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https://www.readbyqxmd.com/read/28822131/metabolic-engineering-of-photorespiration
#1
Martin K M Engqvist, Veronica G Maurino
The introduction of two alternative glycolate catabolic pathways in the chloroplasts of Arabidopsis thaliana rendered plants with increased biomass. To introduce these synthetic pathways, the selected genes were stepwise integrated in the nuclear genome of wild-type plants. These plants were transformed by Agrobacterium tumefaciens carrying the binary vectors using the floral dip method. Selection of transformants was conducted using different selection agents and the expression of the transgenes was confirmed by PCR and enzyme activity measurements...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28822124/measurement-of-enzyme-activities
#2
Hermann Bauwe
The determination of enzyme activities in organ or organellar extracts is an important means of investigating metabolic networks and allows testing the success of enzyme-targeted genetic engineering. It also delivers information on intrinsic enzyme parameters such as kinetic properties or impact of effector molecules. This chapter provides protocols on how to assess activities of the enzymes of the core photorespiratory pathway, from 2-phosphoglycolate phosphatase to glycerate 3-kinase.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28821847/overcoming-the-plasticity-of-plant-specialized-metabolism-for-selective-diterpene-production-in-yeast
#3
Codruta Ignea, Anastasia Athanasakoglou, Aggeliki Andreadelli, Maria Apostolaki, Minas Iakovides, Euripides G Stephanou, Antonios M Makris, Sotirios C Kampranis
Plants synthesize numerous specialized metabolites (also termed natural products) to mediate dynamic interactions with their surroundings. The complexity of plant specialized metabolism is the result of an inherent biosynthetic plasticity rooted in the substrate and product promiscuity of the enzymes involved. The pathway of carnosic acid-related diterpenes in rosemary and sage involves promiscuous cytochrome P450s whose combined activity results in a multitude of structurally related compounds. Some of these minor products, such as pisiferic acid and salviol, have established bioactivity, but their limited availability prevents further evaluation...
August 18, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28820431/high-throughput-screening-assay-for-laccase-engineering-toward-lignosulfonate-valorization
#4
David Rodríguez-Escribano, Felipe de Salas, Isabel Pardo, Susana Camarero
Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin-Ciocalteau reagent (FCR), to detect the decrease in phenolic content produced upon polymerization of lignosulfonate by the enzyme...
August 18, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28818809/differential-oxidative-and-antioxidative-response-of-duckweed-lemna-minor-toward-plant-growth-promoting-inhibiting-bacteria
#5
Hidehiro Ishizawa, Masashi Kuroda, Masaaki Morikawa, Michihiko Ike
Bacteria colonizing the plant rhizosphere are believed to positively or negatively affect the host plant productivity. This feature has inspired researchers to engineer such interactions to enhance crop production. However, it remains to be elucidated whether rhizobacteria influences plant oxidative stress vis-a-vis other environmental stressors, and whether such influence is associated with their growth promoting/inhibiting ability. In this study, two plant growth-promoting bacteria (PGPB) and two plant growth-inhibiting bacteria (PGIB) were separately inoculated into axenic duckweed (Lemna minor) culture under laboratory conditions for 4 and 8 days in order to investigate their effects on plant oxidative stress and antioxidant activities...
August 9, 2017: Plant Physiology and Biochemistry: PPB
https://www.readbyqxmd.com/read/28818426/investigation-of-the-escherichia-coli-membrane-transporters-involved-in-the-secretion-of-d-lactate-based-oligomers-by-loss-of-function-screening
#6
Camila Utsunomia, Chiaki Hori, Ken'ichiro Matsumoto, Seiichi Taguchi
d-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of d-LA and d-3-hydroxybutyrate that are produced and secreted by engineered Escherichia coli grown on glucose. The cells heterologously express LA-polymerizing polyhydroxyalkanoate synthase and monomer-supplying enzymes. In this study, we attempted to identify the D-LAO secretion route in E. coli, which is thought to be mediated by intrinsic membrane proteins. To this end, a loss-of-function screening of D-LAO secretion was carried out using 209 single-gene membrane protein deletants, which are involved in the transport of organic compounds...
August 14, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28818368/review-metabolic-engineering-of-unusual-lipids-in-the-synthetic-biology-era
#7
REVIEW
Jose A Aznar-Moreno, Timothy P Durrett
The plant kingdom produces a variety of fatty acid structures, many of which possess functional groups useful for industrial applications. The species that produce these unusual fatty acids are often not suitable for large scale commercial production. The ability to create genetically modified plants, together with emerging synthetic biology approaches, offers the potential to develop alternative oil seed crops capable of producing high levels of modified lipids. In some cases, by combining genes from different species, non-natural lipids with a targeted structure can be conceived...
