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Biotechnology and Bioengineering

Kyriakos G Varnava, Ron S Ronimus, Vijayalekshmi Sarojini
Archaea was until recently considered as a third domain of life in addition to bacteria and eukarya but recent studies support the existence of only two superphyla (bacteria and archaea). The fundamental differences between archaeal, bacterial and eukaryal cells are probably the main reasons for the comparatively lower susceptibility of archaeal strains to current antimicrobial agents. The possible emerging pathogenicity of archaea and the role of archaeal methanogens in methane emissions, a potent greenhouse gas, has led many researchers to examine the sensitivity patterns of archaea and make attempts to find agents that have significant anti-archaeal activity...
July 22, 2017: Biotechnology and Bioengineering
Shinichi Goto, Kaori Tsukakoshi, Kazunori Ikebukuro
SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labour consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations...
July 22, 2017: Biotechnology and Bioengineering
Martina Baumann, Elisabeth Gludovacz, Natalie Sealover, Scott Bahr, Henry George, Nan Lin, Kevin Kayser, Nicole Borth
Site-specific Recombinase Mediated Cassette Exchange (RMCE) enables the transfer of the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. For the generation of recombinant production cell lines this has the advantage that screening for high transcription rates at the genome integration site would be required only once, with the possibility to reuse the selected site for new products. Here we describe a strategy that aims at the selection of transcriptionally active genome integration sites in Chinese Hamster Ovary (CHO) cells by using alternate start codons in the surface reporter protein CD4, in combination with FACS sorting for high expressers...
July 22, 2017: Biotechnology and Bioengineering
Lucía Martín Pérez, Laura Benítez Casanova, Antonio Javier Moreno Pérez, Dolores Pérez Gómez, Sandra Gavaldá Martín, Laura Ledesma-García, Noelia Valbuena, Bruno Díez García, Francisco Manuel Reyes-Sosa
Thermochemical pretreatment and enzymatic hydrolysis are the areas contributing most to the operational costs of second generation ethanol in lignocellulosic biorefineries. The improvement of lignocellulosic enzyme cocktails has been significant in the recent years. Although the needs for the reduction of the energy intensity and chemical consumption in the pretreatment step are well known, the reduction of the severity of the process strongly affects the enzymatic hydrolysis yield. To explore the formulation requirements of the well known cellulolytic cocktail from Myceliophthora thermophila on mild pretreated raw materials, this cocktail was tested on steam exploded corn stover without acid impregnation...
July 15, 2017: Biotechnology and Bioengineering
T Steele Reynolds, Colleen M Courtney, Keesha E Erickson, Lisa M Wolfe, Anushree Chatterjee, Prashant Nagpal, Ryan T Gill
The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability...
July 15, 2017: Biotechnology and Bioengineering
Zhuangrong Huang, Dong-Yup Lee, Seongkyu Yoon
Chinese hamster ovary (CHO) cells have been widely used for producing many recombinant therapeutic proteins. Constraint-based modeling, such as flux balance analysis (FBA) and metabolic flux analysis (MFA), has been developing rapidly for the quantification of intracellular metabolic flux distribution at a systematic level. Such methods would produce detailed maps of flows through metabolic networks, which contribute significantly to better understanding of metabolism in cells. Although these approaches have been extensively established in microbial systems, their application to mammalian cells is sparse...
July 15, 2017: Biotechnology and Bioengineering
Mo Jiang, Kristen Severson, J Christopher Love, Helena Madden, Patrick Swann, Li Zang, Richard D Braatz
Real-time release testing (RTRT) is defined as "the ability to evaluate and ensure the quality of in-process and/or final drug product based on process data, which typically includes a valid combination of measured material attributes and process controls" (ICH Q8(R2)). This article discusses sensors (process analytical technology, PAT) and control strategies that enable RTRT for the spectrum of critical quality attributes (CQAs) in biopharmaceutical manufacturing. Case studies from the small-molecule and biologic pharmaceutical industry are described to demonstrate how RTRT can be facilitated by integrated manufacturing and multivariable control strategies to ensure the quality of products...
