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Biotechnology and Bioengineering

Byoungjin Kim, Robert Binkley, Hyun Uk Kim, Sang Yup Lee
Despite wide applications of L-tyrosine in the market, microbial overproduction of L-tyrosine has been a great challenge due to the complex gene regulations involved in its biosynthetic pathway. To this end, effects of knocking out tyrR on the L-tyrosine production were further explored during the strain development. Also, blocking cellular uptake of L-tyrosine by knocking out tyrosine transporters was examined with respect to L-tyrosine production. Using feedback resistant aroG and tyrA genes (aroGfbr and tyrAfbr hereafter) as initial overexpression targets, which encode 3-deoxy-7-phosphoheptulonate synthase and chorismate mutase/prephenate dehydrogenase, respectively, various combinations of genes were subsequently overexpressed in the Escherichia coli wild-type and tyrR knockout strain, and their effects on the L-tyrosine production were examined...
July 18, 2018: Biotechnology and Bioengineering
Roland Prielhofer, Michaela Reichinger, Nina Wagner, Katrien Claes, Christoph Kiziak, Brigitte Gasser, Diethard Mattanovich
Protein production in Pichia pastoris is often based on the methanol-inducible PAOX1 promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose-regulated PGTH1 promoter which we reported recently. In order to further increase its potential, we investigated its regulation in more detail by screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of PGTH1 ...
July 17, 2018: Biotechnology and Bioengineering
Lahiru N Jayakody, Jing-Jing Liu, Eun Ju Yun, Timothy Lee Turner, Eun Joong Oh, Yong-Su Jin
Simultaneous saccharification and fermentation (SSF) of cellulose via engineered Saccharomyces cerevisiae is a sustainable solution to valorize cellulose into fuels and chemicals. In this study, we demonstrate the feasibility of direct conversion of cellulose into ethanol and a bio-degradable surfactant, ethyl-β-d-glucoside, via an engineered yeast strain (i.e., strain EJ2) expressing heterologous cellodextrin transporter (CDT-1) and intracellular β-glucosidase (GH1-1) originating from Neurospora crassa. We identified the formation of ethyl-β-d-glucoside in SSF of cellulose by the EJ2 strain owing to transglycosylation activity of GH1-1...
July 16, 2018: Biotechnology and Bioengineering
Akhil Patel, Yingfei Xue, Rebecca Hartley, Vinayak Sant, James Eles, Xinyan Tracy Cui, Donna Beer Stolz, Shilpa Sant
Despite significant interest in developing extracellular matrix (ECM)-inspired biomaterials to recreate native cell-instructive microenvironments, the siginificant challenge in the biomaterial field is to recapitulate the complex structural and biophysical features of native ECM. These biophysical features include multi-scale hierarchy, electrical conductivity, optimum wettability, and mechanical properties. These features are critical to the design of cell-instructive biomaterials for bioengineering applications such as skeletal muscle tissue engineering...
July 16, 2018: Biotechnology and Bioengineering
Che Lin Kim, Mi Yeong Jung, Young Sik Kim, Ju Woong Jang, Gyun Min Lee
Endocytic regulation serves a critical role in modulating the extracellular level of signaling molecules such as bone morphogenetic proteins (BMPs). Unfortunately, endocytosis may result in poor yields of recombinant human BMP-4 (rhBMP-4) from Chinese hamster ovary (CHO) cell cultures. When rhBMP-4 was incubated with CHO cells, rhBMP-4 was actively internalized into cells. Cell surface bound-heparan sulfate proteoglycans (HSPGs) served as the major receptors for rhBMP-4 internalization. Removal of cell surface heparan sulfate (HS) by heparinases or reduction of HSPG synthesis by knockdown of xylosyltransferase2 (xylt2) in CHO cells decreased internalization of rhBMP-4...
July 16, 2018: Biotechnology and Bioengineering
Pyung-Gang Lee, Sang-Hyuk Lee, Eun Young Hong, Stefan Lutz, Byung-Gee Kim
Tyrosinase is a type-3 copper oxygenase that catalyzes a phenol moiety into ortho-diphenol, and subsequently to ortho-quinone. Diverse tyrosinases have been observed across the kingdom including Animalia, Bacteria, Plantae, and Fungi. Among the tyrosinases, bacterial and mushroom tyrosinases have been extensively exploited to prepare melanin, ortho-hydroxy-polyphenols or novel plant secondary metabolites during the past decade. And their use as a biocatalyst to prepare various functional bio-compounds have drawn great attention worldwide...
