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Metabolic engineering

Jacqueline E Gonzalez, Maciek R Antoniewicz
Engineered microbes offer a practical and sustainable alternative to traditional industrial approaches. To increase the economic feasibility of biological processes, microbial isolates are engineered to take up inexpensive feedstocks (including lignocellulosic biomass, syngas, methane, and carbon dioxide), and convert them into substrates of central metabolism and further into value-added products. To trace the metabolism of these feedstocks into products, isotopic tracers are applied together with isotopomer analysis techniques such as (13)C-metabolic flux analysis to provide a detailed picture of pathway utilization...
October 22, 2016: Current Opinion in Biotechnology
Maria Giovanna Sabbieti, Alessandra Dubbini, Fulvio Laus, Emanuele Paggi, Andrea Marchegiani, Melania Capitani, Luigi Marchetti, Fabrizio Dini, Tina Vermonden, Piera Di Martino, Dimitrios Agas, Roberta Censi
The present study reports on the biocompatibility in vivo after intramuscular and subcutaneous administration in Balb/c mice of vinyl sulphone bearing p(HPMAm-lac1-2)-PEG-p(HPMAm-lac1-2)/thiolated hyaluronic acid hydrogels, designed as novel injectable biomaterials for potential application in the fields of tissue engineering and regenerative medicine. Ultrasonography, used as a method to study hydrogel gelation and residence time in vivo, showed that, upon injection, the biomaterial efficiently formed a hydrogel by simultaneous thermal gelation and Michael Addition cross-linking forming a viscoelastic spherical depot at the injection site...
October 24, 2016: Journal of Tissue Engineering and Regenerative Medicine
Xuan Cao, Yu-Bei Lv, Jun Chen, Tadayuki Imanaka, Liu-Jing Wei, Qiang Hua
BACKGROUND: Limonene, a monocyclic monoterpene, is known for its using as an important precursor of many flavoring, pharmaceutical, and biodiesel products. Currently, d-limonene has been produced via fractionation from essential oils or as a byproduct of orange juice production, however, considering the increasing need for limonene and a certain amount of pesticides may exist in the limonene obtained from the citrus industry, some other methods should be explored to produce limonene. RESULTS: To construct the limonene synthetic pathway in Yarrowia lipolytica, two genes encoding neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) were codon-optimized and heterologously expressed in Y...
2016: Biotechnology for Biofuels
Leonid E Fridlyand, Natalia A Tamarina, Andrew V Schally, Louis H Philipson
Growth hormone-releasing hormone (GHRH) is produced by the hypothalamus and stimulates growth hormone synthesis and release in the anterior pituitary gland. In addition, GHRH is an important regulator of cellular functions in many cells and organs. Expression of GHRH G-Protein Coupled Receptor (GHRHR) has been demonstrated in different peripheral tissues and cell types, including pancreatic islets. Among the peripheral activities, recent studies demonstrate a novel ability of GHRH analogs to increase and preserve insulin secretion by beta-cells in isolated pancreatic islets, which makes them potentially useful for diabetes treatment...
2016: Frontiers in Endocrinology
Katsumi Sakata, Toshiyuki Saito, Hajime Ohyanagi, Jun Okumura, Kentaro Ishige, Harukazu Suzuki, Takuji Nakamura, Setsuko Komatsu
Soybean (Glycine max) is sensitive to flooding stress, and flood damage at the seedling stage is a barrier to growth. We constructed two mathematical models of the soybean metabolic network, a control model and a flooded model, from metabolic profiles in soybean plants. We simulated the metabolic profiles with perturbations before and after the flooding stimulus using the two models. We measured the variation of state that the system could maintain from a state-space description of the simulated profiles. The results showed a loss of variation of state during the flooding response in the soybean plants...
