiO2 primer set for Escherichia coli and Pseudomonas aeruginosa samples at 65˚C and 30 min. We detected the end-point colorimetric readouts by the naked eye under day light. We confirmed the specificity and sensitivity of our approach using pure genomic DNA and crude bacterial colonies. We benchmarked our Colony-LAMP detection against Colony PCR. The number of samples tested can easily be modified for higher throughput in our system. We strongly believe that our platform can greatly contribute rapid and reliable diagnosis in versatile operational environments.
Plasmid transfection of mammalian cells is the dominant platform used to produce adeno-associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection-based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation-specific antibody staining and flow cytometry we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5-10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection-based AAV vector manufacturing platforms.
High volumetric productivities can be achieved when perfusion processes are operated at high cell densities. Yet it is fairly challenging to keep high cell density cultures in a steady state over an extended period. Aiming for robust processes, in this study cultures were operated at a constant biomass specific perfusion rate (BSPR). The cell density was monitored with a capacitance probe and a continuous bleed maintained the cell density at the targeted viable cell volume (VCV). Despite our tightly controlled BSPR, a gradual accumulation of ammonium and changes in cell diameter were observed during the production phase for the three different monoclonal antibodies (mAbs). Although a lot of efforts in media optimization have been made to reduce ammonium in fed-batch process, less examples are known about how media components impact the cellular metabolism and thus the quality of monoclonal antibodies in continuous processes. In this work, we show that a continuous Na-pyruvate fed at 2 g/L/day strongly reduced ammonium production and stabilized fucosylation, sialylation and high mannose content for three different mAbs.
Taste is one of the most basic and important sensations that is able to monitor the food quality and avoid intake of potential danger materials. Whether as an inevitable symptom of aging or a complication of cancer treatment, taste loss so seriously affects the patient’s life quality. Taste bud organoids provide a great convenience for the research of taste functions and the underlying mechanisms due to their characteristics of availability, strong maneuverability, and high similarity to the in-vivo taste buds. This review gives a systemic and comprehensive introduction to the preparation and application of taste bud organoids towards chemical sensing mechanisms. For the first, the basic structure and function of taste buds in biomedicine will be brief introduced. Then, the currently available approaches for the preparation of taste bud organoids are summarized and discussed, which are mainly divided into two categories, i.e. stem/progenitor cell-derived approach and tissue-derived approach. For the next, different applications of taste bud organoids in biomedicine are outlined based on their central roles such as disease modeling, biological sensing, gene regulation, and signal transduction. Finally, the current challenges, future development trends and prospects of research in taste bud organoids are proposed and discussed.
Bioconversion process with a single target product often lacks economic competitiveness owing to incomplete use of raw material and high costs of downstream processing (DSP). Here, we show with the microbial conversion of crude glycerol that an integrated strain engineering and catalytic conversion of the so-called byproducts can greatly improve DSP and the process economy. Specifically, Clostridium pasteurianum was first adapted to increased concentration of crude glycerol in a novel automatic laboratory evolution system. At m3 scale bioreactor the strain achieved a simultaneous production of 1,3-propanediol (PDO), acetic and butyric acids at 81.21, 18.72 and 11.09 g/L within only 19 h, respectively, representing the most efficient fermentation of crude glycerol to targeted products. A heterogeneous catalytic step was developed and integrated into the DSP process to obtain high-value methyl esters from acetic and butyric acids at high yields. The co-production of the esters also greatly simplified the recovery of PDO. For example, a cosmetic grade PDO (96% PDO) was easily obtained by a simple single-stage distillation process (with an overall yield more than 77%). This integrated approach provides an industrially attractive route for a complete use of the raw material with the simultaneous production of three appealing products which greatly improve the process economy and ecology.