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Heterologous expression lab

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https://www.readbyqxmd.com/read/28817927/identification-of-biosynthetic-gene-clusters-from-metagenomic-libraries-using-pptase-complementation-in-a-streptomyces-host
#1
J Kipchirchir Bitok, Christophe Lemetre, Melinda A Ternei, Sean F Brady
The majority of environmental bacteria are not readily cultured in the lab, leaving the natural products they make inaccessible using culture-dependent discovery methods. Cloning and heterologous expression of DNA extracted from environmental samples (environmental DNA, eDNA) provides a means of circumventing this discovery bottleneck. To facilitate the identification of clones containing biosynthetic gene clusters, we developed a model heterologous expression reporter strain Streptomyces albus::bpsA ΔPPTase...
September 1, 2017: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/28751387/complete-genome-sequence-of-kluyveromyces-lactis-strain-gg799-a-common-yeast-host-for-heterologous-protein-expression
#2
Léa Chuzel, Mehul B Ganatra, Kelly M Schermerhorn, Andrew F Gardner, Brian P Anton, Christopher H Taron
We report the genome sequence of the dairy yeast Kluyveromyces lactis strain GG799 obtained using the Pacific Biosciences RS II platform. K. lactis strain GG799 is a common host for the expression of proteins at both laboratory and industrial scales.
July 27, 2017: Genome Announcements
https://www.readbyqxmd.com/read/28722114/uv-b-irradiation-differentially-regulates-terpene-synthases-and-terpene-content-of-peach
#3
Hongru Liu, Xiangmei Cao, Xiaohong Liu, Rui Xin, Jiaojiao Wang, Jie Gao, Boping Wu, Liuxiao Gao, Changjie Xu, Bo Zhang, Donald Grierson, Kunsong Chen
Plants generate protective molecules in response to UV light. In laboratory experiments, 48 h UV-B irradiation of peach fruit and leaves reduced the flavor-related monoterpene linalool by 60 %. No isoprene was detected but other terpenoids increased significantly, including a 3-fold accumulation of the sesquiterpene (E,E)-α-farnesene, which was also increased by jasmonic acid treatment. RNA-sequencing revealed altered transcript levels for two terpene synthases: PpTPS1, a TPS-g subfamily member, decreased by 86 % and PpTPS2, a TPS-b subfamily member, increased 80-fold...
July 19, 2017: Plant, Cell & Environment
https://www.readbyqxmd.com/read/28710782/identification-and-functional-analysis-of-two-p450-enzymes-of-gossypium-hirsutum-involved-in-dmnt-and-tmtt-biosynthesis
#4
Danfeng Liu, Xinzheng Huang, Weixia Jing, Xingkui An, Qiang Zhang, Hong Zhang, Jingjiang Zhou, Yongjun Zhang, Yuyuan Guo
The homoterpenes (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) are major herbivore-induced plant volatiles (HIPV) that can attract predatory or parasitic arthropods to protect injured plants from herbivore attack. In the present study, DMNT and TMTT were confirmed to be emitted from cotton (Gossypium hirsutum) plants infested with chewing caterpillars or sucking bugs. Two CYP genes (GhCYP82L1 and GhCYP82L2) involved in homoterpene biosynthesis in G. hirsutum were newly identified and characterized...
July 15, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28697978/functional-characterization-of-bdb1-a-well-conserved-carboxylesterase-among-tephritid-fruit-flies-associated-with-malathion-resistance-in-bactrocera-dorsalis-hendel
#5
Luo-Luo Wang, Xue-Ping Lu, Guy Smagghe, Li-Wei Meng, Jin-Jun Wang
There are many evidences that insect carboxylesterase possess important physiological roles in xenobiotic metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the ongoing resistance development in the oriental fruit fly, Bactrocera dorsalis (Hendel), the molecular basis of carboxylesterase and its ability to confer OP resistance remain largely obscure. This study was initiated to provide a better understanding of carboxylesterase-mediated resistance mechanism in a tephritid pest fly...
