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Biotechnology Journal

Nadiya Romanova, Thomas Noll
Promoters play a pivotal role in integrating and processing the signals related to transcription initiation. Strong natural viral promoters, such as hCMV or SV40E, have been routinely employed to achieve a high rate of gene expression in ubiquitously used Chinese hamster ovary (CHO) cells. However, viral promoters are susceptible to epigenetic silencing and lack precise regulation levers. This has paved the way to more sensible control elements: endogenous, inducible and synthetic promoters. In this review we summarise and discuss the use of natural viral, mammalian and endogenous promoters, as well as recent advances in synthetic promoters and inducible systems for protein expression in CHO cells...
November 16, 2017: Biotechnology Journal
Yidan Wang, Meiyan Wang, Kaili Dong, Haifeng Ye
Synthetic biology applies engineering principles to biological systems and has significantly advanced the design of synthetic gene circuits that can reprogram cell activities to perform new functions. The ability to engineer mammalian designer cells with robust therapeutic behaviors has brought new opportunities for treating metabolic diseases. In this review, we highlight the most recent advances in the development of synthetic designer cells uploaded with open-loop gene circuits or closed-loop gene circuits for the treatment of metabolic disorders including diabetes, hypertension, hyperuricemia and obesity, and discuss the current technologies and future perspectives in applying these designer cells for clinical applications...
November 16, 2017: Biotechnology Journal
Min Suk Lee, Hee Seok Yang
The skeletal muscle consists of highly aligned dense cables of collagen fibers with nanometer feature size to support muscle fibers. The skeletal myocyte can be greatly affected to differentiate by their surrounding topographical structure. To improve myogenic differentiation, we fabricated cell culture platform that sphingosine-1-phosphate (S1P) which regulated myocyte behavior was immobilized on a biomimetic nanopatterned polyurethaneacrylate (PUA) substrate using 3,4-dihydroxyphenylalanine (L-DOPA) for providing topographical and biological cues synergistically...
November 16, 2017: Biotechnology Journal
Spyridon Konstantinidis, Hai-Yuan Goh, José Mauricio Martin Bufájer, Paul de Galbert, Maria Parau, Ajoy Velayudhan
The High Throughput (HT) investigation of chromatographic separations is an important element of downstream bioprocess development due to the importance of chromatography as a technique for achieving stringent regulatory requirements on product purity. Various HT formats for chromatography exist, but the miniature column approach has characteristics resembling large scale packed bed column chromatography the most. The operation of such columns on robotic stations can be automated, but this is not always a straightforward procedure; the robotic manipulations are highly dependent on the settings of each experiment and the standard commands of the supporting software may not provide readily the required flexibility and accessibility for 'plug and play' functionality...
November 13, 2017: Biotechnology Journal
Hiroki Hashi, Yasuyuki Nakamura, Jun Ishii, Akihiko Kondo
Neurotensin receptor type 1 (NTSR1), a member of the G-protein-coupled receptor (GPCR) family, is naturally activated by binding of a neurotensin peptide, leading to a variety of physiological effects. The budding yeast Saccharomyces cerevisiae is a proven host organism for assaying the agonistic activation of human GPCRs. Previous studies showed that yeast cells can functionally express human NTSR1 receptor, permitting the detection of neurotensin-promoted signaling using a ZsGreen fluorescent reporter gene...
November 13, 2017: Biotechnology Journal
Steffen Klamt, Radhakrishnan Mahadevan, Oliver Hädicke
Apart from product yield and titer, volumetric productivity is a key performance indicator for many biotechnological processes. Due to the inherent trade-off between the production of biomass as catalyst and of the actual target product, yield and volumetric productivity cannot be optimized simultaneously. Therefore, in combination with genetic techniques for dynamic regulation of metabolic fluxes, two-stage fermentations (TSFs) with separated growth and production phase have recently gained much interest because of their potential to improve the productivity of bioprocesses while still allowing high product yields...
November 13, 2017: Biotechnology Journal
Irene C Schneider, Jessica Hartmann, Gundula Braun, Jörn Stitz, Thorsten Klamp, Mahjoub Bihi, Ugur Sahin, Christian J Buchholz
Virus-like particles (VLPs) displaying foreign antigens have become an important tool in vaccination including the induction of immune responses against self-antigens. Claudin 6 (CLDN6) has been identified as tumor-associated antigen and is therefore a potential target for tumor vaccination strategies. However, as tetra-membrane spanning protein its incorporation into VLPs while preserving a native fold is challenging. Here, we attempted the incorporation of a panel of engineered CLDN6 variants into the membrane of retrovirus-derived VLPs...