October 2017: Plant Science: An International Journal of Experimental Plant Biology
https://www.readbyqxmd.com/read/28815508/multipart-dna-assembly-using-site-specific-recombinases-from-the-large-serine-integrase-family
#8
Femi J Olorunniji, Christine Merrick, Susan J Rosser, Margaret C M Smith, W Marshall Stark, Sean D Colloms
Assembling multiple DNA fragments into functional plasmids is an important and often rate-limiting step in engineering new functions in living systems. Bacteriophage integrases are enzymes that carry out efficient recombination reactions between short, defined DNA sequences known as att sites. These DNA splicing reactions can be used to assemble large numbers of DNA fragments into a functional circular plasmid in a method termed serine integrase recombinational assembly (SIRA). The resulting DNA assemblies can easily be modified by further recombination reactions catalyzed by the same integrase in the presence of its recombination directionality factor (RDF)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28813599/tetraterpene-synthase-substrate-and-product-specificity-in-the-green-microalga-botryococcus-braunii-race-l
#9
Hem Raj Thapa, Su Tang, James C Sacchettini, Timothy P Devarenne
Recently, the biosynthetic pathway for lycopadiene, a C40 tetraterpenoid hydrocarbon, was deciphered from the L race of Botryococcus braunii, an alga that produces hydrocarbon oils capable of being converted into combustible fuels. The lycopadiene pathway is initiated by the squalene synthase (SS)-like enzyme lycopaoctaene synthase (LOS), which catalyzes the head-to-head condensation of two C20 geranylgeranyl diphosphate (GGPP) molecules to produce C40 lycopaoctaene. LOS shows unusual substrate promiscuity for SS or SS-like enzymes by utilizing C15 farnesyl diphosphate (FPP) and C20 phytyl diphosphate in addition to GGPP as substrates...
August 16, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28811531/the-selective-antifungal-activity-of-drosophila-melanogaster-metchnikowin-reflects-the-species-dependent-inhibition-of-succinate-coenzyme-q-reductase
#10
Mohammad-Reza Bolouri Moghaddam, Thomas Gross, Annette Becker, Andreas Vilcinskas, Mohammad Rahnamaeian
Insect-derived antifungal peptides have a significant economic potential, particularly for the engineering of pathogen-resistant crops. However, the nonspecific antifungal activity of such peptides could result in detrimental effects against beneficial fungi, whose interactions with plants promote growth or increase resistance against biotic and abiotic stress. The antifungal peptide metchnikowin (Mtk) from Drosophila melanogaster acts selectively against pathogenic Ascomycota, including Fusarium graminearum, without affecting Basidiomycota such as the beneficial symbiont Piriformospora indica...
August 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28811118/prediction-and-characterization-of-the-stability-enhancing-effect-of-the-cherry-tag%C3%A2-in-highly-concentrated-protein-solutions-by-complex-rheological-measurements-and-md-simulations
#11
Pascal Baumann, Marie-Therese Schermeyer, Hannah Burghardt, Cathrin Dürr, Jonas Gärtner, Jürgen Hubbuch
Solution stability attributes are one of the key parameters within the production and launching phase of new biopharmaceuticals. Instabilities of active biological compounds can reduce the yield of biopharmaceutical productions, and may induce undesired reactions in patients, such as immunogenic rejections. Protein solution stability thus needs to be engineered and monitored throughout production and storage. In contrast to the gold standard of long-term storage experiments applied in industry, novel experimental and in silico molecular dynamics tools for predicting protein solution stability can be applied within several minutes or hours...
August 12, 2017: International Journal of Pharmaceutics
https://www.readbyqxmd.com/read/28809106/structural-characteristics-and-function-of-a-new-kind-of-thermostable-trehalose-synthase-from-thermobaculum-terrenum
#12
Junqing Wang, Xudong Ren, Ruiming Wang, Jing Su, Feng Wang
Trehalose has important applications in the food industry and pharmaceutical manufacturing. The thermostable enzyme trehalose synthase from Thermobaculum terrenum (TtTS) catalyzes the reversible interconversion of maltose and trehalose. Here, we investigated the structural characteristics of TtTS in complex with the inhibitor TriS. TtTS exhibits the typical three domain glycoside hydrolase family 13 structure. The catalytic cleft consists of Asp202-Glu244-Asp310 and various conserved substrate-binding residues...
August 15, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28809073/biocatalytic-stimuli-responsive-asymmetric-triblock-terpolymer-membranes-for-localized-permeability-gating
#13
Jennifer L Poole, Scott Donahue, David Wilson, Yuk Mun Li, Qi Zhang, Yibei Gu, Rachel Ferebee, Zhao Lu, Rachel Mika Dorin, Lawrence F Hancock, Larry Takiff, Ilhem F Hakem, Michael R Bockstaller, Ulrich Wiesner, Jeremy Walker
The functionalization with phosphotriesterase of poly(isoprene-b-styrene-b-4-vinylpyridine)-based nanoporous membranes fabricated by self-assembly and nonsolvent induced phase separation (SNIPS) is shown to enable dynamically responsive membranes capable of substrate-specific and localized gating response. Integration of the SNIPS process with macroporous nylon support layers yields mechanically robust textile-type films with high moisture vapor transport rates that display rapid and local order-of-magnitude modulation of permeability...