October 24, 2016: Scientific Reports
Karan Malhotra, Mayavan Subramaniyan, Khushboo Rawat, Md Kalamuddin, M Irfan Qureshi, Pawan Malhotra, Asif Mohmmed, Katrina Cornish, Henry Daniell, Shashi Kumar
Artemisinin is highly effective against drug-resistant malarial parasites, which affects nearly half of the global population and kills >500 000 people each year. The primary cost of artemisinin is the very expensive process used to extract and purify the drug from Artemisia annua. Elimination of this apparently unnecessary step will make this potent antimalarial drug affordable to the global population living in endemic regions. Here we reported the oral delivery of a non-protein drug artemisinin biosynthesized (∼0...
October 20, 2016: Molecular Plant
Shi Yun Tong, Zuyong Wang, Poon Nian Lim, Wilson Wang, Eng San Thian
Regeneration of injuries at tendon-to-bone interface (TBI) remains a challenging issue due to the complex tissue composition involving both soft tendon tissues and relatively hard bone tissues. Tissue engineering using polymeric/ceramic composites has been of great interest to generate scaffolds for tissue's healing at TBI. Herein, we presented a novel method to blend polymers and bioceramics for tendon tissue engineering application. A homogeneous composite comprising of nanohydroxyapatite (nHA) particles in poly(ε-caprolactone) (PCL) matrix was obtained using a combination of solvent and mechanical blending process...
January 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
Monica A Schmidt, Ken Pendarvis
Oilseed crops are global commodities for their oil and protein seed content. We have engineered the oilseed Camelina sativa to exhibit increased protein content with a slight decrease in oil content. The introduction of a phytoene synthase gene with an RNAi cassette directed to suppress the storage protein 2S albumin resulted in seeds with an 11-24 % elevation in overall protein. The phytoene synthase cassette alone produced enhanced β-carotene content of an average 275 ± 6.10 μg/g dry seed and an overall altered seed composition of 11 % less protein and comparable nontransgenic amounts of both oil and carbohydrates...
October 22, 2016: Transgenic Research
Andrew J Loder, Yejun Han, Aaron B Hawkins, Hong Lian, Gina L Lipscomb, Gerrit J Schut, Matthew W Keller, Michael W W Adams, Robert M Kelly
The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle fixes CO2 in extremely thermoacidophilic archaea and holds promise for metabolic engineering because of its thermostability and potentially rapid pathway kinetics. A reaction kinetics model was developed to examine the biological and biotechnological attributes of the 3HP/4HB cycle as it operates in Metallosphaera sedula, based on previous information as well as on kinetic parameters determined here for recombinant versions of five of the cycle enzymes (malonyl-CoA/succinyl-CoA reductase, 3-hydroxypropionyl-CoA synthetase, 3-hydroxypropionyl-CoA dehydratase, acryloyl-CoA reductase, and succinic semialdehyde reductase)...
October 19, 2016: Metabolic Engineering
R Balfour Sartor, Gary D Wu
Intestinal microbiota are involved in the pathogenesis of Crohn's disease, ulcerative colitis, and pouchitis. We review the mechanisms by which these gut bacteria, fungi, and viruses mediate mucosal homeostasis, via their composite genes (metagenome) and metabolic products (metabolome). We explain how alterations to their profiles and functions under conditions of dysbiosis contribute to inflammation and effector immune responses that mediate inflammatory bowel diseases (IBD) in humans and enterocolitis in mice...
October 18, 2016: Gastroenterology
Xiaoxue Tong, Tania Triscari Barberi, Catherine H Botting, Sunil V Sharma, Mark J H Simmons, Tim W Overton, Rebecca J M Goss
BACKGROUND: Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis...
October 21, 2016: Microbial Cell Factories
Anne-Sophie Dumas, Ludivine Taconnat, Evangelos Barbas, Guillem Rigaill, Olivier Catrice, Delphine Bernard, Abdelilah Benamar, David Macherel, Abdelhak El Amrani, Richard Berthomé
BACKGROUND: Higher plants have to cope with increasing concentrations of pollutants of both natural and anthropogenic origin. Given their capacity to concentrate and metabolize various compounds including pollutants, plants can be used to treat environmental problems - a process called phytoremediation. However, the molecular mechanisms underlying the stabilization, the extraction, the accumulation and partial or complete degradation of pollutants by plants remain poorly understood. RESULTS: Here, we determined the molecular events involved in the early plant response to phenanthrene, used as a model of polycyclic aromatic hydrocarbons...