July 15, 2017: Biotechnology and Bioengineering
Weifeng Zhang, Dan Xiao, Linlin Shan, Junli Zhao, Qinwen Mao, Haibin Xia
Apoptosis has important functions during pathophysiologic processes. However, from a biopharmaceutical point of view, active apoptosis of host cells is undesirable during viral packaging or protein expression, because it decreases the efficiency of viral or protein production. Here we used the CRISPR/Cas technique to knock out four pro-apoptotic genes, Caspase3, Caspase6, Caspase7 and AIF1, in HEK293 cells, and successfully produced an apoptosis-resistant cell line. Furthermore, this cell line showed higher expression levels of pro-apoptotic proteins and higher packaging efficiency for the virus carrying these proteins than control HEK293 cells...
July 15, 2017: Biotechnology and Bioengineering
Axel Theorell, Samuel Leweke, Wolfgang Wiechert, Katharina Nöh
(13) C Metabolic Fluxes Analysis ((13) C MFA) remains to be the most powerful approach to determine intracellular metabolic reaction rates. Decisions on strain engineering and experimentation heavily rely upon the certainty with which these fluxes are estimated. For uncertainty quantification, the vast majority of (13) C MFA studies relies on confidence intervals from the paradigm of Frequentist statistics. However, it is well known that the confidence intervals for a given experimental outcome are not uniquely defined...
July 11, 2017: Biotechnology and Bioengineering
Ayca Cankorur-Cetinkaya, Duygu Dikicioglu, Stephen G Oliver
Genome-scale metabolic models are valuable tools for the design of novel strains of industrial microorganisms, such as Komagataella phaffii (syn. Pichia pastoris). However, as is the case for many industrial microbes, there is no executable metabolic model for K. phaffiii that confirms to current standards by providing the metabolite and reactions IDs, to facilitate model extension and reuse, and gene-reaction associations to enable identification of targets for genetic manipulation. In order to remedy this deficiency, we decided to reconstruct the genome-scale metabolic model of K...
July 10, 2017: Biotechnology and Bioengineering
Timo van der Zwan, Jinguang Hu, Jack N Saddler
Effective enzyme-mediated viscosity reduction, disaggregation, or 'liquefaction', is required to overcome the rheological challenges resulting from the fibrous, hygroscopic nature of lignocellulosic biomass, particularly at the high solids loadings that will be required for an economically viable process. However, the actual mechanisms involved in enzyme-mediated deconstruction, as determined by viscosity or yield stress reduction, have yet to be fully resolved. Particle fragmentation, interparticle interaction, material dilution and yield stress rheometry were compared for their ability to quantify enzyme-mediated liquefaction of model and more realistic pretreated biomass substrates...
July 10, 2017: Biotechnology and Bioengineering
Amy M Ehrenworth, Mitchell A Haines, Amy Wong, Pamela Peralta-Yahya
Compartmentalization of metabolic pathways into organelles of the yeast Saccharomyces cerevisiae has been used to improve chemical production. Pathway compartmentalization aids chemical production by bringing enzymes into close proximity to one another, placing enzymes near key starting metabolites or essential co-factors, increasing the effective concentration of metabolic intermediates, and providing a more suitable chemical environment for enzymatic activity. Although several translocation tags have been used to localize enzymes to different yeast organelles, their translocation efficiencies have not been quantified...
July 8, 2017: Biotechnology and Bioengineering
M Catherine Bartlett, Chapman Wright, Angel Kuo, Noelle Colant, Matthew Westoby, J Christopher Love
Antibodies are an important class of therapeutics and are predominantly produced in Chinese Hamster Ovary (CHO) cell lines. While this manufacturing platform is sufficiently productive to supply patient populations of currently approved therapies, it is unclear whether or not the current CHO platform can address two significant areas of need: affordable access to biologics for patients around the globe and production of unprecedented quantities needed for very large populations of patients. Novel approaches to recombinant protein production for therapeutic biologic products may be needed, and might be enabled by non-mammalian expression systems and recent advances in bioengineering...
