Escherichia coli coculture for de novo production of esters derived of methyl-branched alcohols and multi-methyl branched fatty acids

Background A broad diversity of natural and non-natural esters have now been made in bacteria, and in other microorganisms, as a result of original metabolic engineering approaches. However, the fact that the properties of these molecules, and therefore their applications, are largely defined by the structural features of the fatty acid and alcohol moieties, has driven a persistent interest in generating novel structures of these chemicals. Results In this research, we engineered Escherichia coli to synthesize de novo esters composed of multi-methyl-branched-chain fatty acids and short branched-chain alcohols (BCA), from glucose and propionate. A coculture engineering strategy was developed to avoid metabolic burden generated by the reconstitution of long heterologous biosynthetic pathways. The cocultures were composed of two independently optimized E. coli strains, one dedicated to efficiently achieve the biosynthesis and release of the BCA, and the other to synthesize the multi methyl-branched fatty acid and the corresponding multi-methyl-branched esters (MBE) as the final products. Response surface methodology, a cost-efficient multivariate statistical technique, was used to empirical model the BCA-derived MBE production landscape of the coculture and to optimize its productivity. Compared with the monoculture strategy, the utilization of the designed coculture improved the BCA-derived MBE production in 45%. Finally, the coculture was scaled up in a high-cell density fed-batch fermentation in a 2 L bioreactor by fine-tuning the inoculation ratio between the two engineered E. coli strains. Conclusion Previous work revealed that esters containing multiple methyl branches in their molecule present favorable physicochemical properties which are superior to those of linear esters. Here, we have successfully engineered an E. coli strain to broaden the diversity of these molecules by incorporating methyl branches also in the alcohol moiety. The limited production of these esters by a monoculture was considerable improved by a design of a coculture system and its optimization using response surface methodology. The possibility to scale-up this process was confirmed in high-cell density fed-batch fermentations.

Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells

Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.

Media optimization for SHuffle T7 Escherichia coli expressing SUMO-Lispro proinsulin by response surface methodology

Background SHuffle is a suitable Escherichia coli (E. coli) strain for high yield cytoplasmic soluble expression of disulfide-bonded proteins such as Insulin due to its oxidative cytoplasmic condition and the ability to correct the arrangement of disulfide bonds. Lispro is an Insulin analog that is conventionally produced in E. coli as inclusion bodies (IBs) with prolonged production time and low recovery. Here in this study, we aimed to optimize cultivation media composition for high cell density fermentation of SHuffle T7 E. coli expressing soluble Lispro proinsulin fused to SUMO tag (SU-INS construct) to obtain high cell density fermentation. Results Factors including carbon and nitrogen sources, salts, metal ions, and pH were screened via Plackett–Burman design for their effectiveness on cell dry weight (CDW) as a measure of cell growth. The most significant variables of the screening experiment were Yeast extract and MgCl2 concentration, as well as pH. Succeedingly, The Central Composite Design was utilized to further evaluate and optimize the level of significant variables. The Optimized media (OM-I) enhanced biomass by 2.3 fold in the shake flask (2.5 g/L CDW) that reached 6.45 g/L (2.6 fold increase) when applied in batch culture fermentation. The efficacy of OM-I media for soluble expression was confirmed in both shake flask and fermentor. Conclusion The proposed media was suitable for high cell density fermentation of E. coli SHuffle T7 and was applicable for high yield soluble expression of Lispro proinsulin.

Capillary-Like Formations of Endothelial Cells in Defined Patterns Generated by Laser Bioprinting

Bioprinting is seen as a promising technique for tissue engineering, with hopes of one day being able to produce whole organs. However, thick tissue requires a functional vascular network, which naturally contains vessels of various sizes, down to capillaries of ~10 µm in diameter, often spaced less than 200 µm apart. If such thick tissues are to be printed, the vasculature would likely need to be printed at the same time, including the capillaries. While there are many approaches in tissue engineering to produce larger vessels in a defined manner, the small capillaries usually arise only in random patterns by sprouting from the larger vessels or from randomly distributed endothelial cells. Here, we investigated whether the small capillaries could also be printed in predefined patterns. For this purpose, we used a laser-based bioprinting technique that allows for the combination of high resolution and high cell density. Our aim was to achieve the formation of closed tubular structures with lumina by laser-printed endothelial cells along the printed patterns on a surface and in bioprinted tissue. This study shows that such capillaries are directly printable; however, persistence of the printed tubular structures was achieved only in tissue with external stimulation by other cell types.

