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Biotechnology and Bioengineering | Page 2

Yu Wang, Ye Liu, Junwei Li, Yi Yang, Xiaomeng Ni, Haijiao Cheng, Teng Huang, Yanmei Guo, Hongwu Ma, Ping Zheng, Meng Wang, Jibin Sun, Yanhe Ma
CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing...
July 17, 2019: Biotechnology and Bioengineering
Caymen Novak, Eric Horst, Charles Taylor, Catherine Liu, Geeta Mehta
Breast cancer cells experience a range of shear stresses in the tumor microenvironment (TME). However most current in vitro three-dimensional (3D) models fail to systematically probe the effects of this biophysical stimuli on cancer cell metastasis, proliferation and chemoresistance. To investigate the roles of shear stress within the mammary and lung pleural effusion TME, a bioreactor capable of applying shear stress to cells within a 3D extracellular matrix was designed and characterized. Breast cancer cells were encapsulated within an interpenetrating network (IPN) hydrogel and subjected to shear stress of 5...
July 17, 2019: Biotechnology and Bioengineering
Sheng-Jie Yue, Chen Song, Song Li, Peng Huang, Shu-Qi Guo, Hong-Bo Hu, Wei Wang, Xue-Hong Zhang
Cinnabarinic acid is a valuable phenoxazinone that has broad applications in the pharmaceutical, chemical and dyeing industries. However, few studies have investigated the production of cinnabarinic acid or its derivatives using genetically engineered microorganisms. Herein, an efficient synthetic pathway of cinnabarinic acid was designed and constructed in Pseudomonas chlororaphis GP72 for the first time, which was more straightforward and robust than the known eukaryotic biosynthetic pathways. First, we screened and identified trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) dehydrogenases from Escherichia coli MG1655 (encoded by entA), Streptomyces sp...
July 17, 2019: Biotechnology and Bioengineering
Emmanuel Anane, Ángel Córcoles García, Benjamin Haby, Sebastian Hans, Niels Krausch, Manuel Krewinkel, Peter Hauptmann, Peter Neubauer, Mariano Nicolas Cruz-Bournazou
Concentration gradients that occur in large industrial-scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities so as to reduce the risk of failure during process scale-up. Although there are various scale-down techniques to study these effects, scale-down strategies are rarely applied in the early developmental phases of a bioprocess as they have not yet been implemented on small scale parallel cultivation devices...
July 17, 2019: Biotechnology and Bioengineering
Daniel A M Pais, Rui M C Portela, Manuel J T Carrondo, Inês A Isidro, Paula M Alves
The process analytical technology (PAT) initiative shifted the bioprocess development mindset towards real-time monitoring and control tools to measure relevant process variables online, and acting accordingly when undesirable deviations occur. Online monitoring is especially important in lytic production systems in which released proteases and changes in cell physiology are likely to affect product quality attributes, as is the case of the insect cell-baculovirus expression vector system (IC-BEVS), a well-established system for production of viral vectors and vaccines...
July 17, 2019: Biotechnology and Bioengineering
Sang Woo Lee, Soyoung Hong, Boyoung Jung, Soo Yeon Jeong, Jaehee Byeon, Gi Seok Jeong, Jaesoon Choi, Chang Mo Hwang
The purpose of this study was to demonstrate self-organizing in vitro multicellular tumor spheroid (MCTS) formation in a microfluidic system and to observe the behavior of MCTSs under controlled microenvironment. The employed microfluidic system was designed for simple and effective formation of MCTSs by generating nutrient and oxygen gradients. The MCTSs were composed of cancer cells, vascular endothelial cells, and type I collagen matrix to mimic the in vivo tumor microenvironment (TME). Cell culture medium was perfused to the microfluidic device loaded with MCTSs by a passive fluidic pump at a constant flow rate...
July 11, 2019: Biotechnology and Bioengineering
Azita Fazelkhah, Samaneh Afshar, Katrin Braasch, Michael Butler, Elham Salimi, Greg Bridges, Douglas Thomson
The ability to monitor the status of cells during nutrient limitation is important for optimizing bioprocess growth conditions in batch and fed-batch cultures. The activity level of Na+ /K+ ATPase pumps and cytoplasm ionic concentrations are directly influenced by the nutrient level, and thus, cytoplasm conductivity can be used as a marker-less indicator of cell status. In this work, we monitored the change in cytoplasm conductivity of Chinese hamster ovary cells during nutrient deprivation and reintroduction...