October 21, 2016: BMC Genomics
Stacy-Anne Morgan, Dana C Nadler, Rayka Yokoo, David F Savage
Metabolic engineering offers the potential to renewably produce important classes of chemicals, particularly biofuels, at an industrial scale. DNA synthesis and editing techniques can generate large pathway libraries, yet identifying the best variants is slow and cumbersome. Traditionally, analytical methods like chromatography and mass spectrometry have been used to evaluate pathway variants, but such techniques cannot be performed with high throughput. Biosensors - genetically encoded components that actuate a cellular output in response to a change in metabolite concentration - are therefore a promising tool for rapid and high-throughput evaluation of candidate pathway variants...
October 18, 2016: Current Opinion in Chemical Biology
Durga Mahor, Anu Priyanka, Gandham S Prasad, Krishan Gopal Thakur
Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway...
2016: PloS One
Po C Liu, Yi T Lee, Chun Y Wang, Ya-Tang Yang
We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility...
September 27, 2016: Journal of Visualized Experiments: JoVE
Whitney D Hollinshead, Sarah Rodriguez, Hector Garcia Martin, George Wang, Edward E K Baidoo, Kenneth L Sale, Jay D Keasling, Aindrila Mukhopadhyay, Yinjie J Tang
BACKGROUND: Glycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism. RESULTS: Overexpression of edd and eda in E...
2016: Biotechnology for Biofuels
Christopher Chukwudi Okonkwo, Victor Ujor, Thaddeus Chukwuemeka Ezeji
Understanding the capacity of Paenibacillus polymyxa DSM 365 to tolerate increasing concentrations of 2,3-butanediol (2,3-BD) is critical to engineering a 2,3-BD-overproducing strain. Hence, we investigated the response of P. polymyxa to high 2,3-BD concentrations. In fed-batch cultures (6-L bioreactor) 2,3-BD was accumulated to a maximum concentration of 47g/L despite the presence of residual 13g/L glucose in the medium. Concomitantly, accumulation of acetoin, the precursor of 2,3-BD increased after maximum 2,3-BD concentration was reached, suggesting that 2,3-BD was reconverted to acetoin after the concentration tolerance threshold of 2,3-BD was exceeded...
October 17, 2016: New Biotechnology
Alessandro Borri, Pasquale Palumbo, Abhyudai Singh
Synthetic biology combines different branches of biology and engineering aimed at designing synthetic biological circuits able to replicate emergent properties useful for the biotechnology industry, human health and environment. The role of negative feedback in noise propagation for a basic enzymatic reaction scheme is investigated. Two feedback control schemes on enzyme expression are considered: one from the final product of the pathway activity, the other from the enzyme accumulation. Both schemes are designed to provide the same steady-state average values of the involved players, in order to evaluate the feedback performances according to the same working mode...
October 2016: IET Systems Biology
Angad P Mehta, Han Li, Sean A Reed, Lubica Supekova, Tsotne Javahishvili, Peter G Schultz
Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E...
October 20, 2016: Journal of the American Chemical Society
Amit Ghosh, David Ando, Jennifer Gin, Weerawat Runguphan, Charles Denby, George Wang, Edward E K Baidoo, Chris Shymansky, Jay D Keasling, Héctor García Martín
Efficient redirection of microbial metabolism into the abundant production of desired bioproducts remains non-trivial. Here, we used flux-based modeling approaches to improve yields of fatty acids in Saccharomyces cerevisiae. We combined (13)C labeling data with comprehensive genome-scale models to shed light onto microbial metabolism and improve metabolic engineering efforts. We concentrated on studying the balance of acetyl-CoA, a precursor metabolite for the biosynthesis of fatty acids. A genome-wide acetyl-CoA balance study showed ATP citrate lyase from Yarrowia lipolytica as a robust source of cytoplasmic acetyl-CoA and malate synthase as a desirable target for downregulation in terms of acetyl-CoA consumption...
2016: Frontiers in Bioengineering and Biotechnology
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