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https://www.readbyqxmd.com/read/28730410/engineering-of-the-cytochrome-p450-monooxygenase-system-for-benzyl-maltol-hydroxylation
#1
Iori Kozono, Kousuke Mihara, Kazuyuki Minagawa, Makoto Hibi, Jun Ogawa
Maltol derivatives are utilized in a variety of fields due to their metal-chelating abilities, and modification of the 2-methyl side chain is known to effectively expand their functional diversity. In the present study, microbial enzymes were screened for hydroxylating activity towards the 2-methyl group in a maltol derivative, 3-benzyloxy-2-methyl-4-pyrone (BMAL). Novosphingobium sp. SB32149 was found to have the ability to convert BMAL into 3-benzyloxy-2-hydroxymethyl-4-pyrone (BMAL-OH). The enzymes responsible, a cytochrome P450 monooxygenase (P450nov), a ferredoxin (FDXnov), and a ferredoxin reductase (FDRnov), were identified in the SB32149 strain...
July 20, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28729404/exploiting-natural-variation-to-uncover-an-alkene-biosynthetic-enzyme-in-poplar
#2
Eliana Gonzales-Vigil, Charles A Hefer, Michelle E von Loessl, Jonathan La Mantia, Shawn D Mansfield
Alkenes are linear hydrocarbons with one or more double bonds. Despite their potential as biofuels and precursors for specialty chemicals, the underlying biochemistry and genetics of alkene biosynthesis in plants remain elusive. Here, we report on a screen of natural accessions of poplar (Populus trichocarpa) revealing that the leaf cuticular waxes are predominantly composed of alkanes and alkenes. Interestingly, the accumulation of alkenes increases with leaf development, is limited to the abaxial side of the leaf, and is impaired in a few accessions...
July 20, 2017: Plant Cell
https://www.readbyqxmd.com/read/28727740/genetically-engineered-suicide-gene-in-mesenchymal-stem-cells-using-a-tet-on-system-for-anaplastic-thyroid-cancer
#3
Senthilkumar Kalimuthu, Ji Min Oh, Prakash Gangadaran, Liya Zhu, Ho Won Lee, Yong Hyun Jeon, Shin Young Jeong, Sang-Woo Lee, Jaetae Lee, Byeong-Cheol Ahn
Anaplastic thyroid cancer (ATC) is the most aggressive malignancy of the thyroid, during which undifferentiated tumors arise from the thyroid follicular epithelium. ATC has a very poor prognosis due to its aggressive behavior and poor response to conventional therapies. Gene-directed enzyme/prodrug therapy using genetically engineered mesenchymal stromal cells (MSC) is a promising therapeutic strategy. The doxycycline (DOX)-controlled Tet inducible system is the most widely utilized regulatory system and could be a useful tool for therapeutic gene-based therapies...
2017: PloS One
https://www.readbyqxmd.com/read/28725483/engineering-glucose-metabolism-of-escherichia-coli-under-nitrogen-starvation
#4
Victor Chubukov, John James Desmarais, George Wang, Leanne Jade G Chan, Edward Ek Baidoo, Christopher J Petzold, Jay D Keasling, Aindrila Mukhopadhyay
A major aspect of microbial metabolic engineering is the development of chassis hosts that have favorable global metabolic phenotypes, and can be further engineered to produce a variety of compounds. In this work, we focus on the problem of decoupling growth and production in the model bacterium Escherichia coli, and in particular on the maintenance of active metabolism during nitrogen-limited stationary phase. We find that by overexpressing the enzyme PtsI, a component of the glucose uptake system that is inhibited by α-ketoglutarate during nitrogen limitation, we are able to achieve a fourfold increase in metabolic rates...