July 9, 2019: Biotechnology and Bioengineering
Youngbin Baek, Parinaz Emami, Nripen Singh, Andrew Ilott, Erinc Sahin, Andrew Zydney
Histidine is a frequently used buffer in the final formulation of many commercialized monoclonal antibodies, with the histidine helping to stabilize the antibody during storage in addition to its buffering function. The objective of this study was to examine the stereospecificity of any histidine-antibody interactions using a combination of experimental studies and molecular dynamic simulations. Isothermal titration calorimetry provided evidence of weak stereospecific interactions, with the antibody showing approximately 2-4 additional interaction sites for D- versus L-histidine...
July 9, 2019: Biotechnology and Bioengineering
Robin Legner, Alexander Wirtz, Tim Koza, Till Tetzlaff, Anna Nickisch-Hartfiel, Martin Jaeger
Compact 1 H NMR and Raman spectrometers were used for real-time process monitoring of an alcoholic fermentation in a continuous flow reactor. Yeast cells catalyzing the sucrose conversion were immobilized in alginate beads floating in the reactor. The spectrometers proved to be robust and could be easily attached to the reaction apparatus. As environmentally friendly analysis methods, 1 H NMR and Raman spectroscopy were selected to match the resource- and energy-saving process. Analyses took only few seconds to minutes compared to chromatographic procedures and were therefore suitable for real-time control realized as a feedback-loop...
July 9, 2019: Biotechnology and Bioengineering
Heejae Kim, Andrew Gaynor, Wilfred Chen
While immunoglobulins find ubiquitous use in biotechnology as static binders, recent developments have created pro-antibodies that enable orthogonal switch-like behavior to antibody function. Previously, peptides with low binding affinity have been genetically fused to antibodies, to proteolytically control binding function by blocking the antigen-binding site. However, development of these artificial blockers requires panning for the right peptide sequences that reversibly affect antigen affinity for each antibody...
July 9, 2019: Biotechnology and Bioengineering
Mohamed A Elkhodiry, Mariève D Boulanger, Omar Bashth, Jean-François Tanguay, Gaétan Laroche, Corinne A Hoesli
The expansion of human peripheral blood endothelial progenitor cells to obtain therapeutically relevant endothelial colony forming cells (ECFCs) has been commonly performed on xeno-derived extracellular matrix proteins. For cellular therapy applications, xeno-free culture conditions are desirable to improve product safety and reduce process variability. We have previously described a novel fluorophore-tagged RGD peptide (RGD-TAMRA) that enhanced the adhesion of mature endothelial cells in vitro. To investigate whether this peptide can replace animal-derived extracellular matrix proteins in the isolation and expansion of ECFCs, peripheral blood mononuclear cells from 22 adult healthy adult donors were seeded on RGD-TAMRA-modified polystyrene culture surfaces...
July 9, 2019: Biotechnology and Bioengineering
Elisa M de Medeiros, John A Posada, Henk Noorman, Rubens Maciel Filho
Syngas fermentation is one of the bets for the future sustainable bio-based economy due to its potential as intermediate step in the conversion of waste carbon to ethanol fuel and other chemicals. Integrated with gasification and suitable downstream processing, it may constitute an efficient and competitive route for the valorization of various waste materials, especially if systems engineering principles are employed targeting process optimization. In this study, a dynamic multi-response model is presented for syngas fermentation with acetogenic bacteria in a continuous stirred tank reactor, accounting for gas-liquid mass transfer, substrate (CO, H2 ) uptake, biomass growth and death, acetic acid re-assimilation and product selectivity...
July 9, 2019: Biotechnology and Bioengineering
Leander A H Petersen, Christian Lieven, Subir K Nandy, John Villadsen, Sten B Jørgensen, Ib Christensen, Krist V Gernaey
The methanotrophic bacterium Methylococcus capsulatus is capable of assimilating methane and oxygen into a protein rich biomass, however the diverse metabolism of the microorganism also allows for several undesired co-metabolic side-reactions to occur. In this study, the ammonia co-metabolism in Methylococcus capsulatus is investigated using pulse experiments. Surprisingly Methylococcus capsulatus oxidizes ammonia to nitrate through a yet unknown mechanism, and fixes molecular nitrogen even at a high dissolved oxygen tension...
