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enzyme engineering

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https://www.readbyqxmd.com/read/28239413/improving-acetyl-coa-biosynthesis-in-saccharomyces-cerevisiae-via-the-overexpression-of-pantothenate-kinase-and-pdh-bypass
#1
Wenshan Liu, Bo Zhang, Rongrong Jiang
BACKGROUND: Acetyl-CoA is an important precursor in Saccharomyces cerevisiae. Various approaches have been adopted to improve its cytosolic level previously with the emphasis on engineering the "acetyl-" part of acetyl-CoA. To the best of our knowledge, there have been no reports on engineering the "-CoA" part so far. RESULTS: In this study, we had tried to engineer S. cerevisiae from both the "-CoA" part via pantothenate kinase overexpression (PanK from S. cerevisiae, the rate-limiting enzyme for CoA synthesis) and the "acetyl-"part through PDH bypass introduction (ALD6 from S...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28239412/genetic-engineering-of-arabidopsis-to-overproduce-disinapoyl-esters-potential-lignin-modification-molecules
#2
Shinyoung Lee, Huaping Mo, Jeong Im Kim, Clint Chapple
BACKGROUND: Monolignol-like molecules can be integrated into lignin along with conventional monolignol units, and it has been shown that the incorporation of non-canonical subunits can be used to generate hydrolysable lignin by introduction of ester linkages into the polymer and that this type of lignin is more easily removable. Disinapoyl esters (DSEs), which to some degree resemble the monolignol sinapyl alcohol, may be promising lignin modifying units for this purpose. As a first step toward determining whether this goal is achievable, we manipulated metabolic flux in Arabidopsis to increase the amounts of DSEs by overexpressing sinapoylglucose:sinapoylglucose sinapoyltransferase (SST) which produces two main DSEs, 1,2-disinapoylglucose, and another compound we identify in this report as 3,4-disinapoyl-fructopyranose...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28239372/identification-by-genome-mining-of-a-type-i-polyketide-gene-cluster-from-streptomyces-argillaceus-involved-in-the-biosynthesis-of-pyridine-and-piperidine-alkaloids-argimycins-p
#3
Suhui Ye, Brian Molloy, Alfredo F Braña, Daniel Zabala, Carlos Olano, Jesús Cortés, Francisco Morís, José A Salas, Carmen Méndez
Genome mining of the mithramycin producer Streptomyces argillaceus ATCC 12956 revealed 31 gene clusters for the biosynthesis of secondary metabolites, and allowed to predict the encoded products for 11 of these clusters. Cluster 18 (renamed cluster arp) corresponded to a type I polyketide gene cluster related to the previously described coelimycin P1 and streptazone gene clusters. The arp cluster consists of fourteen genes, including genes coding for putative regulatory proteins (a SARP-like transcriptional activator and a TetR-like transcriptional repressor), genes coding for structural proteins (three PKSs, one aminotransferase, two dehydrogenases, two cyclases, one imine reductase, a type II thioesterase, and a flavin reductase), and one gene coding for a hypothetical protein...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28239189/pyrrolysyl-trna-synthetase-an-aminoacyl-trna-synthetase-for-genetic-code-expansion
#4
Ana Crnković, Tateki Suzuki, Dieter Söll, Noah M Reynolds
Genetic code expansion (GCE) has become a central topic of synthetic biology. GCE relies on engineered aminoacyl-tRNA synthetases (aaRSs) and a cognate tRNA species to allow codon reassignment by co-translational insertion of non-canonical amino acids (ncAAs) into proteins. Introduction of such amino acids increases the chemical diversity of recombinant proteins endowing them with novel properties. Such proteins serve in sophisticated biochemical and biophysical studies both in vitro and in vivo, they may become unique biomaterials or therapeutic agents, and they afford metabolic dependence of genetically modified organisms for biocontainment purposes...