2017: NPJ Systems Biology and Applications
https://www.readbyqxmd.com/read/28725265/two-bifunctional-enzymes-from-the-marine-protist-thraustochytrium-roseum-biochemical-characterization-of-wax-ester-synthase-acyl-coa-diacylglycerol-acyltransferase-activity-catalyzing-wax-ester-and-triacylglycerol-synthesis
#5
Nannan Zhang, Zejing Mao, Ling Luo, Xia Wan, Fenghong Huang, Yangmin Gong
BACKGROUND: Triacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels. In bacteria, the final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT)...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28724386/engineering-microbial-cell-factories-for-the-production-of-plant-natural-products-from-design-principles-to-industrial-scale-production
#6
REVIEW
Xiaonan Liu, Wentao Ding, Huifeng Jiang
Plant natural products (PNPs) are widely used as pharmaceuticals, nutraceuticals, seasonings, pigments, etc., with a huge commercial value on the global market. However, most of these PNPs are still being extracted from plants. A resource-conserving and environment-friendly synthesis route for PNPs that utilizes microbial cell factories has attracted increasing attention since the 1940s. However, at the present only a handful of PNPs are being produced by microbial cell factories at an industrial scale, and there are still many challenges in their large-scale application...
July 19, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28723577/enabling-tools-for-high-throughput-detection-of-metabolites-metabolic-engineering-and-directed-evolution-applications
#7
REVIEW
Jyun-Liang Lin, James M Wagner, Hal S Alper
Within the Design-Build-Test Cycle for strain engineering, rapid product detection and selection strategies remain challenging and limit overall throughput. Here we summarize a wide variety of modalities that transduce chemical concentrations into easily measured absorbance, luminescence, and fluorescence signals. Specifically, we cover protein-based biosensors (including transcription factors), nucleic acid-based biosensors, coupled enzyme reactions, bioorthogonal chemistry, and fluorescent and chromogenic dyes and substrates as modalities for detection...
July 16, 2017: Biotechnology Advances
https://www.readbyqxmd.com/read/28721734/enantioselective-total-synthesis-of-nigelladine-a-via-late-stage-c-h-oxidation-enabled-by-an-engineered-p450-enzyme
#8
Steven A Loskot, David K Romney, Frances H Arnold, Brian M Stoltz
An enantioselective total synthesis of the norditerpenoid alkaloid nigelladine A is described. Stra-tegically, the synthesis relies on a late stage C-H oxida-tion of an advanced intermediate. While traditional chemical methods failed to deliver the desired outcome, an engineered cytochrome P450 enzyme was employed to effect a chemo- and regioselective allylic C-H oxida-tion in the presence of four oxidizable positions. The enzyme variant was readily identified from a focused library of three enzymes, allowing for completion of the synthesis without the need for extensive screening...
July 19, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28721723/enrichment-of-long-chain-polyunsaturated-fatty-acids-by-coordinated-expression-of-multiple-metabolic-nodes-in-the-oleaginous-microalga-phaeodactylum-tricornutum
#9
Xiang Wang, Yu-Hong Liu, Wei Wei, Xia Zhou, Wasiqi Yuan, Srinivasan Balamurugan, Ting-Bin Hao, Wei-Dong Yang, Jie-Sheng Liu, Hong-Ye Li
Microalgal long-chain polyunsaturated fatty acids (LC-PUFAs) have emerged as promising alternatives to depleting fish oils. However, the overproduction of LC-PUFAs in microalgae has remained challenging. Here, we report a sequential metabolic engineering strategy that systematically overcomes the metabolic bottlenecks and overproduces LC-PUFAs. Malonyl CoA-acyl carrier protein transacylase, catalyzing the first committed step in type II fatty acid synthesis, and desaturase 5b, involved in fatty acid desaturation, were coordinately expressed in Phaeodactylum tricornutum...
July 19, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28720496/substrate-binding-interferes-with-active-site-conformational-dynamics-in-endoglucanase-cel5a-from-thermobifida-fusca
#10
Xukai Jiang, Yuying Wang, Limei Xu, Guanjun Chen, Lushan Wang
The role of protein dynamics in enzyme catalysis is one of the most active areas in current enzymological research. Here, using endoglucanase Cel5A from Thermobifida fusca (TfCel5A) as a model, we applied molecular dynamics simulations to explore the dynamic behavior of the enzyme upon substrate binding. The collective motions of the active site revealed that the mechanism of TfCel5A substrate binding can likely be described by the conformational-selection model; however, we observed that the conformations of active site residues changed differently along with substrate binding...
