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Biotechnology and Bioengineering

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https://www.readbyqxmd.com/read/28941283/intracellular-response-to-process-optimization-and-impact-on-productivity-and-product-aggregates-for-a-high-titer-cho-cell-process
#1
Michael W Handlogten, Allison Lee-O'Brien, Gargi Roy, Sophia V Levitskaya, Raghavan Venkat, Shailendra Singh, Sanjeev Ahuja
A key goal in process development for antibodies is to increase productivity while maintaining or improving product quality. During process development of an antibody, titers were increased from 4 to 10 g/L while simultaneously decreasing aggregates. Process development involved optimization of media and feed formulations, feed strategy, and process parameters including pH and temperature. To better understand how CHO cells respond to process changes, the changes were implemented in a stepwise manner. The first change was an optimization of the feed formulation, the second was an optimization of the medium, and the third was an optimization of process parameters...
September 23, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28941282/engineering-of-cell-membrane-to-enhance-heterologous-production-of-hyaluronic-acid-in-bacillus-subtilis
#2
Adam W Westbrook, Xiang Ren, Murray Moo-Young, C Perry Chou
Hyaluronic acid (HA) is a high-value biopolymer used in the biomedical, pharmaceutical, cosmetic, and food industries. Current methods of HA production, including extraction from animal sources and streptococcal cultivations, are associated with high costs and health risks. Accordingly, the development of bioprocesses for HA production centered on robust 'Generally Recognized as Safe (GRAS)' organisms such as Bacillus subtilis is highly attractive. Here, we report the development of novel strains of B. subtilis in which the membrane cardiolipin (CL) content and distribution has been engineered to enhance the functional expression of heterologously expressed hyaluronan synthase of Streptococcus equisimilis (SeHAS), in turn, improving the culture performance for HA production...
September 23, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28941275/reconstruction-of-lignin-and-hemicelluloses-by-aqueous-ethanol-anti-solvents-to-improve-the-ionic-liquid-acid-pretreatment-performance-of-arundo-donax-linn
#3
Tingting You, Ruizhen Wang, Xueming Zhang, Shri Ramaswamy, Feng Xu
Ionic liquid (IL)-acid pretreatment is known to not only enhance the enzymatic hydrolysis efficiency of lignocellulose but also to generate deposits on the surface of fiber by conventional water regeneration, which retard the increment. In this study, ethanol aqueous solution regeneration was developed as a new method to change the substrates characteristics for IL-acid pretreatment and their effects on the enzymatic hydrolysis were evaluated. Following the IL-acid reaction, the biomass slurry was subjected to ethanol aqueous solution at various concentration...
September 23, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28941274/advancements-in-the-design-and-scalable-production-of-viral-gene-transfer-vectors
#4
REVIEW
David Sharon, Amine Kamen
The last 10 years have seen a rapid expansion in the use of viral gene transfer vectors, with approved therapies and late stage clinical trials underway for the treatment of genetic disorders, and multiple forms of cancer, as well as prevention of infectious diseases through vaccination. With this increased interest and widespread adoption of viral vectors by clinicians and biopharmaceutical industries, there is an imperative to engineer safer and more efficacious vectors, and develop robust, scalable and cost-effective production platforms for industrialization...
September 23, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28941272/burkholderia-cepacia-lipase-a-versatile-catalyst-in-synthesis-reactions
#5
REVIEW
Daniel Alberto Sánchez, Gabriela Marta Tonetto, María Luján Ferreira
The lipase from Burkholderia cepacia, formerly known as Pseudomonas cepacia lipase, is a commercial enzyme in both soluble and immobilized forms widely recognized for its thermal resistance and tolerance to a large number of solvents and short-chain alcohols. The main applications of this lipase are in transesterification reactions and in the synthesis of drugs (because of the properties mentioned above). This review intends to show the features of this enzyme and some of the most relevant aspects of its use in different synthesis reactions...
September 23, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28926680/formic-acid-as-a-secondary-substrate-for-succinic-acid-production-by-metabolically-engineered-mannheimia-succiniciproducens
#6
Jung Ho Ahn, Junho Bang, Won Jun Kim, Sang Yup Lee
There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. (13) C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL)...
September 19, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28922469/one-pot-synthesis-of-gdp-mannose-by-a-multi-enzyme-cascade-for-enzymatic-assembly-of-lipid-linked-oligosaccharides
#7
Thomas F T Rexer, Anna Schildbach, Jan Klapproth, Angelika Schierhorn, Reza Mahour, Markus Pietzsch, Erdmann Rapp, Udo Reichl
Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars...
