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Biotechnology and Bioengineering

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https://www.readbyqxmd.com/read/28436027/production-and-characterization-of-active-recombinant-human-factor-ii-with-consistent-sialylation
#1
Jeong H Lee, Jason Reier, Kelley M Heffner, Christopher Barton, David Spencer, Albert E Schmelzer, Raghavan Venkat
Coagulation factor II (prothrombin; FII) is the pre-proteolyzed precursor to thrombin in the coagulation cascade. It has ten sites of gamma-carboxylation, which are required for its bioactivity, and is N-glycosylated at three of four putative sites. Production of recombinant human FII (rhFII) using a platform fed-batch process designed for monoclonal antibody production resulted in low levels of gamma-carboxylation and sialylation. There have not been any prior reports of successful process development and clinical manufacture of rhFII with optimal, consistent gamma-carboxylation and sialylation...
April 24, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28436007/overexpression-of-the-regulatory-subunit-of-glutamate-cysteine-ligase-enhances-monoclonal-antibody-production-in-cho-cells
#2
Camila A Orellana, Esteban Marcellin, Peter P Gray, Lars K Nielsen
For decades, Chinese hamster ovary (CHO) cells have been the preferred host for therapeutic monoclonal antibody (mAb) production; however, increasing mAb titer by rational engineering remains a challenge. Our previous proteomic analysis in CHO cells suggested that a higher content of glutathione (GSH) might be related to higher productivity. GSH is an important antioxidant, cell detoxifier, and is required to ensure the formation of native disulfide bonds in proteins. To investigate the involvement of GSH in mAb production, we generated stable CHO cell lines overexpressing genes involved in the first step of GSH synthesis; namely the glutamate-cysteine ligase catalytic subunit (Gclc) and the glutamate-cysteine ligase modifier subunit (Gclm)...
April 24, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28436005/fast-and-reliable-strain-characterization-of-streptomyces-lividans-through-micro-scale-cultivation
#3
Joachim Koepff, Matthias Keller, C Konstantinos Tsolis, Tobias Busche, Christian Rückert, Mohamed B Hamed, Jozef Anné, Joern Kalinowski, Wolfgang Wiechert, Anastassios Economou, Marco Oldiges
Filamentous organisms of the genus Streptomyces play an important role in industrial production processes, due to their extensive secondary metabolism variability, as well as their ability to secrete efficiently large amounts of (heterologous) proteins. While genetic engineering tools are available to rapidly build up large strain libraries, the subsequent strain screening and bioprocess development still constitutes a bottleneck. This is due to the lack of reliable parallelized and accelerated cultivation techniques for morphologically challenging organisms...
April 24, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28436004/engineering-of-an-h2-o2-auto-scavenging-in-vivo-cascade-for-pinoresinol-production
#4
Yongkun Lv, Xiaozhong Cheng, Guocheng Du, Jingwen Zhou, Jian Chen
Pinoresinol is a natural lignan with a high market value that has potential pharmacological and food supplement applications. Pinoresinol is currently isolated from plants, which suffers from low efficiency and yield. To produce pinoresinol from inexpensive and industrially available eugenol, an in vivo enzymatic cascade composed of vanillyl alcohol oxidase and peroxidase was designed, which scavenges H2 O2 automatically and eliminates protein purification and cofactor addition. Two peroxidases were screened and identified from Escherichia coli BL21 (DE3), and tested in the enzymatic cascade...
April 24, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28436003/bio-coda-a-new-analysis-class-to-ensure-accurate-and-precise-monoclonal-antibody-cqa-estimation-and-control
#5
Anthony Lonardo, Babita Saxena Parekh, Arvind Srivastava
Monoclonal antibody production processes control Critical Quality Attributes (CQAs) which are the measures that provide proof of a product's identity and quality. Critical decisions rely on the accuracy and precision of these measures, as well as their appropriate statistical treatment. Many measures require special attention. For example the charge heterogeneity CQA measured by Ionic Exchange Chromatography (IEC) reports proportions or percentages of the total integrated peak area of known species. Since proportions sum to a constant (1 or 100%) they fall into a special class of data called compositional data which have a unit sum constraint and therefore an inherent correlation...
April 24, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28436002/characterization-of-the-chondrocyte-secretome-in-photoclickable-poly-ethylene-glycol-hydrogels
#6
Margaret C Schneider, Christopher A Barnes, Stephanie J Bryant
Poly(ethylene glycol) (PEG) hydrogels are highly tunable platforms that are promising cell delivery vehicles for chondrocytes and cartilage tissue engineering. In addition to characterizing the type of extracellular matrix (ECM) that forms, understanding the types of proteins that are secreted by encapsulated cells may be important. Thus, the objectives for this study were to characterize the secretome of chondrocytes encapsulated in PEG hydrogels and determine whether the secretome varies as a function of hydrogel stiffness and culture condition...