August 15, 2017: Macromolecular Rapid Communications
https://www.readbyqxmd.com/read/28808436/ewald-an-extended-wide-angle-laue-diffractometer-for-the-second-target-station-of-the-spallation-neutron-source
#14
Leighton Coates, Lee Robertson
Visualizing hydrogen atoms in biological materials is one of the biggest remaining challenges in biophysical analysis. While X-ray techniques have unrivaled capacity for high-throughput structure determination, neutron diffraction is uniquely sensitive to hydrogen atom positions in crystals of biological materials and can provide a more complete picture of the atomic and electronic structures of biological macromolecules. This information can be essential in providing predictive understanding and engineering control of key biological processes, for example, in catalysis, ligand binding and light harvesting, and to guide bioengineering of enzymes and drug design...
August 1, 2017: Journal of Applied Crystallography
https://www.readbyqxmd.com/read/28805804/a-genetically-encoded-tool-for-manipulation-of-nadp-nadph-in-living-cells
#15
Valentin Cracan, Denis V Titov, Hongying Shen, Zenon Grabarek, Vamsi K Mootha
The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD(+)/NADH ratio in human cells...
August 7, 2017: Nature Chemical Biology
https://www.readbyqxmd.com/read/28805373/inefficient-ribosomal-skipping-enables-simultaneous-secretion-and-display-of-proteins-in-saccharomyces-cerevisiae
#16
Carlos A Cruz-Teran, Karthik Tiruthani, Adam Mischler, Balaji M Rao
The need for recombinant expression of soluble protein slows the validation of engineered proteins isolated from combinatorial libraries and limits the number of protein variants evaluated. To overcome this bottleneck, we describe a system for simultaneous cell surface display and soluble secretion of proteins in Saccharomyces cerevisiae based on inefficient ribosomal skipping. Ribosomal skipping mediated by "self-cleaving" 2A peptides produces two proteins from a single open reading frame. Incorporation of the F2A peptide sequence-with ∼50% efficiency of ribosomal skipping-between the protein of interest and the yeast cell wall protein Aga2 results in simultaneous expression of both the solubly secreted protein and the protein-Aga2 fusion that is tethered to the yeast cell surface...
August 14, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28803776/mechanism-of-enzyme-repair-by-the-aaa-chaperone-rubisco-activase
#17
Javaid Y Bhat, Goran Miličić, Gabriel Thieulin-Pardo, Andreas Bracher, Andrew Maxwell, Susanne Ciniawsky, Oliver Mueller-Cajar, John R Engen, F Ulrich Hartl, Petra Wendler, Manajit Hayer-Hartl
How AAA+ chaperones conformationally remodel specific target proteins in an ATP-dependent manner is not well understood. Here, we investigated the mechanism of the AAA+ protein Rubisco activase (Rca) in metabolic repair of the photosynthetic enzyme Rubisco, a complex of eight large (RbcL) and eight small (RbcS) subunits containing eight catalytic sites. Rubisco is prone to inhibition by tight-binding sugar phosphates, whose removal is catalyzed by Rca. We engineered a stable Rca hexamer ring and analyzed its functional interaction with Rubisco...
July 20, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28803132/quantum-chemical-study-of-mechanism-and-stereoselectivity-of-secondary-alcohol-dehydrogenase
#18
Sara Moa, Fahmi Himo
Secondary alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) is a Zn- and NADP-dependent enzyme that catalyses the reversible transformation of secondary alcohols into ketones. It is of potential biocatalytic interest as it can be used in the synthesis of chiral alcohols by asymmetric reduction of ketones. In this paper, density functional theory calculations are employed to elucidate the origins of the enantioselectivity of TbSADH using a large model of the active site and considering two different substrates, 2-butanol and 3-hexanol...
July 24, 2017: Journal of Inorganic Biochemistry
https://www.readbyqxmd.com/read/28802264/crystal-structure-and-substrate-specificity-modification-of-acetyl-xylan-esterase-from-aspergillus-luchuensis
#19
Dai Komiya, Akane Hori, Takuya Ishida, Kiyohiko Igarashi, Masahiro Samejima, Takuya Koseki, Shinya Fushinobu
Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis (AlAXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AlAXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that AlAXEA has conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120)...
August 11, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28801651/discovery-of-novel-highly-active-and-stable-aspartate-dehydrogenases
#20
Hao Li, Taicheng Zhu, Liangtian Miao, Dan Zhang, Yongxian Li, Qi Li, Yin Li
Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications. Most if not all AFAAs are produced under aerobic conditions which is energy-intensive. To establish a cost-effective anaerobic process for production of AFAAs, it holds great promise to develop a new pathway enabling the conversion of oxoloacetate into aspartate through direct amination which is catalyzed by aspartate dehydrogenase (AspDH). Compared with the well studied aspartate aminotransferase and aspartate ammonia-lyase, only a few AspDHs are characterized till date, and failure to reproduce the high activity of AspDH from Rastonia eutropha documented in the literature encouraged us to screen and characterize novel AspDHs from different origins...
August 11, 2017: Scientific Reports
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