July 8, 2017: Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP
https://www.readbyqxmd.com/read/28672185/utilization-of-biodiesel-by-product-as-substrate-for-high-production-of-%C3%AE-farnesene-via-relatively-balanced-mevalonate-pathway-in-escherichia-coli
#6
Shengping You, Qingdian Yin, Jianye Zhang, Chengyu Zhang, Wei Qi, Lan Gao, Zhiping Tao, Rongxin Su, Zhimin He
Farnesene has been identified as suitable jet fuel substitutes and metabolic engineering for microbial production of farnesene is an alternative and attractive route. In this study, due to accumulation of toxic intermediate isopentenyl pyrophosphate (IPP), an engineered Escherichia coli strain harboring heterologous mevalonate pathway produced only 4.11mg/L β-farnesene. Through higher-level expression of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase to minimize the accumulated IPP, another engineered strain with relatively balanced mevalonate pathway was constructed and had the highest production of β-farnesene to date (8...
June 13, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28650175/bioactive-peptide-natural-products-as-lead-structures-for-medicinal-use
#7
Tam Dang, Roderich D Süssmuth
The need for new drugs for the treatment of various diseases is enormous. From the previous century until the present, numerous peptide and peptide-derived natural products have been isolated from bacteria and fungi. Hence, microorganisms play a pivotal role as sources for novel drugs with an emphasis on anti-infective agents. Various disciplines from biology, chemistry, and medicine are involved in early stages of the search for peptide natural products including taxonomy, microbiology, bioanalytics, bioinformatics, and medicinal chemistry...
June 26, 2017: Accounts of Chemical Research
https://www.readbyqxmd.com/read/28539920/technological-microbiology-development-and-applications
#8
REVIEW
Luciana C Vitorino, Layara A Bessa
Over thousands of years, modernization could be predicted for the use of microorganisms in the production of foods and beverages. However, the current accelerated pace of new food production is due to the rapid incorporation of biotechnological techniques that allow the rapid identification of new molecules and microorganisms or even the genetic improvement of known species. At no other time in history have microorganisms been so present in areas such as agriculture and medicine, except as recognized villains...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28331545/metabolic-engineering-of-saccharomyces-cerevisiae-to-produce-a-reduced-viscosity-oil-from-lignocellulose
#9
Tam N T Tran, Rebecca J Breuer, Ragothaman Avanasi Narasimhan, Lucas S Parreiras, Yaoping Zhang, Trey K Sato, Timothy P Durrett
BACKGROUND: Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers...
2017: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/28321412/recombinant-lactococcus-lactis-expressing-haemagglutinin-from-a-polish-avian-h5n1-isolate-and-its-immunological-effect-in-preliminary-animal-trials
#10
Agnieszka K Szczepankowska, Katarzyna Szatraj, Przemysław Sałański, Agnieszka Rózga, Roman K Górecki, Jacek K Bardowski
Lactic acid bacteria (LAB) are Gram-positive, nonpathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in various LAB species, including Lactococcus lactis. Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of a Polish avian H5H1 influenza isolate and examining its effect on animals...
2017: BioMed Research International
https://www.readbyqxmd.com/read/28315210/recombinant-allergens-production-in-yeast
#11
Maria Neophytou, Marcos Alcocer
The methylotropic yeast Pichia pastoris has been extensively used in large-scale production of properly folded recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins such as post-translational modifications, protein processing, and a reasonably sophisticated quality control of protein folding when compared against Escherichia coli. In this chapter, we describe the modified lab procedure for cloning and expression in Pichia pastoris of common food allergens sequences from the raw fruit to the fully folded biotinylated protein product...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28288336/breaking-the-silence-new-strategies-for-discovering-novel-natural-products
#12
REVIEW
Hengqian Ren, Bin Wang, Huimin Zhao
Natural products have been a prolific source of antibacterial and anticancer drugs for decades. One of the major challenges in natural product discovery is that the vast majority of natural product biosynthetic gene clusters (BGCs) have not been characterized, partially due to the fact that they are either transcriptionally silent or expressed at very low levels under standard laboratory conditions. Here we describe the strategies developed in recent years (mostly between 2014-2016) for activating silent BGCs...
March 10, 2017: Current Opinion in Biotechnology
https://www.readbyqxmd.com/read/28265993/real-time-genetic-manipulations-of-the-cytokinin-pathway-a-tool-for-laboratory-and-field-studies
#13
Martin Schäfer, Stefan Meldau
Although many established tools for cytokinin (CK) pathway manipulations are well suitable for the analysis of molecular interactions, their use on a whole plant scale is often limited by the induction of severe developmental defects. To circumvent this problem, different methods were developed that allow for a more precise manipulation of the CK pathway. Here we present one of these systems, the pOp6/LhGR system for chemically inducible gene expression. This system allows regulation on a spatial, temporal, and quantitative scale and therefore provides a superior tool for analyzing the role of CKs in the interactions of plants with their environment...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28210796/optogenetic-switches-for-light-controlled-gene-expression-in-yeast
#14
REVIEW
Francisco Salinas, Vicente Rojas, Verónica Delgado, Eduardo Agosin, Luis F Larrondo
Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms...