September 18, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921555/improved-performance-of-pseudomonas-putida-in-a-bioelectrochemical-system-through-overexpression-of-periplasmic-glucose-dehydrogenase
#8
Shiqin Yu, Bin Lai, Manuel R Plan, Mark P Hodson, Endah A Lestari, Hao Song, Jens O Krömer
It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD...
September 18, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921549/%C3%AE-flucs-brighter-photinus-pyralis-firefly-luciferases-identified-by-surveying-consecutive-single-amino-acid-deletion-mutations-in-a-thermostable-variant
#9
Lisa M Halliwell, Amit P Jathoul, Jack P Bate, Harley L Worthy, James C Anderson, Dafydd D Jones, James A H Murray
The bright bioluminescence catalysed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bioimaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within 6 loop structures of the bright and thermostable x11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes...
September 16, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921540/imaging-stem-cell-distribution-growth-migration-and-differentiation-in-3-d-scaffolds-for-bone-tissue-engineering-using-mesoscopic-fluorescence-tomography
#10
Qinggong Tang, Charlotte Piard, Jonathan Lin, Kai Nan, Ting Guo, John Caccamese, John Fisher, Yu Chen
Regenerative medicine has emerged as an important discipline that aims to repair injury or replace damaged tissues or organs by introducing living cells or functioning tissues. Successful regenerative medicine strategies will likely depend upon a simultaneous optimization strategy for the design of biomaterials, cell-seeding methods, cell-biomaterial interactions and molecular signaling within the engineered tissues. It remains a challenge to image three-dimensional (3-D) structures and functions of the cell-seeded scaffold in mesoscopic scale (>2∼3 mm)...
September 16, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921534/exploring-cellular-behaviour-under-transient-gene-expression-and-its-impact-on-mab-productivity-and-fc-glycosylation
#11
Si Nga Sou, Ken Lee, Kalpana Nayyar, Karen M Polizzi, Christopher Sellick, Cleo Kontoravdi
Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced to overcome the low yield typically achieved with TGE and improve specific protein productivity. It is therefore of interest to examine the impact of mild hypothermic temperatures on both the yield and quality of transiently-expressed proteins and the relationship to changes in cellular processes and metabolism. In this study, we focus on the ability of a Chinese hamster ovary cell line to galactosylate a recombinant monoclonal antibody (mAb) product...
September 16, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921524/karyotype-variation-of-cho-host-cell-lines-over-time-in-culture-characterized-by-chromosome-counting-and-chromosome-painting
#12
Sabine Vcelar, Vaibhav Jadhav, Michael Melcher, Norbert Auer, Astrid Hrdina, Rebecca Sagmeister, Kelley Heffner, Anja Puklowski, Michael Betenbaugh, Till Wenger, Friedrich Leisch, Martina Baumann, Nicole Borth
Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO-S, CHO-K1 8mM glutamine and CHO-K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture...
September 16, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28921510/development-of-a-formaldehyde-biosensor-with-application-to-synthetic-methylotrophy
#13
Benjamin M Woolston, Timothy Roth, Ishwar Kohale, David Liu, Gregory Stephanopoulos
Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is therefore of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of E. coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM...
September 16, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28898391/recoding-of-synonymous-genes-to-expand-evolutionary-landscapes-requires-control-of-secondary-structure-affecting-translation
#14
Jose Antonio Escudero, Aleksandra Nivina, Guillaume Cambray, Rocío López-Igual, Celine Loot, Didier Mazel
Synthetic DNA design needs to harness the many information layers embedded in a DNA string. We previously developed the Evolutionary Landscape Painter (ELP), an algorithm that exploits the degeneracy of the code to increase protein evolvability. Here, we have used ELP to recode the integron integrase gene (intI1) in two alternative alleles. Although synonymous, both alleles yielded less IntI1 protein and were less active in recombination assays than intI1. We spliced the three alleles and mapped the activity decrease to the beginning of alternative sequences...