July 9, 2019: Biotechnology and Bioengineering
Luis Caspeta, Jesús Coronel, Arturo Montes de Oca, Eduardo Abarca, Lidia González, Alfredo Martínez
Thermal damage, high osmolarity and ethanol toxicity in the yeast Saccharomyces cerevisiae limit titer and productivity in fermentations to produce ethanol. We show that long-term adaptive laboratory evolution (ALE) at 39.5°C generates thermotolerant yeast strains, which increased ethanol yield and productivity by 10% and 70%, in 2% glucose fermentations. From these strains, which also tolerate elevated-osmolarity, we selected a stable one, namely a strain lacking chromosomal duplications. This strain (TTY23) showed reduced mitochondrial metabolism and high proton efflux, and therefore lower ethanol tolerance...
July 8, 2019: Biotechnology and Bioengineering
Julien Robin, Marc Gueroult, Randa Cheikhrouhou, Marie Guicherd, Vinciane Borsenberger, Alain Marty, Florence Bordes
Claviceps purpurea bifunctional Δ12-hydroxylase/desaturase, CpFAH12, and monofunctional desaturase CpFAD2, share 86% of sequence identity. In order to identify the underlying determinants of the hydroxylation/desaturation specificity, chimeras of these two enzymes were tested for their fatty acid production in an engineered Yarrowia lipolytica strain. It reveals that transmembrane helices are not involved in the hydroxylation/desaturation specificity whereas all cytosolic domains have an impact on it. Especially, replacing the CpFAH12 cytosolic part near the second histidine-box by the corresponding CpFAD2 part annihilates all hydroxylation activity...
July 8, 2019: Biotechnology and Bioengineering
Maria Nikmanesh, Limary M Cancel, Zhong-Dong Shi, John M Tarbell
It is widely believed that differentiation of embryonic stem cells (ESC) into viable endothelial cells (EC) for use in vascular tissue engineering can be enhanced by mechanical forces. In our previous work (Nikmanesh, Shi et al. 2012) we reported that shear stress enhanced important EC functional genes on a CD31+ /CD45- cell population derived from mouse ESC committed to the EC lineage. In the present study, in contrast to the effects of shear stress on this cell population, we observed that cyclic strain significantly reduced the expression of EC-specific marker genes (vWF, VE-cadherin, PECAM-1), tight junction protein genes (ZO-1, OCLD, CLD5), and vasoactive genes (eNOS, ET1), while it didn't alter the expression of COX-2...
July 8, 2019: Biotechnology and Bioengineering
Elisabete C Costa, Daniel N Silva, André F Moreira, Ilídio J Correia
Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a light scattering phenomenon that limits the light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely i) simple immersion in an aqueous solution with high RI; ii) delipidation and dehydration followed by RI matching; iii) delipidation and hyperhydration followed by RI matching; and iv) hydrogel embedding followed by delipidation and RI matching...
July 8, 2019: Biotechnology and Bioengineering
Jie Hu, Kang Xiao, Birui Jin, Xuyang Zheng, Fanpu Ji, Dan Bai
Bridging the unmet need of efficient point-of-care testing (POCT) in biomedical engineering research and practice with the emerging development in artificial synthetic xeno nucleic acids (XNAs), this review summarized the recent development in paper-based POCT using XNAs as sensing probes. Alongside the signal transducing mode and immobilization methods of XNA probes, detailed evaluation of probe performance was disclosed. With these new aspects, both researchers in synthetic chemistry / biomedical engineering and physicians in clinical practice could gain new insights in designing, manufacturing and choosing suitable reagents and techniques for POCT...
July 8, 2019: Biotechnology and Bioengineering
Li-Gong Zou, Srinivasan Balamurugan, Tian-Bao Zhou, Jia-Wen Chen, Da-Wei Li, Wei-Dong Yang, Jie-Sheng Liu, Hong-Ye Li
There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes...
July 8, 2019: Biotechnology and Bioengineering
Youneng Tang, Zhiming Zhang, Bruce E Rittmann, Hyung-Sool Lee
Anaerobic oxidation of methane coupled to denitrification (AOM-D) in a membrane biofilm reactor (MBfR), a platform used for efficiently coupling gas delivery and biofilm development, has attracted attention in recent years due to the low cost and high availability of methane. However, experimental studies have shown that the nitrate removal flux in the CH4 -based MBfR (<1.0 g N/m2 -day) is about one order of magnitude smaller than that in the H2 -based MBfR (1.1-6.7 g N/m2 -day). A one-dimensional multispecies biofilm model predicts that the nitrate-removal flux in the CH4 -based MBfR is limited to <1...
June 26, 2019: Biotechnology and Bioengineering
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