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Biotechnology and Bioengineering

Li-Yu Sung, Meng-Ying Wu, Mei-Wei Lin, Mu-Nung Hsu, Vu Anh Truong, Chih-Che Shen, Yi Tu, Kuen-Yuan Hwang, An-Pang Tu, Yu-Han Chang, Yu-Chen Hu
CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for E. coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate sgRNA, this study aimed to explore alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a)...
January 13, 2019: Biotechnology and Bioengineering
Alexandra Westbrook, Xun Tang, Ryan Marshall, Colin S Maxwell, James Chappell, Deepak K Agrawal, Mary J Dunlop, Vincent Noireaux, Chase L Beisel, Julius Lucks, Elisa Franco
To build complex genetic networks with predictable behaviours, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely employed to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and CRISPR interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression...
January 13, 2019: Biotechnology and Bioengineering
Meng-Yao Li, Bruno Ebel, Fabrice Blanchard, Cédric Paris, Emmanuel Guedon, Annie Marc
The cell specific growth rate (µ) is a critical process parameter for antibody production processes performed by animal cell cultures, as it describes the cell growth and reflects the cell physiological state. When there are changes in these parameters, which are indicated by variations of µ, the synthesis and the quality of antibodies are often affected. Therefore, it is essential to monitor and control the variations of µ in order to assure the antibody production and achieve high product quality. In this study, a novel approach for on-line estimation of µ was developed based on the Process Analytical Technology (PAT) initiative by using an in situ dielectric spectroscopy...
January 13, 2019: Biotechnology and Bioengineering
Chieh Mei, Chih-Wei Chao, Che-Wei Lin, Shing Tak Li, Kuan-Han Wu, Kai-Chiang Yang, Jiashing Yu
Microenvironmental factors including physical and chemical cues can regulate stem cells as well as terminally differentiated cells to modulate their biological function and differentiation. However, one of the physical cues, the substrate's dimensionality , has not studied extensively. In this study, the flow-focusing method with a microfluidic device was used to generate gelatin bubbles to fabricate highly ordered three-dimensional (3D) scaffolds. Rat H9c2 myoblasts were seeded into the 3D gelatin bubble-based scaffolds and compared to those grown on 2D gelatin-coating substrates to demonstrate the influences of spatial cues on cell behaviours...
January 13, 2019: Biotechnology and Bioengineering
Carolyn R Shurer, Shelby E Head, Marc C Goudge, Matthew J Paszek
Optimization of host-cell production systems with improved yield and production reliability is desired in order to meet the increasing demand for biologics with complex post-translational modifications. Aggregation of suspension-adapted mammalian cells remains a significant problem that can limit the cellular density and per volume yield of bio-reactors. Here, we propose a genetically encoded technology that directs the synthesis of anti-adhesive and protective coatings on the cellular surface. Inspired by the natural ability of mucin glycoproteins to resist cellular adhesion and hydrate and protect cell and tissue surfaces, we genetically encode new cell-surface coatings through the fusion of engineered mucin domains to synthetic transmembrane anchors...
January 13, 2019: Biotechnology and Bioengineering
Jong Hyun Lee, Jihoon Kim, Jong-Ho Park, Won Do Heo, Gyun Min Lee
Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable CHO cell clones expressing pHluorin2, a ratiometric pH-sensitive fluorescent protein (FP), in the cis- and trans-Golgi, were constructed by fusing pHluorin2 with specific targeting proteins, acetylglucosaminyltransferase and galactosyltransferase, respectively. Stable CHO cell clones expressing pHluorin2 in cytoplasm were also constructed...
January 13, 2019: Biotechnology and Bioengineering
Alican Ozkan, Neda Ghousifam, P Jack Hoopes, Marissa Nichole Rylander
This paper presents the development of a vascularized breast tumor and healthy or tumorigenic liver microenvironments-on-a-chip connected in series. This is the first description of a vascularized multi tissue-on-a-chip microenvironment for modeling cancerous breast and cancerous/healthy liver microenvironments, to allow for the study of dynamic and spatial transport of particles. This device enables the dynamic determination of vessel permeability, the measurement of drug and nanoparticle transport, and the assessment of the associated efficacy and toxicity to the liver...