MPC-7 Clinical features of Diffuse hemispheric glioma, H3 G34-mutant in children and young adults

INTRODUCTION: H3F3A G34R/V mutated gliomas are seen predominantly in children and young adults, and have been proposed as “Diffuse hemispheric glioma, H3 G34-mutant” in cIMPACT-NOW Update 6. However, the clinical features of the tumor have not been fully elucidated. METHODS: We retrospectively reviewed 4 cases with H3G34R mutation among 40 cases diagnosed as glioblastoma under 30 years old or primitive neuroectodermal tumor (PNET) in our hospital. RESULTS: There were one male and three female patients with a median age of 21.5 years (range: 17–27 years). All lesions were localized in the cerebral hemispheres, and the initial symptoms were headache in two cases and seizures in two cases. On imaging, there was one case with poor contrast, and unlike the infiltrative growth pattern of the other three contrasted cases, it showed a well-defined mass lesion. DWI showed high signal in all four cases, reflecting the high cell density in histopathology. All cases were IDH-wildtype. CONCLUSION: Although the patient background and genetic characteristics of the glioma with H3 G34R/V mutation at our institution were generally consistent with previous reports, there were some cases with atypical imaging findings. Further investigation is required for a deeper understanding of the clinical features of this tumor.

High Cell Density Cultivation of Escherichia coli DH5α in Shake Flasks with a New Formulated Medium

High cell density cultivation necessitates cell division and biomass formation, the mechanisms of which remain poorly understood, especially from the cellular energetics perspective. Specifically, the sensing of energy abundance and the channelling of nutritional energy into biomass formation and cell maintenance remains enigmatic at the sensory, effector and decision levels. Thus, optimization of cell growth remains an iterative trial and error process where the principal parameters are growth medium composition and incubation temperature. In this study, a new semidefined formulated medium was shown to be useful for high cell density cultivation of Escherichia coli DH5α (ATCC 53868). Comprising K2HPO4, 12.54; KH2PO4, 2.31; D-Glucose, 4.0; NH4Cl, 1.0; Yeast extract, 12.0; NaCl, 5.0; MgSO4, 0.24; the medium possessed a high capacity phosphate buffer able to moderate pH fluctuations during cell growth known to be detrimental to biomass formation. With glucose and NH4Cl providing the nutrients for initial growth, followed by a lag phase of 3 hours, a maximal optical density of 12.0 was obtained after 27 hours of cultivation at 37 oC and 230 rpm. Yeast extract provides a secondary source of carbon and nitrogen. Maximal optical density obtained in formulated medium was higher than the 10.1, 4.2, and 3.4 obtained in Tryptic Soy Broth, M9 with 1 g/L of yeast extract, and LB Lennox, respectively. Cultivation of E. coli DH5α in formulated medium with 6 g/L of glucose resulted in a longer lag phase of 8 hours and a longer time (68 hours) to attainment of maximal optical density, which marked the upper limit of glucose concentration beyond which biomass formation would be reduced. Specifically, glucose concentration above 6 g/L markedly reduced biomass formation possibly due to the environmental stress arising from low pH in the culture broth. Glucose concentration below 4 g/L, on the other hand, reduced biomass formation through a smaller pool of nutrients serving as biomass building blocks. Deviation from 1:1 molar ratio between glucose and NH4Cl was not detrimental to biomass formation and growth rates. Collectively, a semi-defined formulated medium could increase optical density of E. coli DH5α beyond that of LB Lennox and Tryptic Soy Broth, and may find use in cultivation of cells for applied microbiology research.