Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α-D-glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. Using G1P at a concentration excluding the substrate-bound E-Glc, the product ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group transfer reactions by hydrolytic enzymes.
Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms due to the ability to produce complex biologics, use of simple chemically-defined, animal component-free media, robustness of host cells, and biosafety. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous techno-economic models or analyses have been published. Here we present techno-economic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4-11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes.
Acidithiobacillus ferrooxidans cells can oxidize iron and sulfur and are key members of the microbial biomining communities that are exploited in the large-scale bioleaching of metal sulfide ores. Some minerals are recalcitrant to bioleaching due to presence of other inhibitory materials in the ore bodies. Additives are intentionally included in processed metals to reduce environmental and microbially influenced corrosion. We have previously reported a new aerobic corrosion mechanism where A. ferrooxidans cells combined with pyrite and chloride can oxidize low grade stainless steel (SS304) with a thiosulfate-mediated mechanism. Here we explore process conditions and genetic engineering of the cells to enable corrosion of a higher grade steel (SS316). The addition of elemental sulfur and an increase in the cell loading resulted in a 74% increase in the corrosion of SS316 as compared to sulfur- and cell-free control experiments. The overexpression of the endogenous rus gene, which is involved in the cellular iron oxidation pathway, led to further 85% increase in the corrosion of the steel. Thus, the modification of the culturing conditions and cell line, led to a more than 3-fold increase in the corrosion of SS316 stainless steel, such that 15% of the metal coupons was dissolved in just 2 weeks. This work demonstrates how the engineering of cells and the optimization of their cultivation conditions can be used to discover conditions that lead to the corrosion of a complex metal target.
Biofilms commonly develop in flowing aqueous environments, where the flow causes the biofilm to deform. Because biofilm deformation affects the flow regime, and because biofilms behave as complex heterogeneous viscoelastic materials, few models are able to predict biofilm deformation. In this study, a phase field continuum model coupled with the Oldroyd-B constitutive equation was developed and used to simulate biofilm deformation. The accuracy of the model was evaluated using two types of biofilms: a synthetic biofilm, made from alginate mixed with bacterial cells, and a Pseudomonas aeruginosa biofilm. Shear rheometry was used to experimentally determine the mechanical parameters for each biofilm, as inputs for the model. Biofilm deformation under fluid flow was monitored experimentally using optical coherence tomography. The fit between the experimental and modeling geometries after fluid-driven deformation was very good, with relative errors of 12.8% for synthetic biofilm and 22.2% for homogenized P. aeruginosa biofilm. This is the first demonstration of the effectiveness of a viscoelastic phase field biofilm model. This model provides an important tool for predicting biofilm viscoelastic deformation. It also can benefit the design and control of biofilms in engineering systems.
Abstract This study describes the response of Arthrospira platensis to a variety of temperature conditions as reflected in variations of photosynthetic parameters, pigmentation, and biomass productivity in indoor photobioreactor (PBR) cultivations. These experiments are designed to better understand the impact of temperature, seasonal variations, and acclimation effects on outdoor biomass production. The irradiance level and temperature range (20 – 39°C) are chosen to enable modeling of semi-continuous operation of large-scale outdoor PBR deployments. Overall, the cultivations were quite stable with some pigment-related instabilities after prolonged high temperature exposure. Changes in productivity with temperature, as reflected in measured photosynthetic parameters, are immediate and mainly attributable to the temperature dependence of the photosaturation parameter, a secondary factor being variation in pigment content on a longer time scale corresponding to turnover of the culture population. Though pigment changes have minimum impact on productivity, prolonged exposure at 35°C and above yields a clear degradation in performance. Productivities in a semi-continuous operation are quantitatively reproduced with a productivity model incorporating photosynthetic parameters measured herein. This study confirms the importance of temperature for biomass and pigment production in Arthrospira cultivations and provides a basis for risk assessments related to temperature mitigation for large-scale outdoor cultivations. Keywords: Arthrospira Platensis, photosynthetic parameters, pigment production, productivity modeling, photobioreactors
Vaccines provide effective protection against many infectious diseases as well as therapeutics for some serious diseases, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, that was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a higher doubling time in both 2D and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.
Previously, our lab developed high molecular weight (MW) tense (T) state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this work, we prepared PolybHb in the T- and relaxed (R) quaternary state with ultrahigh MW (> 500 kDa) with varying cross-link densities and investigated the effect of MW on key biophysical properties (i.e., O2 affinity, cooperativity coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, autoxidation, and haptoglobin-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e. cross-linker:bovine Hb (bHb) molar ratio, gas/liquid exchange time, temperature during dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T-state and R-state PolybHbs. Our results showed that, the duration of the fast-step autoxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHb exhibited significantly higher biomolecular rate constants for binding to haptoglobin and unimoleular O2 offloading rate constants compared to R-state PolybHb. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHb. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.