January 13, 2019: Biotechnology and Bioengineering
David R Wilson, Mark P Suprenant, John H Michel, Ellen B Wang, Stephany Y Tzeng, Jordan J Green
Intracellular delivery of nucleic acids to mammalian cells using polyplex nanoparticles remains a challenge both in vitro and in vivo, with transfections often suffering from variable efficacy. To improve reproducibility and efficacy of transfections in vitro using a next-generation polyplex transfection material poly(beta-amino ester)s (PBAEs), the influence of multiple variables in the preparation of these nanoparticles on their transfection efficacy was explored. The results indicate that the even though PBAE/pDNA polyplex nanoparticles are formed by self-assembly of polyelectrolytes, their transfection is not affected by the manner in which the components are mixed, facilitating self-assembly in a single step, but a timing for self-assembly of 5-20 min is optimal...
January 13, 2019: Biotechnology and Bioengineering
Daniel Burgstaller, Alois Jungbauer, Peter Satzer
Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG 6000 and Zn++ in a tubular reactor integrated with a two-stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor re-solubilization. We developed a new two-stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with wash buffer...
January 13, 2019: Biotechnology and Bioengineering
Jan Volmer, Martin Lindmeyer, Julia Seipp, Andreas Schmid, Bruno Bühler
Solvent-tolerant bacteria represent an interesting option to deal with substrate and product toxicity in bioprocesses. Recently, constitutive solvent tolerance was achieved for Pseudomonas taiwanensis VLB120 via knockout of the regulator TtgV, making tedious adaptation unnecessary. Remarkably, ttgV knockout increased styrene epoxidation activities of P. taiwanensis VLB120ΔC. With the aim to characterize and exploit the biocatalytic potential of P. taiwanensis VLB120ΔC and VLB120ΔCΔttgV, we investigated and correlated growth physiology, native styrene monooxygenase (StyAB) gene expression, whole-cell bioconversion kinetics, and epoxidation performance...
January 13, 2019: Biotechnology and Bioengineering
Kentaro Kamata, Yoshihiro Toya, Hiroshi Shimizu
Mevalonate is a useful metabolite synthesized from three molecules of acetyl-CoA, consuming two molecules of NADPH. Escherichia coli catabolizes glucose to acetyl-CoA via several routes, such as the Embden-Meyerhof-Parnas (EMP) and the oxidative pentose phosphate (oxPP) pathways. Although the oxPP pathway supplies NADPH, it is disadvantageous in terms of acetyl-CoA supply, compared to the EMP pathway. In this study, the optimal flux ratio between the EMP and oxPP pathways on the mevalonate yield was investigated...
January 13, 2019: Biotechnology and Bioengineering
Hayley K Charlton Hume, João Vidigal, Manuel J T Carrondo, Anton P J Middelberg, António Roldão, Linda H L Lua
Vaccination is the most effective method of disease prevention and control. Many viruses and bacteria that once caused catastrophic pandemics (e.g., smallpox, poliomyelitis, measles, and diphtheria) are either eradicated or effectively controlled through routine vaccination programs. Nonetheless, vaccine manufacturing remains incredibly challenging. Viruses exhibiting high antigenic diversity and high mutation rates cannot be fairly contested using traditional vaccine production methods and complexities surrounding the manufacturing processes, which impose significant limitations...
December 31, 2018: Biotechnology and Bioengineering
Anh Nguyen Dang, Melissa Mun, Christopher M Rose, Patrick Ahyow, Angela Meier, Wendy Sandoval, Inn H Yuk
The extent of afucosylation, which refers to the absence of core fucose on Fc glycans, can correlate positively with the antibody-dependent cellular cytotoxicity (ADCC) activity of a monoclonal antibody (mAb). Therefore, it is important to maintain consistent afucosylation during cell culture process scale-up in bioreactors for a mAb with ADCC activity. However, there is currently a lack of understanding about the impact of pCO2 -a parameter that can vary with bioreactor scale-on afucosylation. Using a small-scale (3-L) bioreactor model that can modulate pCO2 levels through modified configurations and gassing strategies, we identified three cell culture process parameters that influence afucosylation of a mAb produced by a recombinant CHO cell line: pCO2 , media hold duration (at 37°C), and manganese...
