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https://www.readbyqxmd.com/read/28624535/atp-free-biosynthesis-of-a-high-energy-phosphate-metabolite-fructose-1-6-diphosphate-by-in-vitro-metabolic-engineering
#1
Wei Wang, Meixia Liu, Chun You, Zhimin Li, Yi-Heng Percival Zhang
Fructose 1,6-diphosphate (FDP) is a widely used medicine and is also a precursor of two important three-carbon phosphates -- glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) for the biosynthesis of numerous fine chemicals. An in vitro synthetic cofactor-free enzymatic pathway comprised of four hyperthermophilic enzymes was designed to produce FDP from starch and pyrophosphate. All of four hyperthermophilic enzymes (i.e., alpha-glucan phosphorylase from Thermotaga maritima, phosphoglucomutase from Thermococcus kodakarensis, glucose 6-phosphate isomerase from Thermus thermophilus, and pyrophosphate phosphofructokinase from T...
June 14, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28606145/three-steps-in-one-pot-biosynthesis-of-4-hydroxycinnamyl-alcohols-using-immobilized-whole-cells-of-two-genetically-engineered-escherichia-coli-strains
#2
Shuxin Liu, Jiabin Liu, Jiayin Hou, Nan Chao, Ying Gai, Xiangning Jiang
BACKGROUND: 4-Hydroxycinnamyl alcohols are a class of natural plant secondary metabolites that include p-coumaryl alcohol, caffeyl alcohol, coniferyl alcohol and sinapyl alcohol, and have physiological, ecological and biomedical significance. While it is necessary to investigate the biological pathways and economic value of these alcohols, research is hindered because of their limited availability and high cost. Traditionally, these alcohols are obtained by chemical synthesis and plant extraction...
June 12, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28590138/vibrational-probes-of-molybdenum-cofactor-protein-interactions-in-xanthine-dehydrogenase
#3
Chao Dong, Jing Yang, Stefan Reschke, Silke Leimkühler, Martin L Kirk
The pyranopterin dithiolene (PDT) ligand is an integral component of the molybdenum cofactor (Moco) found in all molybdoenzymes with the sole exception of nitrogenase. However, the roles of the PDT in catalysis are still unknown. The PDT is believed to be bound to the proteins by an extensive hydrogen-bonding network, and it has been suggested that these interactions may function to fine-tune Moco for electron- and atom-transfer reactivity in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe Moco-protein interactions using heavy-atom congeners of lumazine, molecules that bind tightly to both wild-type xanthine dehydrogenase (wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation to the corresponding violapterin product molecules...
June 7, 2017: Inorganic Chemistry
https://www.readbyqxmd.com/read/28587256/biochemical-and-computational-insights-on-a-novel-acid-resistant-and-thermal-stable-glucose-1-dehydrogenase
#4
Haitao Ding, Fen Gao, Yong Yu, Bo Chen
Due to the dual cofactor specificity, glucose 1-dehydrogenase (GDH) has been considered as a promising alternative for coenzyme regeneration in biocatalysis. To mine for potential GDHs for practical applications, several genes encoding for GDH had been heterogeneously expressed in Escherichia coli BL21 (DE3) for primary screening. Of all the candidates, GDH from Bacillus sp. ZJ (BzGDH) was one of the most robust enzymes. BzGDH was then purified to homogeneity by immobilized metal affinity chromatography and characterized biochemically...
June 5, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28552632/structural-basis-for-the-magnesium-dependent-activation-of-transketolase-from-chlamydomonas-reinhardtii
#5
Miriam Pasquini, Simona Fermani, Daniele Tedesco, Chiara Sciabolini, Pierre Crozet, Marina Naldi, Julien Henri, Ute Vothknecht, Carlo Bertucci, Stéphane D Lemaire, Mirko Zaffagnini, Francesco Francia
BACKGROUND: In photosynthetic organisms, transketolase (TK) is involved in the Calvin-Benson cycle and participates to the regeneration of ribulose-5-phosphate. Previous studies demonstrated that TK catalysis is strictly dependent on thiamine pyrophosphate (TPP) and divalent ions such as Mg(2+). METHODS: TK from the unicellular green alga Chlamydomonas reinhardtii (CrTK) was recombinantly produced and purified to homogeneity. Biochemical properties of the CrTK enzyme were delineated by activity assays and its structural features determined by CD analysis and X-ray crystallography...