Inflammatory breast cancer (IBC), a rare form of breast cancer associated with increased angiogenesis and metastasis, is largely driven by tumor-stromal interactions with the vasculature and the extracellular matrix (ECM). However, there is currently a lack of understanding of the role these interactions play in initiation and progression of the disease. In this study, we developed the first three-dimensional, in vitro, vascularized, IBC platform to quantify the spatial and temporal dynamics of tumor-vasculature and tumor-ECM interactions specific to IBC. Platforms consisting of collagen type 1 ECM with an endothelialized blood vessel were cultured with IBC cells, MDA-IBC3 (HER2+) or SUM149 (triple negative), and for comparison to non-IBC cells, MDA-MB-231 (triple negative). An acellular collagen platform with an endothelial blood vessel served as control. SUM149 and MDA-MB-231 platforms exhibited a significantly (p<0.05) higher vessel permeability and decreased endothelial coverage of the vessel lumen compared to the control. Both IBC platforms, MDA-IBC3 and SUM149, expressed higher levels of VEGF (p<0.05) and increased collagen ECM porosity compared to non-IBC MDA-MB-231 (p<0.05) and control (p<0.01) platforms. Additionally, unique to the MDA-IBC3 platform, we observed progressive sprouting of the endothelium over time resulting in viable vessels with lumen. The newly sprouted vessels encircled clusters of MDA-IBC3 cells replicating a feature of in vivo IBC. The IBC in vitro vascularized platforms introduced in this study model well-described in vivo and clinical IBC phenotypes and provide an adaptable, high throughout tool for systematically and quantitatively investigating tumor-stromal mechanisms and dynamics of tumor progression.
Fluorescent in situ hybridization (FISH) has been extensively used in the past decades for the detection and localization of nucleic acid sequences or of the microorganisms themselves within samples. However, a mechanistic approach of the whole FISH process is still missing, and the main limiting steps for the hybridization to occur remain unclear. In here, FISH is approached as a particular case of a diffusion-reaction kinetics, where molecular probes move from the hybridization solution to the target RNA site within the cells. Based on literature models, the characteristic times taken by different molecular probes to diffuse across multiple cellular barriers, and the reaction time associated with the formation of the duplex molecular probe-RNA were estimated. Structural and size differences at the membrane level of bacterial and animal cells were considered. For bacterial cells, the limiting step for diffusion is likely to be the peptidoglycan layer (characteristic time of 2700-4524 s), whereas for animal cells the limiting step should be the diffusion of the probe through the bulk (1.8-5.0 s) followed by the diffusion through the lipid membrane (1 s). The information provided here may serve as a basis to optimize FISH protocols.
Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this work, a rapid, highly efficient and easily manipulated base editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base editing window could be readily and efficiently achieved by the pCBEso system without requiring double strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5. With this tool, the roles of the key genes involving in N-acetyl-glucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild type when glucose or GlcNAc was used as the sole carbon source. Such a base editing system could be readily applied to other EEB. This work not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1, but also accelerates the robust construction of engineered strains for environmental bioremediation.
Tissue constructs of physiologically relevant scale require a vascular system to maintain cell viability. However, in vitro vascularization of engineered tissues is still a major challenge. Successful approaches are based on a feeder layer (FL) to support vascularization. Here, we investigated whether the supporting effect on the self-assembled formation of vascular-like structures by microvascular endothelial cells (mvECs) originates from the FL itself or from its extracellular matrix (ECM). Therefore, we compared the influence of ECM, either derived from adipose-derived stem cells (ASCs) or adipogenic differentiated ASCs, with the classical approaches based on a cellular FL. All cell-derived ECM (cdECM) substrates enable mvEC growth with high viability. Vascular-like structure formation was visualized by immunofluorescence staining of endothelial surface protein CD 31 and can be observed on all cdECM and FL substrates but not on control substrate collagen I. On adipogenic differentiated ECM longer and higher branched structures can be found compared to stem cell cdECM. An increased concentration of pro-angiogenic factors can be found in cdECM substrates and FL approaches compared to controls. Finally, expression of proteins associated with tube formation (E-selectin and thrombomodulin) was confirmed. These results highlight cdECM as promising biomaterial for in vitro vascularization in adipose tissue engineering.
We report on the development of a new model of alveolar air-tissue interface consisting of an array of suspended hexagonal monolayers of gelatin nanofibers supported by microframes and a microfluidic device for the patch integration. The suspended monolayers are deformed to a central displacement of 40-80 μm at the air-liquid interface by application of air pressure in the range of 200-1000 Pa. With respect to the diameter of the monolayers that is 500 μm, this displacement corresponds to a linear strain of 2-10% in agreement with the physiological strain range in the lung alveoli. The culture of A549 cells on the monolayers for an incubation time 1-3 days showed viability in the model. We exerted a periodic strain of 5% at a frequency of 0.2 Hz during 1 hour to the cells. We found that the cells were strongly coupled to the nanofibers, but the strain reduced the coupling and induced remodeling of the actin cytoskeleton, which led to a better tissue formation. Our model can serve as a versatile tool in lung investigations such as in inhalation toxicology and therapy.