July 16, 2018: Biotechnology and Bioengineering
Sultan Khetani, Mehdi Mohammadi, Amir Sanati Nezhad
Isolation of Circulating Tumor Cells (CTCs) from blood has long been a challenge due to the rarity and heterogeneity of these cells. Detection technologies have predominantly focused on different molecular or physical properties of CTCs. Size-based isolation approach using microfilters have been widely used to capture CTCs because of the difference in size and stiffness of the cells compared to other hemocytes. Isolation of rare cells based on their size was the original CTC enrichment technique and it demonstrated a simple yet rapid method that enhanced the recovery of cells with high throughput...
July 10, 2018: Biotechnology and Bioengineering
D Geoghegan, C Arnall, D Hatton, J Noble-Longster, C Sellick, T Senussi, D C James
Amino acid transporters (AATs) represent a key interface between the cell and its environment, critical for all cellular processes; energy generation, redox control, and synthesis of cell and product biomass. However, very little is known about the activity of different functional classes of amino acid transporters in Chinese hamster ovary (CHO) cells, how they support cell growth and productivity, and the potential for engineering their activity and/or the composition of amino acids in growth media to improve CHO cell performance in vitro...
July 10, 2018: Biotechnology and Bioengineering
Annika Wenz, Iva Tjoeng, Isabelle Schneider, Petra J Kluger, Kirsten Borchers
The co-culture of osteogenic and angiogenic cells and the resulting paracrine signaling via soluble factors is supposed to be crucial for successfully engineering vascularized bone tissue equivalents. In this study, a co-culture system combining primary human adipose-derived stem cells (hASCs) and primary human dermal microvascular endothelial cells (HDMECs) within two types of hydrogels based on methacryl-modified gelatin (GM) as threedimensional (3D) scaffolds was examined regarding its support of tissue specific cell functions...
July 7, 2018: Biotechnology and Bioengineering
Bing-Ming Ou, Carolina Garcia, Ye-Jun Wang, Wei-Ping Zhang, Guo-Qiang Zhu
Due to the inherent expression stability and low metabolic burden to the host cell, the expression of heterologous proteins in the bacterial chromosome in a precise and efficient manner is highly desirable for metabolic engineering and live bacterial applications. However, obtaining suitable chromosome expression levels is particularly challenging. In this mini-review, we briefly present the technologies available for the integration of heterologous genes into Escherichia coli chromosomes and strategies to optimize the expression levels of heterologous proteins...
July 7, 2018: Biotechnology and Bioengineering
Joseph T Decker, Matthew S Hall, Rachel B Blaisdell, Kallen Schwark, Jacqueline S Jeruss, Lonnie D Shea
MicroRNAs (miRNA) are implicated in numerous physiologic and pathologic processes, such as the development of resistance to chemotherapy. Determining the role of miRNAs in these processes is often accomplished through measuring miRNA abundance by PCR, sequencing, or microarrays. We have developed a system for the large-scale monitoring of dynamic miRNA activity, and have applied this system to identify the contribution miRNA activity to the development of trastuzumab resistance in a cell model of HER2+ breast cancer...
July 7, 2018: Biotechnology and Bioengineering
Seung Yeon Lee, Jong Hwan Sung
Hepatic steatosis is a process of abnormal lipid deposition within the liver cells, often caused by excessive alcohol uptake or obesity. A conventional in vitro model for hepatic steatosis uses a liver cell culture, treated with fatty acids and measures accumulation of lipids within the cells. This model does not recapitulate the complex process of absorption and metabolism of digestive lipids. Here, we introduce a gut-liver chip, which mimics the gut-absorption and hepatic metabolism in a microfluidic chip...
July 7, 2018: Biotechnology and Bioengineering
Adam W Westbrook, Xiang Ren, Murray Moo-Young, C Perry Chou
Bacillus subtilis has been commonly applied to industrial enzyme production due to its genetic tractability, 'Generally Recognized as Safe (GRAS)' status, and robust growth characteristics. In spite of its ideal attributes as a biomanufacturing platform, B. subtilis has seen limited use in the production of other value-added biochemicals. Here, we report the derivation of engineered strains of B. subtilis for L-valine overproduction using our recently developed CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated [protein] 9) toolkit...
July 7, 2018: Biotechnology and Bioengineering
Jonathan M Conway, James R Crosby, Bennett S McKinley, Nathaniel L Seals, Michael W W Adams, Robert M Kelly
Six multi-domain glycoside hydrolases (GHs), CelA (Athe_1867), CelB (Athe_1859), CelC (Athe_1857), CelD (Athe_1866), CelE (Athe_1865), and CelF (Athe_1860) are encoded in the Caldicellulosiruptor bescii Glucan Degradation Locus (GDL). Each GH was affinity-tagged, over-expressed and purified from recombinant C. bescii for side-by-side characterization in vitro and to examine the contribution of each of these enzymes to microcrystalline cellulose hydrolysis in vivo. All six recombinant GDL GHs were glycosylated, and deletion of glycosyltransferase Athe_1864 eliminated this post-translational modification...