April 24, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28425561/creating-homogenous-strain-distribution-within-3d-cell-encapsulated-constructs-using-a-simple-and-cost-effective-uniaxial-tensile-bioreactor-design-and-validation-study
#7
Gayathri Subramanian, Mostafa Elsaadany, Callan Bialorucki, Eda Yildirim-Ayan
Mechanical loading bioreactors capable of applying uniaxial tensile strains are emerging to be a valuable tool to investigate physiologically-relevant cellular signaling pathways and biochemical expression. In this study, we have introduced a simple and cost-effective uniaxial tensile strain bioreactor for the application of precise and homogenous uniaxial strains to 3D cell-encapsulated collagen constructs at physiological loading strains (0-12%) and frequencies (0.01-1 Hz). The bioreactor employs silicone-based loading chambers specifically designed to stretch constructs without direct gripping to minimize stress concentration at the ends of the construct and preserve its integrity...
April 20, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28409846/an-in-vitro-synthetic-biology-platform-for-the-industrial-biomanufacturing-of-myo-inositol-from-starch
#8
Chun You, Ting Shi, Yunjie Li, Pingping Han, Xigui Zhou, Yi-Heng Percival Zhang
myo-Inositol (vitamin B8) is widely used in the drug, cosmetic, and food & feed industries. Here we present an in vitro non-fermentative enzymatic pathway that converts starch to inositol in one vessel. This in vitro pathway is comprised of four enzymes that operate without ATP or NAD(+) supplementation. All enzyme BioBricks are carefully selected from hyperthermophilic microorganisms, that is, alpha-glucan phosphorylase from Thermotoga maritima, phosphoglucomutase from Thermococcus kodakarensis, inositol 1-phosphate synthase from Archaeoglobus fulgidus, and Inositol monophosphatase from T...
April 14, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28409838/modulation-and-modeling-of-monoclonal-antibody-n-linked-glycosylation-in-mammalian-cell-perfusion-reactors
#9
Daniel J Karst, Ernesto Scibona, Elisa Serra, Jean-Marc Bielser, Jonathan Souquet, Matthieu Stettler, Hervé Broly, Miroslav Soos, Massimo Morbidelli, Thomas K Villiger
Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity...
April 14, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28409824/a-comparative-genomic-hybridization-approach-to-study-gene-copy-number-variations-among-chinese-hamster-cell-lines
#10
Nandita Vishwanathan, Arpan Bandyopadhyay, Hsu-Yuan Fu, Kathryn C Johnson, Nathan M Springer, Wei-Shou Hu
Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines...
April 14, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28409822/non-hazardous-biocatalytic-oxidation-in-nylon-9-monomer-synthesis-on-a-40-gram-scale-with-efficient-downstream-processing
#11
Sofia Milker, Michael J Fink, Florian Rudroff, Marko D Mihovilovic
This paper describes the development of a biocatalytic process on the multi-dozen gram scale for the synthesis of a precursor to Nylon-9, a specialty polyamide. Such materials are growing in demand, but their corresponding monomers are often difficult to synthesize, giving rise to biocatalytic approaches. Here, we implemented cyclopentadecanone monooxygenase as an Escherichia coli whole-cell biocatalyst in a defined medium, together with a substrate feeding-product removal concept, and an optimized downstream processing (DSP)...
April 14, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28409820/identification-and-control-of-novel-growth-inhibitors-in-fed-batch-cultures-of-chinese-hamster-ovary-cells
#12
Bhanu Chandra Mulukutla, Jaitashree Kale, Taylor Kalomeris, Michaela Jacobs, Gregory W Hiller
Chinese hamster ovary (CHO) cells in culture are known to consume large amounts of nutrients and divert most of them towards byproducts, some of which, including lactate and ammonia, are known to be toxic in nature. Glucose limitation strategies can successfully control lactate accumulation in fed-batch cultures yielding higher peak cell densities and titers. Interestingly, even in such optimized cultures, cell growth slows and eventually stops, indicating the emergence of other factors that negatively affect cell growth...
April 14, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28401637/alcohol-tolerant-mutants-of-cyanobacterium-synechococcus-elongatus-pcc-7942-obtained-by-single-cell-mutant-screening-system
#13
Sayuri Arai, Kayoko Hayashihara, Yuki Kanamoto, Kazunori Shimizu, Yasutaka Hirokawa, Taizo Hanai, Akio Murakami, Hiroyuki Honda
Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO2 , low productivity and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported.n this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10 g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10 g/L IPA...
April 12, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28401536/autodisplay-of-glucose-6-phosphate-dehydrogenase-for-redox-cofactor-regeneration-at-the-cell-surface
#14
Jan Schüürmann, Paul Quehl, Fabian Lindhorst, Kristina Lang, Joachim Jose
Inherent cofactor regeneration is a pivotal feature of whole cell biocatalysis. For specific biotechnological applications, surface display of enzymes is emerging as a tool to circumvent mass transfer limitations or enzyme stability problems. Even complex reactions can be accomplished applying displayed enzymes. Yet, industrial utilization of the technique is still impeded by lacking cofactor regeneration at the cell surface. Here, we report on the surface display of a glucose-6-phoshate dehydrogenase (G6PDH) via Autodisplay to address this limitation and regenerate NADPH directly at the cell surface...