June 2016: Croatica Chemica Acta. Arhiv za Kemiju
https://www.readbyqxmd.com/read/28238910/engineering-a-chimeric-acid-stable-%C3%AE-amylase-glucoamylase-amy-glu-for-one-step-starch-saccharification
#5
Deepak Parashar, T Satyanarayana
For saccharifying starch in one step, a chimeric biocatalyst (Amy-Glu) was generated from engineered α-amylase (Ba-Gt-amy) of Bacillus acidicola and glucoamylase (Glu) gene of Aspergillus niger. In order to join two enzymes, a linker peptide of 25 amino acids was used. Chimeric Amy-Glu was expressed in E. coli. Glu is of 75kDa, while Amy-Glu is of 145kDa. Both Amy-Glu and Glu displayed similar pH profile with good activity in the acidic pH range like that of Ba-Gt-amy with optimum at pH 4.0. All three enzymes (Ba-Gt-amy, Amy-Glu and glucoamylase) exhibited activity in the temperature range between 40 and 70°C with optimum at 60°C...
February 23, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28238759/in-vitro-metabolic-engineering-for-the-production-of-%C3%AE-ketoglutarate
#6
Barbara Beer, André Pick, Volker Sieber
α-Ketoglutarate (aKG) represents a central intermediate of cell metabolism. It is used for medical treatments and as a chemical building block. Enzymatic cascade reactions have the potential to sustainably synthesize this natural product. Here we report a systems biocatalysis approach for an in vitro reaction set-up to produce aKG from glucuronate using the oxidative pathway of uronic acids. Because of two dehydrations, a decarboxylation, and reaction conditions favoring oxidation, the pathway is driven thermodynamically towards complete product formation...
February 22, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28238262/a-cell-free-protein-synthesis-approach-to-biosensing-htrb-specific-endocrine-disruptors
#7
Amin S M Salehi, Miriam J Shakalli Tang, Mark Thomas Smith, Jeremy M Hunt, Robert A Law, David W Wood, Bradley Charles Bundy
Here we introduce a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting ligands that interact with NHRs. The RAPID platform can be adapted for field use, allowing rapid evaluation of EDC presence or absence in environmental samples, and could also be applied for drug screening. The biosensor is based on an engineered, allosterically-activated fusion protein, which contains the ligand-binding domain (LBD) from a target NHR (human thyroid receptor in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output...
February 26, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28238040/biocalcite-and-carbonic-acid-activators
#8
Xiaohong Wang, Meik Neufurth, Emad Tolba, Shunfeng Wang, Heinz C Schröder, Werner E G Müller
Based on evolution of biomineralizing systems and energetic considerations, there is now compelling evidence that enzymes play a driving role in the formation of the inorganic skeletons from the simplest animals, the sponges, up to humans. Focusing on skeletons based on calcium minerals, the principle enzymes involved are the carbonic anhydrase (formation of the calcium carbonate-based skeletons of many invertebrates like the calcareous sponges, as well as deposition of the calcium carbonate bioseeds during human bone formation) and the alkaline phosphatase (providing the phosphate for bone calcium phosphate-hydroxyapatite formation)...
2017: Progress in Molecular and Subcellular Biology
https://www.readbyqxmd.com/read/28236756/electrical-activity-of-cellobiose-dehydrogenase-adsorbed-on-thiols-influence-of-charge-and-hydrophobicity
#9
P Lamberg, J Hamit-Eminovski, M D Toscano, O Eicher-Lorka, G Niaura, T Arnebrant, S Shleev, T Ruzgas
The interface between protein and material surface is of great research interest in applications varying from implants, tissue engineering to bioelectronics. Maintaining functionality of bioelements depends greatly on the immobilization process. In the present study direct electron transfer of cellobiose dehydrogenase from Humicola insolens (HiCDH), adsorbed on four different self-assembled monolayers (SAMs) formed by 5-6 chain length carbon thiols varying in terminal group structure was investigated. By using a combination of quartz crystal microbalance with dissipation, ellipsometry and electrochemistry the formation and function of the HiCDH film was studied...