August 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28551440/osmolyte-induced-enhancement-of-expression-and-solubility-of-human-dihydrofolate-reductase-an-in-vivo-study
#6
Naira Rashid, Charu Thapliyal, Pratima Chaudhuri Chattopadhyay
The process of recombinant protein production in E. coli system is often hampered by the formation of insoluble aggregates. Human Dihydrofolate reductase (hDHFR), an enzyme involved in the synthesis of purine, thymidilate and several other amino acids like glycine, methionine and serine is highly aggregation prone. It catalyzes the reduction of dihydrofolate (H2F) in order to regenerate tetrahydrofolate (H4F) utilizing NADPH as a cofactor. We have attempted to ameliorate the production of soluble and functional protein by growing and inducing the cells under osmotic stress condition, in the presence of various osmolytes like glycerol, sorbitol, TMAO, proline and glycine at 37°C...
May 25, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28545904/reductive-amination-of-ketones-catalyzed-by-whole-cell-biocatalysts-containing-imine-reductases-ireds
#7
Zaira Maugeri, Dörte Rother
The asymmetric reductive amination of ketones represents an elegant and convenient way to obtain chiral amines. Recently, several examples have been reported in which isolated imine reductases (IREDs) have been used for this type of reaction leading to promising results. In this work we focus on the applicability of whole cell biocatalysts (recombinant E.coli cells heterologously overexpressing an IRED) to simplify its preparation and to cut on catalyst production costs. Thirteen IREDs were screened towards six different ketones, using methylamine as amine donor...
May 22, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28527827/in%C3%A2-vitro-bioconversion-of-chitin-to-pyruvate-with-thermophilic-enzymes
#8
Kohsuke Honda, Keisuke Kimura, Pham Huynh Ninh, Hironori Taniguchi, Kenji Okano, Hisao Ohtake
Chitin is the second most abundant organic compound on the planet and thus has been regarded as an alternative resource to petroleum feedstocks. One of the key challenges in the biological conversion of biomass-derived polysaccharides, such as cellulose and chitin, is to close the gap between optimum temperatures for enzymatic saccharification and microbial fermentation and to implement them in a single bioreactor. To address this issue, in the present study, we aimed to perform an in vitro, one-pot bioconversion of chitin to pyruvate, which is a precursor of a wide range of useful metabolites...
May 17, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28526345/facile-fabrication-of-3d-porous-hybrid-sphere-by-co-immobilization-of-multi-enzyme-directly-from-cell-lysates-as-an-efficient-and-recyclable-biocatalyst-for-asymmetric-reduction-with-coenzyme-regeneration-in-situ
#9
Mingan Yu, Duqiang Liu, Lili Sun, Jing Li, Qian Chen, Lijun Pan, Jingchuan Shang, Shurong Zhang, Wei Li
Ni(2+)-agarose bead-wrapped multi-enzyme/inorganic hybrid sphere composed of the immobilized enzymes as organic component and NaH2PO4 and NaCl as inorganic component was developed by co-immobilizing extracellular His-tagged 3-quinuclidinone reductases and glucose dehydrogenase without pre-purification. The resulting biocatalysts has 3D porous architectures as confirmed by SEM and FESEM, and it enabled the continuous biotransformation of 3-quinuclidone to (R)-3-quinuclidinol with cofactor regeneration in situ...
May 17, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28512473/vitamin-c-in-stem-cell-biology-impact-on-extracellular-matrix-homeostasis-and-epigenetics
#10
REVIEW
Cristina D'Aniello, Federica Cermola, Eduardo Jorge Patriarca, Gabriella Minchiotti
Transcription factors and signaling molecules are well-known regulators of stem cell identity and behavior; however, increasing evidence indicates that environmental cues contribute to this complex network of stimuli, acting as crucial determinants of stem cell fate. l-Ascorbic acid (vitamin C (VitC)) has gained growing interest for its multiple functions and mechanisms of action, contributing to the homeostasis of normal tissues and organs as well as to tissue regeneration. Here, we review the main functions of VitC and its effects on stem cells, focusing on its activity as cofactor of Fe(+2)/αKG dioxygenases, which regulate the epigenetic signatures, the redox status, and the extracellular matrix (ECM) composition, depending on the enzymes' subcellular localization...