April 12, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28401535/nano-hydroxyapatite-blasted-titanium-surface-affects-pre-osteoblast-morphology-by-modulating-critical-intracellular-pathways
#15
Fábio Bezerra, Marcel R Ferreira, Giselle N Fontes, Célio da Costa Fernandes, Denise C Andia, Nilson C Cruz, Rodrigo A da Silva, Willian F Zambuzzi
Since intracellular signalling pathways are proposed to predict the quality of cell-surface relationship, this study addressed pre-osteoblast behaviour in response to nano hydroxyapatite (HA)-blasted titanium (Ti) surface by exploring critical intracellular pathways and pre-osteoblast morphological change. Physicochemical properties were evaluated by atomic force microscopy and wettability considering water contact angle of three differently texturized Ti surfaces: Machined (Mac), Dual acid-etching (DAE) and nano hydroxyapatite-blasted (nHA)...
April 12, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28401530/a-plug-and-play-pathway-refactoring-workflow-for-natural-product-research-in-escherichia-coli-and-saccharomyces-cerevisiae
#16
Hengqian Ren, Pingfan Hu, Huimin Zhao
Pathway refactoring serves as an invaluable synthetic biology tool for natural product discovery, characterization and engineering. However, the complicated and laborious molecular biology techniques largely hinder its application in natural product research, especially in a high-throughput manner. Here we report a plug-and-play pathway refactoring workflow for high-throughput, flexible pathway construction and in both Escherichia coli and Saccharomyces cerevisiae. Biosynthetic genes were firstly cloned into preassembled helper plasmids with promoters and terminators, resulting in a series of expression cassettes...
April 12, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28398656/a-biomimetic-hydrogel-functionalized-with-adipose-ecm-components-as-a-microenvironment-for-the-3d-culture-of-human-and-murine-adipocytes
#17
Fiona Louis, Pauline Pannetier, Zied Souguir, Didier Le Cerf, Philippe Valet, Jean-Pierre Vannier, Guillaume Vidal, Elise Demange
The lack of relevant in vitro models for adipose tissue makes necessary the development of a more physiological environment providing spatial and chemical cues for the effective maturation of adipocytes. We developed a biofunctionalized hydrogel with components of adipose extracellular matrix: collagen I, collagen VI and the cell binding domain of fibronectin and we compared it to usual 2D cultures on plastic plates. This scaffold allowed 3D culture of mature adipocytes from the preadipocytes cell lines 3T3-L1 and 3T3-F442A, as well as primary Human White Preadipocytes (HWP), acquiring in vivo-like organization, with spheroid shaped adipocytes forming multicellular aggregates...
April 11, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28398594/rapid-in-vitro-screening-for-the-location-dependent-effects-of-unnatural-amino-acids-on-protein-expression-and-activity
#18
Song-Min Schinn, William Bradley, Ashtyn Groesbeck, Jeffrey C Wu, Andrew Broadbent, Bradley C Bundy
The incorporation of unnatural amino acids (uAA) can introduce novel functional groups into proteins site-specifically, with important applications in basic sciences and protein engineering. However, uAA incorporation can impact protein expression and functional activity depending on its location within the protein - a process that is not yet completely understood and difficult to predict. Therefore, practical applications often necessitate a time-consuming optimization of uAA location by individual gene cloning, expressions, purification and evaluations for each location tested...
April 11, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28369757/development-of-rapid-bioconversion-with-integrated-recycle-technology-for-ethanol-production-from-extractive-ammonia-pretreated-corn-stover
#19
Mingjie Jin, Yanping Liu, Leonardo da Costa Sousa, Bruce E Dale, Venkatesh Balan
High enzyme loading and low productivity are two major issues impeding low cost ethanol production from lignocellulosic biomass. This work applied Rapid Bioconversion with Integrated recycle Technology (RaBIT) and Extractive Ammonia (EA) pretreatment for conversion of corn stover (CS) to ethanol at high solids loading. Enzymes were recycled via recycling unhydrolyzed solids. Enzymatic hydrolysis with recycled enzymes and fermentation with recycled yeast cells were studied. Both enzymatic hydrolysis time and fermentation time were shortened to 24 h...
April 1, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28369753/effects-of-plant-density-on-recombinant-hemagglutinin-yields-in-an-agrobacterium-mediated-transient-gene-expression-system-using-nicotiana-benthamiana-plants
#20
Naomichi Fujiuchi, Ryo Matsuda, Nobuyuki Matoba, Kazuhiro Fujiwara
Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM(-1) ) and recombinant protein productivity per unit area-time (g m(-2) /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA)...
April 1, 2017: Biotechnology and Bioengineering
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