November 13, 2017: Biotechnology Journal
Horst Lechner, Pablo Soriano, Roman Poschner, Helen C Hailes, John M Ward, Wolfgang Kroutil
Norcoclaurine synthases (NCS), catalyzing a Pictet-Spengler reaction in plants as one of the first enzymes in the biosynthetic benzylisoquinoline pathway, were investigated for biocatalytic transformations. The library of norcoclaurine synthases available was extended by two novel NCSs from Argemone mexicana (AmNCS1, AmNCS2) and one new NCS from Corydalis saxicola (CsNCS); furthermore, it was shown that the NCS from Papaver bracteatum (PbNCS) is a highly productive catalyst leading to the isoquinoline product with up to >99% e...
November 10, 2017: Biotechnology Journal
Rui M C Portela, Thomas Vogl, Katharina Ebner, Rui Oliveira, Anton Glieder
Unravelling the core promoter sequence-function relationship is fundamental for engineering transcription initiation and thereby a feasible 'tuning knob' for fine-tuning expression in synthetic biology and metabolic engineering applications. Here a systematic replacement studies of the core promoter and 5' untranslated region (5'UTR) of the exceptionally strong and tightly methanol regulated Komagataella phaffii (syn. Pichia pastoris) alcohol oxidase 1 (AOX1) promoter at unprecedented resolution was performed...
November 10, 2017: Biotechnology Journal
Tae Kwang Ha, Anders Holmgaard Hansen, Stefan Kol, Helene Faustrup Kildegaard, Gyun Min Lee
Oxidative stress that naturally accumulates in the endoplasmic reticulum (ER) as a result of mitochondrial energy metabolism and protein synthesis can disturb the ER function. Because ER has a responsibility on the protein synthesis and quality control of the secreted proteins, ER homeostasis has to be well maintained. When H2O2, an oxidative stress inducer, was added to recombinant Chinese hamster ovary (rCHO) cell cultures, it reduced cell growth, monoclonal antibody (mAb) production, and galactosylated form of mAb in a dose-dependent manner...
November 10, 2017: Biotechnology Journal
Cristina Quintelas, Eugénio C Ferreira, João A Lopes, Clara Sousa
The sustained emergence of new declared bacterial species makes typing a continuous challenge for microbiologists. Molecular biology techniques have a very significant role in the context of bacterial typing, but they are often very laborious, time consuming, and eventually fail when dealing with very closely related species. Spectroscopic-based techniques appear in some situations as a viable alternative to molecular methods with advantages in terms of analysis time and cost. Infrared and mass spectrometry are among the most exploited techniques in this context: particularly, infrared spectroscopy emerged as a very promising method with multiple reported successful applications...
November 1, 2017: Biotechnology Journal
Sarah Zimmermann, Sarah Gretzinger, Philipp K Zimmermann, Are Bogsnes, Mattias Hansson, Jürgen Hubbuch
The availability of clinical-scale downstream processing strategies for cell-based products presents a critical juncture between basic research and clinical development. Aqueous two-phase systems (ATPS) facilitate the label-free, scalable, and cost-effective separation of cells, and are a versatile tool for downstream processing of cell-based therapeutics. Here, we report the application of a previously developed robotic screening platform, here extended to enable a multiplexed high-throughput cell partitioning analysis in ATPS...
October 31, 2017: Biotechnology Journal
Helene Lunde Robertsen, Tilmann Weber, Hyun Uk Kim, Sang Yup Lee
Streptomycetes are known for their inherent ability to produce pharmaceutically relevant secondary metabolites. Discovery of medically useful, yet novel compounds has become a great challenge due to frequent rediscovery of known compounds and a consequent decline in the number of relevant clinical trials in the last decades. A paradigm shift took place when the first whole genome sequences of streptomycetes became available, from which silent or "cryptic" biosynthetic gene clusters (BGCs) were discovered. Cryptic BGCs reveal a so far untapped potential of the microorganisms for the production of novel compounds, which has spurred new efforts in understanding the complex regulation between primary and secondary metabolism...