September 12, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28892134/inhibition-analysis-of-inhibitors-derived-from-lignocellulose-pretreatment-on-the-metabolic-activity-of-zymomonas-mobilis-biofilm-and-planktonic-cells-and-the-proteomic-responses
#15
Tatsaporn Todhanakasem, Supanika Yodsanga, Apinya Sowatad, Pattanop Kanokratana, Pornthep Thanonkeo, Verawat Champreda
Lignocellulose pretreatment produces various toxic inhibitors that affect microbial growth, metabolism and fermentation. Zymomonas mobilis is an ethanologenic microbe that has been demonstrated to have potential to be used in lignocellulose biorefineries for bioethanol production. Z. mobilis biofilm has previously exhibited high potential to enhance ethanol production by presenting a higher viable cell number and higher metabolic activity than planktonic cells or free cells when exposed to lignocellulosic hydrolysate containing toxic inhibitors...
September 11, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28892124/systematic-optimization-of-l-tryptophan-riboswitches-for-efficient-monitoring-of-the-metabolite-in-escherichia-coli
#16
Sungho Jang, Gyoo Yeol Jung
Riboswitches form a class of genetically-encoded sensor-regulators and are considered as promising tools for monitoring various metabolites. Functional parameters of a riboswitch, like dynamic or operational range, should be optimized before it is implemented in a specific application for monitoring the target molecule efficiently. However, optimization of a riboswitch was not straightforward and required detailed studies owing to its complex sequence-function relationship. Here, we present three approaches for tuning and optimization of functional parameters of a riboswitch using an artificial L-tryptophan riboswitch as an example...
September 11, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28872660/fabricating-plga-microparticles-with-high-loads-of-the-small-molecule-antioxidant-n-acetylcysteine-that-rescue-oligodendrocyte-progenitor-cells-from-oxidative-stress
#17
Nicholas P Murphy, Kyle J Lampe
Reactive oxygen species (ROS), encompassing all oxygen radical or non-radical oxidizing agents, play key roles in disease progression. Controlled delivery of antioxidants is therapeutically relevant in such oxidant-stressed environments. Encapsulating small hydrophilic molecules into hydrophobic polymer microparticles via traditional emulsion methods has long been a challenge due to rapid mass transport of small molecules out of particle pores. We have developed a simple alteration to the existing water-in-oil-in-water (W/O/W) drug encapsulation method that dramatically improves loading efficiency: doping external water phases with drug to mitigate drug diffusion out of the particle during fabrication...
September 5, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28868788/constructing-arabinofuranosidases-for-dual-arabinoxylan-debranching-activity
#18
Weijun Wang, Nikola Andric, Cody Sarch, Bruno Teixeira Silva, Maija Tenkanen, Emma R Master
Enzymatic conversion of arabinoxylan requires α-L-arabinofuranosidases able to remove α-L-arabinofuranosyl residues (α-L-Araf) from both mono- and double-substituted D-xylopyranosyl residues (Xylp) in xylan (i.e., AXH-m and AXH-d activity). Herein, SthAbf62A (a family GH62 α-L-arabinofuranosidase with AXH-m activity) and BadAbf43A (a family GH43 α-L-arabinofuranosidase with AXH-d3 activity), were fused to create SthAbf62A-BadAbf43A and BadAbf43A-SthAbf62A. Both fusion enzymes displayed dual AXH-m,d and synergistic activity towards native, highly branched wheat arabinoxylan (WAX)...
September 4, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28865130/temperature-dependent-dynamic-control-of-the-tca-cycle-increases-volumetric-productivity-of-itaconic-acid-production-by-escherichia-coli
#19
Björn-Johannes Harder, Katja Bettenbrock, Steffen Klamt
Based on the recently constructed E. coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two-stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ▵aceA ▵pta ▵pykF ▵pykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 h(-1) at 37°C, thus representing an ideal phenotype for the first stage, the growth phase. Subsequently we implemented a synthetic genetic control allowing the downregulation of the TCA cycle and thus the switch from growth to itaconic acid production in the second stage...
September 2, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28865125/countercurrent-staged-diafiltration-for-formulation-of-high-value-proteins
#20
Anirudh Nambiar, Ying Li, Andrew L Zydney
A number of groups have studied the application of continuous bioreactors and continuous chromatographic systems as part of efforts to develop an integrated continuous biomanufacturing process. The objective of this study was to examine the feasibility of using a countercurrent staged diafiltration process for continuous protein formulation with reduced buffer requirements. Experiments were performed using a polyclonal immunoglobulin (IgG) with Cadence(TM) Inline Concentrators. Model equations were developed for the product yield, impurity removal, and buffer requirements as a function of the number of stages and the stage conversion (ratio of permeate to feed flow rate)...
September 2, 2017: Biotechnology and Bioengineering
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