2017: Stem Cells International
https://www.readbyqxmd.com/read/28498544/metabolic-engineering-of-cofactor-flavin-adenine-dinucleotide-fad-synthesis-and-regeneration-in-escherichia-coli-for-production-of-%C3%AE-keto-acids
#11
Ying Hou, Gazi Sakir Hossain, Jianghua Li, Hyun-Dong Shin, Guocheng Du, Jian Chen, Long Liu
Cofactor flavin adenine dinucleotide (FAD) plays a vital role in many FAD-dependent enzymatic reactions; therefore, how to efficiently accelerate FAD synthesis and regeneration is an important topic in biocatalysis and metabolic engineering. In this study, a system involving the synthesis pathway and regeneration of FAD was engineered in Escherichia coli to improve α-keto acid production-from the corresponding L-amino acids-catalyzed by FAD-dependent L-amino acid deaminase (L-AAD). First, key genes, ribH, ribC, and ribF, were overexpressed and fine-tuned for FAD synthesis...
May 12, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28461497/folate-rescues-vitamin-b12-depletion-induced-inhibition-of-nuclear-thymidylate-biosynthesis-and-genome-instability
#12
Ashley M Palmer, Elena Kamynina, Martha S Field, Patrick J Stover
Clinical vitamin B12 deficiency can result in megaloblastic anemia, which results from the inhibition of DNA synthesis by trapping folate cofactors in the form of 5-methyltetrahydrofolate (5-methylTHF) and subsequent inhibition of de novo thymidylate (dTMP) biosynthesis. In the cytosol, vitamin B12 functions in the remethylation of homocysteine to methionine, which regenerates THF from 5-methylTHF. In the nucleus, THF is required for de novo dTMP biosynthesis, but it is not understood how 5-methylTHF accumulation in the cytosol impairs nuclear dTMP biosynthesis...
May 16, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28427921/enhancement-of-ethyl-s-4-chloro-3-hydroxybutanoate-production-at-high-substrate-concentration-by-in-situ-resin-adsorption
#13
Li-Feng Chen, Hai-Yang Fan, Yi-Ping Zhang, Wei Wei, Jin-Ping Lin, Dong-Zhi Wei, Hua-Lei Wang
Asymmetric reduction of ethyl 4-chloro-3-oxobutyrate (COBE) by carbonyl reductases presents an efficient way to produce Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important chiral intermediate for the synthesis of hydroxymethylglutaryl-CoA reductase inhibitors such as Lipitor(®). In this study, an NADPH-dependent carbonyl reductase (SrCR) from Synechocystis sp. was characterized to demonstrate a broad substrate spectrum, and the highest activity (53.1U/mg protein) with COBE. To regenerate the cofactor NADPH, Bacillus subtilis glucose dehydrogenase was successfully coexpressed with SrCR...
April 17, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28423958/strategies-and-perspectives-of-assembling-multi-enzyme-systems
#14
Shi-Zhen Wang, Yong-Hui Zhang, Hong Ren, Ya-Li Wang, Wei Jiang, Bai-Shan Fang
Multi-enzyme complexes have the potential to achieve high catalytic efficiency for sequence reactions due to their advantages in eliminating product inhibition, facilitating intermediate transfer and in situ regenerating cofactors. Constructing functional multi-enzyme systems to mimic natural multi-enzyme complexes is of great interest for multi-enzymatic biosynthesis and cell-free synthetic biotransformation, but with many challenges. Currently, various assembly strategies have been developed based on the interaction of biomacromolecules such as DNA, peptide and scaffolding protein...