October 27, 2017: Biotechnology Journal
Gino Stolfa, Matthew T Smonskey, Ryan Boniface, Anna-Barbara Hachmann, Paul Gulde, Atul D Joshi, Anson P Pierce, Scott J Jacobia, Andrew Campbell
CHO cells are the most prevalent platform for modern bio-therapeutic production. Currently, there are several CHO cell lines used in bioproduction with distinct characteristics and unique genotypes and phenotypes. These differences limit advances in productivity and quality that can be achieved by the most common approaches to bioprocess optimization and cell line engineering. Incorporating omics based approaches into current bioproduction processes will complement traditional methodologies to maximize gains from CHO engineering and bioprocess improvements...
October 26, 2017: Biotechnology Journal
Jae Woong Choi, Sung Sun Yim, Ki Jun Jeong
In the cell surface display, the choice of host cell and anchoring motif are the most crucial for the efficient display of passenger proteins. Corynebacterium glutamicum has mycolic acid layer in outer membrane and the use of protein in the mycolic acid layer as an anchoring motif can provide a potential platform for surface display in C. glutamicum. All 19 mycolic acid layer proteins of C. glutamicum are analyzed, and two proteins, NCgl0535 and NCgl1337, which have a signal peptide and predicted O-mycoloylation site, are selected as anchoring motifs candidates...
October 26, 2017: Biotechnology Journal
Howard C Tribe, Josephine McEwan, Heath Taylor, Richard O C Oreffo, Rahul S Tare
Given articular cartilage has a limited repair potential, untreated osteochondral lesions of the ankle can lead to debilitating symptoms and joint deterioration necessitating joint replacement. While a wide range of reparative and restorative surgical techniques have been developed to treat osteochondral lesions of the ankle, there is no consensus in the literature regarding which is the ideal treatment. Tissue engineering strategies, encompassing stem cells, somatic cells, biomaterials and stimulatory signals (biological and mechanical), have a potentially valuable role in the treatment of osteochondral lesions...
October 25, 2017: Biotechnology Journal
Moritz Ebeler, Florian Pilgram, Kai Wolz, Gunnar Grim, Matthias Franzreb
The growing market of biopharmaceuticals and the constant developments in upstream fermentation have generated a strong demand for new downstream purification methods. Magnetic separation in combination with functional magnetic particles has been known for many years as a promising candidate for a direct capturing tool in protein purification but the lack of suitable GMP-compliant purification equipment has prevented the launch of this technology in large scale bioprocessing. To tackle this bottle-neck, the principle of a 'rotor-stator' high-gradient magnetic separator was fully redesigned to meet the rigorous requirements of modern cGMP biotechnology purification processes...
October 23, 2017: Biotechnology Journal
Tomás Aparicio, Víctor de Lorenzo, Esteban Martínez-García
While adoption of single-stranded (ss) DNA recombineering techniques has greatly eased genetic design of the platform strain Pseudomonas putida KT2440, available methods still produce the desired modifications/deletions at low frequencies. This makes isolation of mutants that do not display selectable or conspicuous phenotypes considerably difficult. To overcome this limitation, we have merged ssDNA recombineering with CRISPR/Cas9 technology in this bacterium for efficient killing of unmodified cells and thus non-phenotypic selection of bacteria bearing the mutations of interest...
October 23, 2017: Biotechnology Journal
Elisabete C Costa, Duarte de Melo-Diogo, André F Moreira, Marco P Carvalho, Ilídio J Correia
Scalable and reproducible production of 3D cellular spheroids is highly demanded, by pharmaceutical companies, for drug screening purposes during the pre-clinical evaluation phase. These 3D cellular constructs, unlike the monolayer culture of cells, can mimic different features of human tissues, including cellular organization, cell-cell and cell-extracellular matrix (ECM) interactions. Up to now, different techniques (scaffold-based and scaffold-free) have been used for spheroids formation, being the Liquid Overlay Technique (LOT) one of the most explored methodologies, due to its low cost and easy handling...
October 23, 2017: Biotechnology Journal
Feng Li, Changji Yin, Liming Sun, Yuanxiu Li, Xuewu Guo, Hao Song
Microbial fuel cell (MFC) is an eco-friendly bio-electrochemical system that uses mi-croorganism as biocatalyst to convert biomass into electricity. Glycerol, as a waste in the biodiesel refinery processes, is an appealing substrate for MFC. Nevertheless, glycerol cannot be utilized as carbon source by well-known exoelectrogens such as Shewanella oneidensis. Herein, to generate electricity by rapidly harnessing glycerol, we rationally constructed a Klebsiella pneumoniae-Shewanella oneidensis microbial consortium to efficiently harvest electricity from glycerol, in which K...
October 18, 2017: Biotechnology Journal
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