February 16, 2017: Bioelectrochemistry
https://www.readbyqxmd.com/read/28231515/computational-tools-for-enzyme-improvement-why-everyone-can-and-should-use-them
#10
REVIEW
Maximilian Ccjc Ebert, Joelle N Pelletier
This review presents computational methods that experimentalists can readily use to create smart libraries for enzyme engineering and to obtain insights into protein-substrate complexes. Computational tools have the reputation of being hard to use and inaccurate compared to experimental methods in enzyme engineering, yet they are essential to probe datasets of ever-increasing size and complexity. In recent years, bioinformatics groups have made a huge leap forward in providing user-friendly interfaces and accurate algorithms for experimentalists...
February 20, 2017: Current Opinion in Chemical Biology
https://www.readbyqxmd.com/read/28230977/extracellular-self-assembly-of-functional-and-tunable-protein-conjugates-from-bacillus-subtilis
#11
Charlie Gilbert, Mark Howarth, Colin R Harwood, Tom Ellis
The ability to stably and specifically conjugate recombinant proteins to one another is a powerful approach for engineering multifunctional enzymes, protein therapeutics and novel biological materials. While many of these applications have been illustrated through in vitro and in vivo intracellular protein conjugation methods, extracellular self-assembly of protein conjugates offers unique advantages: simplifying purification, reducing toxicity and burden, and enabling tunability. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically-encoded to covalently conjugate in culture media following programmable secretion from Bacillus subtilis...
February 23, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28229928/an-expanded-genetic-code-for-probing-the-role-of-electrostatics-in-enzyme-catalysis-by-vibrational-stark-spectroscopy
#12
REVIEW
Jan-Stefan Völler, Hernan Biava, Peter Hildebrandt, Nediljko Budisa
BACKGROUND: To find experimental validation for electrostatic interactions essential for catalytic reactions represents a challenge due to practical limitations in assessing electric fields within protein structures. SCOPE OF REVIEW: This review examines the applications of non-canonical amino acids (ncAAs) as genetically encoded probes for studying the role of electrostatic interactions in enzyme catalysis. MAJOR CONCLUSIONS: ncAAs constitute sensitive spectroscopic probes to detect local electric fields by exploiting the vibrational Stark effect (VSE) and thus have the potential to map the protein electrostatics...
February 14, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28228535/genome-wide-prediction-of-metabolic-enzymes-pathways-and-gene-clusters-in-plants
#13
Pascal Schlapfer, Peifen Zhang, Chuan Wang, Taehyong Kim, Michael Banf, Lee Chae, Kate Dreher, Arvind K Chavali, Ricardo Nilo-Poyanco, Thomas Bernard, Daniel Kahn, Seung Y Rhee
Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways and gene clusters from a sequenced genome...
February 22, 2017: Plant Physiology
https://www.readbyqxmd.com/read/28226780/modeling-of-coupled-differential-equations-for-cellular-chemical-signaling-pathways-implications-for-assay-protocols-utilized-in-cellular-engineering
#14
George D O'Clock, George D O'Clock, George D O'Clock
Cellular engineering involves modification and control of cell properties, and requires an understanding of fundamentals and mechanisms of action for cellular derived product development. One of the keys to success in cellular engineering involves the quality and validity of results obtained from cell chemical signaling pathway assays. The accuracy of the assay data cannot be verified or assured if the effect of positive feedback, nonlinearities, and interrelationships between cell chemical signaling pathway elements are not understood, modeled, and simulated...
August 2016: Conference Proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society
https://www.readbyqxmd.com/read/28224920/genetic-engineering-of-the-biosynthesis-of-glycinebetaine-leads-to-alleviate-salt-induced-potassium-efflux-and-enhances-salt-tolerance-in-tomato-plants
#15
Dandan Wei, Wen Zhang, Cuicui Wang, Qingwei Meng, Gang Li, Tony H H Chen, Xinghong Yang
Tomato (Solanum lycopersicum cv. 'Moneymaker') was transformed with the choline oxidase gene codA from Arthrobacter globiformis, which was modified to allow for targeting to both chloroplasts and the cytosol. Glycine betaine (GB) was accumulated in transformed plants, while no detectable GB was found in wild-type (WT) plants. Compared to WT plants, transgenic lines showed significantly higher photosynthetic rates (Pn) and antioxidant enzyme activities and lower reactive oxygen species (ROS) accumulation in the leaves when exposed to salt stress...