April 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28117773/xenopus-oocytes-optimized-methods-for-microinjection-removal-of-follicular-cell-layers-and-fast-solution-changes-in-electrophysiological-experiments
#15
Maria C Maldifassi, Nisa Wongsamitkul, Roland Baur, Erwin Sigel
The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.(1) and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels(4). This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels. The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments...
December 31, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28075554/an-engineered-allele-of-afsq1-facilitates-the-discovery-and-investigation-of-cryptic-natural-products
#16
Martin Daniel-Ivad, Nabeela Hameed, Stephanie Tan, Rachna Dhanjal, Daniel Socko, Patricia Pak, Tomas Gverzdys, Marie A Elliot, Justin R Nodwell
New approaches to antimicrobial discovery are needed to address the growing threat of antibiotic resistance. The Streptomyces genus, a proven source of antibiotics, is recognized as having a large reservoir of untapped secondary metabolic genes, many of which are likely to produce uncharacterized compounds. However, most of these compounds are currently inaccessible, as they are not expressed under standard laboratory conditions. Here, we present a novel methodology for activating these "cryptic" metabolites by heterologously expressing a constitutively active pleiotropic regulator...
March 17, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/27981234/coordinate-regulation-of-antimycin-and-candicidin-biosynthesis
#17
Thomas C McLean, Paul A Hoskisson, Ryan F Seipke
Streptomyces species produce an incredible array of high-value specialty chemicals and medicinal therapeutics. A single species typically harbors ~30 biosynthetic pathways, but only a few them are expressed in the laboratory; thus, poor understanding of how natural-product biosynthesis is regulated is a major bottleneck in drug discovery. Antimycins are a large family of anticancer compounds widely produced by Streptomyces species, and their regulation is atypical compared to that of most other natural products...
November 2016: MSphere
https://www.readbyqxmd.com/read/27906033/the-role-of-the-backbone-torsion-in-protein-folding
#18
EDITORIAL
Irina Sorokina, Arcady Mushegian
BACKGROUND: The set of forces and sequence of events that govern the transition from an unfolded polypeptide chain to a functional protein with correct spatial structure remain incompletely known, despite the importance of the problem and decades of theory development, computer simulations, and laboratory experiments. Information about the correctly folded state of most proteins is likely to be present in their sequences, and yet many proteins fail to attain native structure after overexpression in a non-native environment or upon experimental denaturation and refolding...
December 1, 2016: Biology Direct
https://www.readbyqxmd.com/read/27888410/genome-sequence-prevalence-and-quantification-of-the-first-iflavirus-identified-in-a-phytoplasma-insect-vector
#19
Simona Abbà, Luciana Galetto, Marta Vallino, Marika Rossi, Massimo Turina, Anne Sicard, Cristina Marzachì
The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CY) and an efficient vector of flavescence dorée phytoplasma (FD) under laboratory conditions. During a transcriptome sequencing (RNA-seq) project aimed at investigating the interactions between the insect and the two phytoplasmas, a 10,616-nucleotide-long contig with high sequence similarity to known picorna-like viruses was identified among the assembled insect transcripts. The discovery came totally unexpected, because insects from the laboratory colony did not show any evident symptom that could be related to the presence of a virus...
March 2017: Archives of Virology
https://www.readbyqxmd.com/read/27830495/cloning-a-novel-endo-1-4-%C3%AE-d-glucanase-gene-from-trichoderma-virens-and-heterologous-expression-in-e-coli
#20
Rong Zeng, Qiao Hu, Xiao-Yan Yin, Hao Huang, Jia-Bao Yan, Zhi-Wei Gong, Zhong-Hua Yang
Endo-1,4-β-D-glucanase (EG), as a key constituent of cellulase taking the responsibility of cutting β-1,4 glycosidic bonds, plays the essential role in the process of degrading cellulose by cellulase. Cloning and expressing the EG gene is important to the cellulase research and application. In this work, a novel EG gene was cloned from Trichoderma virens ZY-01, which was a cellulase secreting microbe isolated by our laboratory. The DNA sequence showed that the length of the cloned EG is 1069 bp, which had 95...
December 2016: AMB Express
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