April 20, 2017: Critical Reviews in Biotechnology
https://www.readbyqxmd.com/read/28401536/autodisplay-of-glucose-6-phosphate-dehydrogenase-for-redox-cofactor-regeneration-at-the-cell-surface
#15
Jan Schüürmann, Paul Quehl, Fabian Lindhorst, Kristina Lang, Joachim Jose
Inherent cofactor regeneration is a pivotal feature of whole cell biocatalysis. For specific biotechnological applications, surface display of enzymes is emerging as a tool to circumvent mass transfer limitations or enzyme stability problems. Even complex reactions can be accomplished applying displayed enzymes. Yet, industrial utilization of the technique is still impeded by lacking cofactor regeneration at the cell surface. Here, we report on the surface display of a glucose-6-phoshate dehydrogenase (G6PDH) via Autodisplay to address this limitation and regenerate NADPH directly at the cell surface...
April 12, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28389394/kinetically-accessible-yield-kay-for-redirection-of-metabolism-to-produce-exo-metabolites
#16
Jimmy G Lafontaine Rivera, Matthew K Theisen, Po-Wei Chen, James C Liao
The product formation yield (product formed per unit substrate consumed) is often the most important performance indicator in metabolic engineering. Until now, the actual yield cannot be predicted, but it can be bounded by its maximum theoretical value. The maximum theoretical yield is calculated by considering the stoichiometry of the pathways and cofactor regeneration involved. Here we found that in many cases, dynamic stability becomes an issue when excessive pathway flux is drawn to a product. This constraint reduces the yield and renders the maximal theoretical yield too loose to be predictive...
April 5, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28387366/elucidating-dynamic-metabolic-physiology-through-network-integration-of-quantitative-time-course-metabolomics
#17
Aarash Bordbar, James T Yurkovich, Giuseppe Paglia, Ottar Rolfsson, Ólafur E Sigurjónsson, Bernhard O Palsson
The increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course absolute quantitative metabolomics. This approach, termed "unsteady-state flux balance analysis" (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic metabolic flux states for red blood cells, platelets, and Saccharomyces cerevisiae...
April 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28357799/what-about-the-detoxification-mechanisms-underlying-ozone-sensitivity-in-liriodendron-tulipifera
#18
Elisa Pellegrini, Alessandra Campanella, Lorenzo Cotrozzi, Mariagrazia Tonelli, Cristina Nali, Giacomo Lorenzini
Liriodendron tulipifera (known as the tulip tree) is a woody species that has been previously classified as sensitive to ozone (O3) in terms of visible leaf injuries and photosynthetic primary reactions. The objective of this work is to give a thorough description of the detoxification mechanisms that are at the basis of O3 sensitivity. Biochemical and molecular markers were used to characterize the response of 1-year-old saplings exposed to O3 (120 ppb, 5 h day(-1), for 45 consecutive days) under controlled conditions...
March 29, 2017: Environmental Science and Pollution Research International
https://www.readbyqxmd.com/read/28346008/wnt-%C3%AE-catenin-signaling-is-required-for-distraction-osteogenesis-in-rats
#19
Xuemei Wang, En Luo, Ruiye Bi, Bin Ye, Jing Hu, Shujuan Zou
OVERVIEW: The Wnt signaling pathway plays crucial roles in embryonic skeletal development and postnatal bone regeneration. However, mechanisms of Wnt signaling functioning in distraction osteogenesis (DO) haven't been well characterized. MATERIALS AND METHODS: We established a DO model using Sprague-Dawley rat tibia. And a Wnt signaling blocking agent, recombinant rat Dickkopf-related protein 1 (rrDkk1), was locally applied in the distracted gap to study the role of Wnt signaling during DO process...
March 1, 2017: Connective Tissue Research
https://www.readbyqxmd.com/read/28345146/evidence-for-the-generation-of-myristylated-fmn-by-bacterial-luciferase
#20
Chaitanya R Tabib, Eveline Brodl, Peter Macheroux
The genes responsible for the light production in bioluminescent bacteria are present as an operon, luxCDABEG. Many strains of Photobacteria carry an additional gene, termed luxF. X-ray crystallographic analysis of LuxF revealed the presence of four molecules of a flavin derivative, i.e. 6-(3'-(R)-myristyl) flavin adenine mononucleotide (myrFMN) non-covalently bound to the homodimer. In the present study, we exploited the binding of myrFMN to recombinant apo-LuxF to explore the occurrence of myrFMN in various bioluminescent bacteria...
June 2017: Molecular Microbiology
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