July 15, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28719738/an-activator-blocker-pair-provides-a-controllable-on-off-switch-for-a-ketosteroid-isomerase-active-site-mutant
#11
Vandana Lamba, Filip Yabukarski, Daniel Herschlag
Control of enzyme activity is fundamental to biology and represents a long-term goal in bioengineering and precision therapeutics. While several powerful molecular strategies have been developed, limitations remain in their generalizability and dynamic range. We demonstrate a control mechanism via separate small molecules that turn on the enzyme (activator) and turn off the activation (blocker). We show that a pocket created near the active site base of the enzyme ketosteriod isomerase (KSI) allows efficient and saturable base rescue when the enzyme's natural general base is removed...
July 18, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28719578/de-novo-active-sites-for-resurrected-precambrian-enzymes
#12
Valeria A Risso, Sergio Martinez-Rodriguez, Adela M Candel, Dennis M Krüger, David Pantoja-Uceda, Mariano Ortega-Muñoz, Francisco Santoyo-Gonzalez, Eric A Gaucher, Shina C L Kamerlin, Marta Bruix, Jose A Gavira, Jose M Sanchez-Ruiz
Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties...
July 18, 2017: Nature Communications
https://www.readbyqxmd.com/read/28718514/aldehyde-dehydrogenase-1-a1-regulates-energy-metabolism-in-adipocytes-from-different-species
#13
Kefeng Yang, Christopher Adin, Qiwen Shen, Ly James Lee, Lianbo Yu, Paolo Fadda, Arpad Samogyi, Kathleen Ham, Lu Xu, Chen Gilor, Ouliana Ziouzenkova
BACKGROUND: Survival and longevity of xenotransplants depend on immune function and ability to integrate energy metabolism between cells from different species. However, mechanisms for interspecies cross talk in energy metabolism are not well understood. White adipose tissue stores energy and is capable of mobilization and dissipation of energy as heat (thermogenesis) by adipocytes expressing uncoupling protein 1 (Ucp1). Both pathways are under the control of vitamin A metabolizing enzymes...
July 17, 2017: Xenotransplantation
https://www.readbyqxmd.com/read/28715793/monitoring-in-vivo-metabolic-flux-with-a-designed-whole-cell-metabolite-biosensor-of-shikimic-acid
#14
Heng Li, Chaoning Liang, Wei Chen, Jian-Ming Jin, Shuang-Yan Tang, Yong Tao
Knowledge of intracellular metabolite levels is important for the understanding of metabolic flux distributions. Whole-cell biosensors of key metabolites are ideal for the monitoring of carbon flow in important metabolic pathways, thus guiding metabolic engineering for microbial improvement. However, lack of biosensors for metabolites of interests has limited their applications. In this study, a genetically encoded whole-cell biosensor specifically responding to shikimic acid has been developed by screening a site-saturation mutagenesis library of the binding pocket of a uric acid-responsive regulatory protein...
July 11, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/28715702/engineering-carbon-fixation-in-e-coli-from-heterologous-rubisco-expression-to-the-calvin-benson-bassham-cycle
#15
REVIEW
Niv Antonovsky, Shmuel Gleizer, Ron Milo
Carbon fixation is the gateway of inorganic carbon into the biosphere. Our ability to engineer carbon fixation pathways in living organisms is expected to play a crucial role in the quest towards agricultural and energetic sustainability. Recent successes to introduce non-native carbon fixation pathways into heterotrophic hosts offer novel platforms for manipulating these pathways in genetically malleable organisms. Here, we focus on past efforts and future directions for engineering the dominant carbon fixation pathway in the biosphere, the Calvin-Benson cycle, into the well-known model organism Escherichia coli...