July 3, 2018: Biotechnology and Bioengineering
Erik Arango Gutierrez, Anne-Maria Wallraf, Alexandra Balaceanu, Marco Bocola, Mehdi D Davari, Thomas Meier, Hartmut Duefel, Ulrich Schwaneberg
Glucose oxidase is of high industrial interest for glucose sensing due to its high β-D-glucose specificity. The efficient and specific electrochemical communication between the redox center and electrodes is crucial to ensure accurate glucose determination. The efficiency of the electron transfer rates with glucose oxidase (GOx) together with quinone diamine-based mediators is low and differs even among mediator derivatives. In order to design optimized enzyme-mediator couples and to describe a mediator binding model, a joint experimental and computational study was performed based on an oxygen-independent GOx variant V7 and two quinone diimine based electron mediators (QDM-1 and QDM-2), which differ in polarity and size, and ferrocenemethanol (FM)...
June 30, 2018: Biotechnology and Bioengineering
Chen Chen, Iris Pla-Palacín, Pedro M Baptista, Peng Shang, Loes A Oosterhoff, Monique E van Wolferen, Louis C Penning, Niels Geijsen, Bart Spee
Direct reprogramming represents an easy technique to generate hepatocyte-like cells (iHeps) from somatic cells. However, current protocols are accompanied by several drawbacks as iHeps are heterogenous and lack fully mature phenotypes of primary hepatocytes. Here, we established a polycistronic expression system to induce the direct reprogramming of mouse embryonic fibroblasts towards hepatocytes. The resulting iHeps are homogenous and display key properties of primary hepatocytes, such as expression of hepatocyte markers, albumin secretion, and presence of liver transaminases...
June 30, 2018: Biotechnology and Bioengineering
Qiang Yan, Stephen S Fong
Chitin is an abundant, biorenewable, nitrogen-rich biomass feedstock that can be potentially developed for biochemical production, however, efficient bioprocesses have yet to be established. Here, we demonstrate an engineered bioprocess to produce N-acetylneuraminic acid (Neu5Ac) directly from chitin using the chitinolytic bacterium, Serratia marcescens by selecting and characterizing promoters, characterization of heterologous enzyme activity, and optimization of pathway fluxes. By generating RNASeq data for S...
June 30, 2018: Biotechnology and Bioengineering
Patrick Kaiser, Melanie Werner, Valérie Jérôme, Ruth Freitag
A number of evolving medical therapies call for the controlled expansion of primary human T lymphocytes. After encapsulation in sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride polyelectrolyte capsules, T lymphocytes can be expanded without persisting activation. Here, the challenge of scaling up this process is addressed. Encapsulated T lymphocytes were cultured in spinner flasks as well as in several types of bioreactor, including fixed and fluidized beds, a waved cell bag, and a standard stirred tank reactor (1 L scale)...
June 30, 2018: Biotechnology and Bioengineering
Yaozhun Huang, Yuanye Bao, Hoi Kwan Kwong, Ting-Hsuan Chen, Miu Ling Lam
Cells cultured in micropatterns exhibit chiral orientation, which may underlie the development of left-right asymmetry in tissue microarchitectures. To investigate this phenomenon, fluorescence staining of nuclei has been used to reveal such orientation. However, for images with high cell density, analysis has been difficult because of overlapping nuclei. Here we report an image processing method that can acquire cell orientations within dense cell populations. After initial separation based on Boolean addition of binarized images using global and adaptive thresholds, the overlapping nucleus contours in the binarized images were segmented by iteratively etching the outlines of nuclei, which allowed the orientations of each cell to be extracted from densely packed cell clusters...
June 30, 2018: Biotechnology and Bioengineering
Ziyuan Zhou, Fuhou Lei, Pengfei Li, Jianxin Jiang
Lignocellulosic biomass is one of the most potential feedstock to produce second generation cellulosic ethanol and biochemicals. In order to enhance the enzymatic digestibility of lignocellulosic biomass for efficient enzymatic saccharification, a variety of pretreatment methods have been studied. Among those, an organosolv pretreatment using ethanol is a promising pretreatment method owing to its inherent advantages, for example, low solvent cost, lack of toxicity, the ability to retain most cellulose fraction in substrate for enzymatic hydrolysis, co-production of high-purity lignin and hemicellulosic sugars, easy solvent recovery and reuse...
June 30, 2018: Biotechnology and Bioengineering
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