July 8, 2017: Biotechnology and Bioengineering
Eik Czarnotta, Mariam Dianat, Marcel Korf, Fabian Granica, Juliane Merz, Jérôme Maury, Simo Abdessamad Baallal Jacobsen, Jochen Förster, Birgitta E Ebert, Lars M Blank
Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into S. cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors...
July 8, 2017: Biotechnology and Bioengineering
R Adam Thompson, Cong T Trinh
As a model thermophilic bacterium for the production of second-generation biofuels, the metabolism of Clostridium thermocellum has been widely studied. However, most studies have characterized C. thermocellum metabolism for growth at relatively low substrate concentrations. This outlook is not industrially relevant, however, as commercial viability requires substrate loadings of at least 100 g/L cellulosic materials. Recently, a wild-type C. thermocellum DSM1313 was cultured on high cellulose loading batch fermentations and reported to produce a wide range of fermentative products not seen at lower substrate concentrations, opening the door for a more in-depth analysis of how this organism will behave in industrially relevant conditions...
July 3, 2017: Biotechnology and Bioengineering
Doris Ribitsch, Altijana Hromic, Sabine Zitzenbacher, Barbara Zartl, Caroline Gamerith, Alessandro Pellis, Alois Jungbauer, Andrzej Łyskowski, Georg Steinkellner, Karl Gruber, Rupert Tscheliessnig, Enrique Herrero Acero, Georg M Guebitz
We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated...
July 3, 2017: Biotechnology and Bioengineering
Ehecatl Antonio Del Rio-Chanona, Fabio Fiorelli, Dongda Zhang, Nur Rashid Ahmed, Keju Jing, Nilay Shah
Lutein is a high-value bioproduct synthesised by microalga Desmodesmus sp.. It has great potential for the food, cosmetics, and pharmaceutical industries. However, in order to enhance its productivity and to fulfil its ever-increasing global market demand, it is vital to construct accurate models capable of simulating the entire behaviour of the complicated dynamics of the underlying biosystem. To this aim, in this study two highly robust artificial neural networks are designed for the first time. Contrary to conventional artificial neural networks, these networks model the rate of change of the dynamic system, which makes them highly relevant in practice...
July 3, 2017: Biotechnology and Bioengineering
Suryang Kwak, Soo Rin Kim, Haiqing Xu, Guo-Chang Zhang, Stephan Lane, Heejin Kim, Yong-Su Jin
Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites...
July 1, 2017: Biotechnology and Bioengineering
Laura Cervera, Irene González-Domínguez, Maria Mercedes Segura, Francesc Gòdia
Transient transfection is a fast, flexible, and cost-effective approach to produce biological products. Despite the continued interest in transient transfection, little is known regarding the transfection process at the intracellular level, particularly for complex products, such as virus-like particles (VLPs). The kinetics of PEI-mediated transfection following an established in-house protocol is reported in this work with the aim of characterizing and understanding the complete process leading to VLP generation and identifying important events driving process improvement...
July 1, 2017: Biotechnology and Bioengineering
Shelby Steinmeyer, Daniel P Howsmon, Robert C Alaniz, Juergen Hahn, Arul Jayaraman
Adoptive transfer of anti-inflammatory FOXP3+ Tregs has gained attention as a new therapeutic strategy for auto-inflammatory disorders such as Inflammatory Bowel Disease. The isolated cells are conditioned in vitro to obtain a sufficient number of anti-inflammatory FOXP3+ Tregs that can be reintroduced into the patient to potentially reduce the pathologic inflammatory response. Previous evidence suggests that microbiota metabolites can potentially condition cells during the in vitro expansion/differentiation step...
July 1, 2017: Biotechnology and Bioengineering
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