Hypertension is a major risk factor for cardiovascular diseases, with high prevalence in low- and high-income countries. Among the various antihypertensive therapeutic strategies, synthetic Angiotensin I-converting enzyme inhibitors (ACEI) are one of the most used pharmacological agents. However, their use in hypertension therapy has been linked to various side effects. In recent years considerable research has been performed on the use of food-derived ACEI peptides (ACEIp) as antihypertensive agents. Although promising, the industrial production of these ACEIp through conventional methods, such as chemical synthesis and enzymatic hydrolysis of food proteins, has been proven troublesome and expensive. Limitations to the large-scale production of ACEIp for functional foods and supplements can be overcome by producing the precursors of these peptides in heterologous hosts. Bacterial hosts have been the privileged choice, particularly to test the success of the genetic engineering strategies, but new platforms based on plants and microalgae have also been emerging. This work provides an overview of the state of antihypertensive therapy, focusing on ACEI, illustrates the latest advances on ACEIp research, and describes current genetic engineer-based approaches for the heterologous production of ACEIp for antihypertensive therapy.
Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5-kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with specific sites of DNA double strand breaks (DSBs) by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and 3 clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at small guide RNA (sgRNA) target sites. Our results indicated the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammal cells, but also inherently caused mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.
The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a non-encapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for a bacterial vaccine production. Batch, fed-batch and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least 4-fold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by Western blot and animal immunization assays, indicating that, so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole cell vaccine production, since it can be integrated to the cell separation process keeping the same vaccine quality.
Mussel adhesive proteins (MAPs) have great potential as bioglues, in particular in wet conditions. Although in vivo residue-specific incorporation of 3,4-dihydroxyphenylalanine (Dopa) in tyrosine-auxotrophic Escherichia coli cells allows production of bioengineered MAPs (bMAPs), the low production yield hinders the practical application of bMAPs. Such low production yield of Dopa-incorporated bMAPs (Dopa-bMAPs) was known to be caused by low translational activity of a noncanonical amino acid, Dopa, in E. coli cells. Herein, in order to enhance the production yield of Dopa-bMAPs, we investigated the coexpression of Dopa-recognizing tyrosyl-tRNA synthetases (TyrRSs). In order to use the Dopa-specific Methanococcus jannascii TyrRS (MjTyrRS-Dopa), we altered the anti-codon of tyrosyl-tRNA amber suppressor into AUA (MjtRNATyrAUA) to recgonize a tyrosine codon (MjtRNATyrAUA). Co-overexpression of MjTyrRS-Dopa and MjtRNATyrAUA increased the production yield of Dopa-MAP by 57%. Similarly, overexpression of E. coli TyrRS (EcTyrRS) led to a 72% higher production yield of Dopa-incorporated bMAP. Even with coexpression of Dopa-recognizing TyrRSs, Dopa-bMAPs have a high Dopa incorporation yield (over 90%) compared to Dopa-bMAPs prepared without any coexpression of TyrRS.
Clinical use of pancreatic beta islets for regenerative medicine applications requires mass production of functional cells. Current technologies are insufficient for large-scale production in a cost-efficient manner. Here, we evaluate advantages of a porous cellulose scaffold and demonstrate scale-up to a wicking-matrix bioreactor as a platform for culture of human endocrine cells. Scaffold modifications were evaluated in a multi-well platform to find the optimum surface condition for pancreatic cell expansion followed by bioreactor culture to confirm suitability. Preceding scale-up, cell morphology, viability and proliferation of primary pancreatic cells were evaluated. Two optimal surface modifications were chosen and evaluated further for insulin secretion, cell morphology and viable cell density for human induced pluripotent stem cell-derived pancreatic cells at different stages of differentiation. Scale-up was accomplished with uncoated, amine-modified cellulose in a miniature bioreactor, and insulin secretion and cell metabolic profiles were determined for 13 days. We achieved 10-fold cell expansion in the bioreactor along with a significant increase in insulin secretion compared with cultures on tissue-culture plastic. Our findings define a new method for expansion of pancreatic cells on wicking-matrix cellulose platform to advance cell therapy biomanufacturing for diabetes.
The rare ginsenosides are recognized as the functionalized molecules after oral administration of Panax ginseng and its products. The sources of rare ginsenosides are extremely limited because of low ginsenoside contents in wild plants, hindering their application in functional foods and drugs. We developed an effective combinatorial biotechnology approach including tissue culture, immobilization, and hydrolyzation methods. Rh2 and nine other rare ginsenosides were produced by MeJA-induced culture of adventitious roots in a 10 L bioreactor associated with enzymatic hydrolysis using six β-glycosidases and their combination with yields ranging from 5.54-32.66 mg L-1. The yield of Rh2 was furthermore increased 7% by using immobilized BglPm and Bgp1 in optimized pH and temperature condition, with the highest yield reaching 51.17 mg L-1 (17.06% of PPD-type ginsenosides mixture). Our combinatorial biotechnology method provides a highly efficient approach to acquiring diverse rare ginsenosides, replacing direct extraction from Panax plants, and can also be used to supplement yeast cell factories.