July 14, 2017: Current Opinion in Biotechnology
https://www.readbyqxmd.com/read/28715126/an-engineered-photoswitchable-mammalian-pyruvate-kinase
#16
Stefanie Gehrig, Jamie A Macpherson, Paul C Driscoll, Alastair Symon, Stephen R Martin, James I MacRae, Jens Kleinjung, Franca Fraternali, Dimitrios Anastasiou
Changes in allosteric regulation of glycolytic enzymes have been linked to metabolic reprogramming involved in cancer. Remarkably, allosteric mechanisms control enzyme function at significantly shorter time-scales compared to the long-term effects of metabolic reprogramming on cell proliferation. It remains unclear if and how the speed and reversibility afforded by rapid allosteric control of metabolic enzymes is important for cell proliferation. Tools that allow specific, dynamic modulation of enzymatic activities in mammalian cells would help address this question...
July 17, 2017: FEBS Journal
https://www.readbyqxmd.com/read/28714570/human-osteoarthritic-chondrons-outnumber-patient-and-joint-matched-chondrocytes-in-hydrogel-culture-future-application-in-autologous-cell-based-oa-cartilage-repair
#17
Miriam Rothdiener, Tatiana Uynuk-Ool, Norbert Südkamp, Matthias Aurich, Alan J Grodzinsky, Bodo Kurz, Bernd Rolauffs
Autologous chondrocyte implantation (ACI) is used in 34-60% for osteoarthritic (OA) cartilage defects, although ACI is neither recommended nor designed for OA. Envisioning a hydrogel-based ACI for OA that uses chondrons instead of classically used chondrocytes, we hypothesized that human OA-chondrons may outperform OA-chondrocytes. We compared patient- and joint surface-matched human OA-chondrons vs. OA-chondrocytes cultured for the first time in a hydrogel, using a self-assembling peptide system. We determined yield, viability, cell numbers, mRNA expression, GAPDH mRNA enzyme activity, collagen II synthesis (CPII) and degradation (C2C), and sGAG...
July 17, 2017: Journal of Tissue Engineering and Regenerative Medicine
https://www.readbyqxmd.com/read/28714313/artificial-metalloenzymes-reaction-scope-and-optimization-strategies
#18
Fabian Schwizer, Yasunori Okamoto, Tillmann Heinisch, Yifan Gu, Michela M Pellizzoni, Vincent Lebrun, Raphael Reuter, Valentin Köhler, Jared C Lewis, Thomas R Ward
The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970's. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000's...
July 17, 2017: Chemical Reviews
https://www.readbyqxmd.com/read/28712952/a-robust-high-throughput-fungal-biosensor-assay-for-the-detection-of-estrogen-activity
#19
Christoph Zutz, Karen Wagener, Desislava Yankova, Stefanie Eder, Erich Möstl, Marc Drillich, Kathrin Rychli, Martin Wagner, Joseph Strauss
Estrogenic active compounds are present in a variety of sources and may alter biological functions in vertebrates. Therefore, it is crucial to develop innovative analytical systems that allow us to screen a broad spectrum of matrices and deliver fast and reliable results. We present the adaptation and validation of a fungal biosensor for the detection of estrogen activity in cow derived samples and tested the clinical applicability for pregnancy diagnosis in 140 mares and 120 cows. As biosensor we used a previously engineered genetically modified strain of the filamentous fungus Aspergillus nidulans, which contains the human estrogen receptor alpha and a reporter construct, in which β-galactosidase gene expression is controlled by an estrogen-responsive-element...
July 13, 2017: Steroids
https://www.readbyqxmd.com/read/28711855/exploiting-and-engineering-hemoproteins-for-abiological-carbene-and-nitrene-transfer-reactions
#20
REVIEW
Oliver F Brandenberg, Rudi Fasan, Frances H Arnold
The surge in reports of heme-dependent proteins as catalysts for abiotic, synthetically valuable carbene and nitrene transfer reactions dramatically illustrates the evolvability of the protein world and our nascent ability to exploit that for new enzyme chemistry. We highlight the latest additions to the hemoprotein-catalyzed reaction repertoire (including carbene Si-H and C-H insertions, Doyle-Kirmse reactions, aldehyde olefinations, azide-to-aldehyde conversions, and intermolecular nitrene C-H insertion) and show how different hemoprotein scaffolds offer varied reactivity and selectivity...
July 13, 2017: Current Opinion in Biotechnology
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