December 29, 2018: Biotechnology and Bioengineering
Jing-Jing Liu, Jae Won Lee, Eun Ju Yun, Sang-Min Jung, Jin-Ho Seo, Yong-Su Jin
L-fucose (6-deoxy-L-galactose) is a major constituent of glycans and glycolipids in mammals. Fucosylation of glycans can confer unique functional properties, and may be an economic way to manufacture of L-fucose. Researches can extract L-fucose directly from brown algae, or by enzymatic hydrolysis of L-fucose-rich microbial exopolysaccharides. However, these L-fucose production methods are not economical or scalable for various applications. We engineered an Escherichia coli strain to produce L-fucose. Specifically, we modified the strain genome to eliminate endogenous L-fucose metabolism, produce 2-fucosyllactose (2-FL), and to liberate L-fucose from 2-FL...
December 29, 2018: Biotechnology and Bioengineering
Seep Arora, Adele Jing Ying Lam, Christine Cheung, Evelyn Kf Yim, Toh
Human pluripotent stem cell derived endothelial cells (hPSC-EC) present an attractive alternative to primary EC sources for vascular grafting but there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arterio-venous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses is still not well understood...
December 29, 2018: Biotechnology and Bioengineering
Jiangang Yang, Chaoyu Tian, Tong Zhang, Chenxi Ren, Yueming Zhu, Yan Zeng, Yan Men, Yuanxia Sun, Yanhe Ma
D-allulose 3-epimerase (DAE) has been applied to produce D-allulose, a low-calorie and functional sweetener. In this study, a new DAE from Paenibacillus senegalensis was characterized in Escherichia coli. Furthermore, we presented a tandem isoenzyme gene expression strategy to express multiple DAEs in one cell and construct food-grade expression systems based on Corynebacterium glutamicum. Seventeen expression cassettes based on three DAE genes from different organisms were constructed. Among all recombinant strains, DAE16 harbouring three DAE genes in an expression vector exhibited the highest enzyme activity with 22...
December 29, 2018: Biotechnology and Bioengineering
Jared Lynn Dopp, Samuel Michael Rothstein, Thomas Joseph Mansell, Nigel Forest Reuel
In this work, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hours from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell-based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost-optimized, scalable-manufactured E...
December 29, 2018: Biotechnology and Bioengineering
Anne Mouré, Elodie Bacou, Steffi Bosch, Dominique Jegou, Apolline Salama, David Riochet, Olivier Gauthier, Gilles Blancho, Jean-Paul Soulillou, Denis Poncelet, Eric Olmos, Jean-Marie Bach, Mathilde Mosser
The bio-artificial pancreas encapsulating pancreatic islets in immuno-protective hydrogel is a promising therapy for type 1 diabetes. As pancreatic islets are highly metabolically active and exquisitely sensitive to hypoxia, maintaining O2 supply after transplantation remains a major challenge. In this study, we address the O2 limitation by combining silicone-encapsulated CaO2 (silicone-CaO2 ) to generate O2 with an extracellular hemoglobin O2 -carrier co-encapsulated with islets. We showed that the hemoglobin improved by 37 % the O2 -diffusivity through an alginate hydrogel and displayed anti-oxidant properties neutralizing deleterious reactive O2 species produced by silicone-CaO2 ...
December 29, 2018: Biotechnology and Bioengineering
Junaid Ali, Qasim Rafiq, Elizabeth Ratcliffe
Therapeutic bacteriophages are emerging as a potential alternative to antibiotics and synergistic treatment for antimicrobial resistant infections. This is reflected by their use in an increasing number of recent clinical trials. Many more therapeutic bacteriophage are being investigated in pre-clinical research and due to the bespoke nature of these products with respect to their limited infection spectrum, translation to the clinic requires combined understanding of the biology underpinning the bioprocess and how this can be optimised and streamlined for efficient methods of scalable manufacture...
December 29, 2018: Biotechnology and Bioengineering
Xiulai Chen, Yang Li, Tian Tong, Liming Liu
Fumarate is a naturally occurring organic acid that is an intermediate of the TCA cycle, and has numerous applications in food, pharmaceutical, and chemical industries. However, microbial fumarate production from renewable feedstock is limited by the intrinsic inefficiency of its synthetic pathway caused by week metabolites transportation and cofactor imbalance. In this study, spatial modulation and cofactor engineering of key pathway enzymes in the reductive TCA pathway were performed for the development of a Candida glabrata strain capable of efficiently producing fumarate...
December 24, 2018: Biotechnology and Bioengineering
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