April 2017: Plant Science: An International Journal of Experimental Plant Biology
https://www.readbyqxmd.com/read/28224230/newly-isolated-bacillus-sp-g51-from-patagonian-wool-produces-an-enzyme-combination-suitable-for-felt-resist-treatments-of-organic-wool
#16
Martín S Iglesias, Cynthia Sequeiros, Sebastián García, Nelda L Olivera
Bacteria from Patagonian Merino wool were isolated to assess their wool-keratinolytic activity and potential for felt-resist treatments. Strains from Bacillus, Exiguobacterium, Deinococcus, and Micrococcus produced wool-degrading enzymes. Bacillus sp. G51 showed the highest wool-keratinolytic activity. LC-MS/MS analysis revealed that G51 secreted two serine proteases belonging to the peptidase family S8 (MEROPS) and a metalloprotease associated with Bacillolysin, along with other enzymes (γ-glutamyltranspeptidase and dihydrolipoyl dehydrogenases) that could be involved in reduction of keratin disulfide bonds...
February 21, 2017: Bioprocess and Biosystems Engineering
https://www.readbyqxmd.com/read/28223726/dendritic-cells-engineered-to-secrete-anti-dcr3-antibody-augment-cytotoxic-t-lymphocyte-response-against-pancreatic-cancer-in-vitro
#17
Jiang Chen, Xiao-Zhong Guo, Hong-Yu Li, Jia-Jun Zhao, Wen-Da Xu
AIM: To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS: DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology...
February 7, 2017: World Journal of Gastroenterology: WJG
https://www.readbyqxmd.com/read/28222675/identification-and-characterization-of-an-efficient-acyl-coa-diacylglycerol-acyltransferase-1-dgat1-gene-from-the-microalga-chlorella-ellipsoidea
#18
Xuejie Guo, Chengming Fan, Yuhong Chen, Jingqiao Wang, Weibo Yin, Richard R C Wang, Zanmin Hu
BACKGROUND: Oil in the form of triacylglycerols (TAGs) is quantitatively the most important storage form of energy for eukaryotic cells. Diacylglycerol acyltransferase (DGAT) is considered the rate-limiting enzyme for TAG accumulation. Chlorella, a unicellular eukaryotic green alga, has attracted much attention as a potential feedstock for renewable energy production. However, the function of DGAT1 in Chlorella has not been reported. RESULTS: A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2...
February 21, 2017: BMC Plant Biology
https://www.readbyqxmd.com/read/28222177/phospholipid-binding-to-the-fak-catalytic-domain-impacts-function
#19
Jessica E Hall, Michael D Schaller
Focal adhesion kinase is an essential nonreceptor tyrosine kinase that plays an important role in development, in homeostasis and in the progression of human disease. Multiple stimuli activate FAK, which requires a change in structure from an autoinhibited to activated conformation. In the autoinhibited conformation the FERM domain associates with the catalytic domain of FAK and PI(4,5)P2 binding to the FERM domain plays a role in the release of autoinhibition, activating the enzyme. An in silico model of FAK/PI(4,5)P2 interaction suggests that residues on the catalytic domain interact with PI(4,5)P2, in addition to the known FERM domain PI(4,5)P2 binding site...
2017: PloS One
https://www.readbyqxmd.com/read/28220960/a-comparative-investigation-of-bombyx-mori-silk-fibroin-hydrogels-generated-by-chemical-and-enzymatic-crosslinking
#20
Traian V Chirila, Shuko Suzuki, Chloé Papolla
Fibroin, the major proteinaceous component of the silk fiber produced by larvae of the domesticated silk moth (Bombyx mori), has been widely investigated as a biomaterial for potential applications in tissue engineering and regenerative medicine. Following sol-gel transition, silk fibroin solutions can generate hydrogels that present certain advantages when employed as biomaterials, especially if they are crosslinked. The subject of this study was the self-crosslinking of silk fibroin through a process induced by the enzyme horseradish peroxidase (HRP) in the presence of hydrogen peroxide, a method only recently proposed and scarcely reported...
February 21, 2017: Biotechnology and Applied Biochemistry
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