Slowly co-fluctuating gene expression patterns are heritable and associated with stress resistance and improved productivity in CHO cell line development

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(How) is formulaic language universal? Insights from Korean, German and English

The existence of common expressions, also referred to as formulaic language or phraseological units, has been evidenced in a very large number of languages. However, the extent to which languages feature such formulaic material, how formulaicity may be understood across typologically different languages and whether indeed there is a concept of formulaic language that applies across languages, are questions that have been less commonly discussed. Using a novel data set consisting of topically matched corpora in three typologically different languages (Korean, German and English), this study proposes an empirically founded universal concept for formulaic language and discusses what the shape of this concept suggests for the theoretical understanding of formulaic language going forward. In particular, it is argued that the nexus of the concept of formulaic language cannot be fixed at any particular structural level (such as the phrase or the level of polylexicality) and incorporates elements specified at varying levels of abstraction (or schematicity). This means that a cross-linguistic concept of formulaic language fits in well with a constructionist view of linguistic structure

Engineering heterologous enzyme secretion in Yarrowia lipolytica

Abstract Background Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species, Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion in Y. lipolytica include codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme. Results By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and $$\alpha$$ α -amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion. Conclusions Overall, our combined strategies not only proved effective in improving the protein production in Yarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast

Engineering Yarrowia lipolytica for the biosynthesis of geraniol

Geraniol is a monoterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Microbial production has largely used model organisms lacking favorable properties for monoterpene production. In this work, we produced geraniol in metabolically engineered Yarrowia lipolytica. First, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were tested based on previous reports of activity. Both wild type and truncated mutants of GES (without signal peptide targeting chloroplast) were examined by co-expressing with MVA pathway enzymes tHMG1 and IDI1. Truncated CrGES (tCrGES) produced the most geraniol and thus was used for further experimentation. The initial strain was obtained by overexpression of the truncated HMG1, IDI and tCrGES. The acetyl-CoA precursor pool was enhanced by overexpressing mevalonate pathway genes such as ERG10, HMGS or MVK, PMK. The final strain overexpressing 3 copies of tCrGES and single copies of ERG10, HMGS, tHMG1, IDI produced approximately 1 g/L in shake-flask fermentation. This is the first demonstration of geraniol production in Yarrowia lipolytica and the highest de novo titer reported to date in yeast

Polyplex Formation Influences Release Mechanism of Mono- and Di-Valent Ions from Phosphorylcholine Group Bearing Hydrogels

The release of monovalent potassium and divalent calcium ions from zwitterionic phosphorylcholine containing poly(2-hydroxyethyl methacrylate) (pHEMA)-based hydrogels was studied and the effects of polymer swelling, ion valence and temperature were investigated. For comparison, ions were loaded during hydrogel formulation or loaded by partitioning following construct synthesis. Using the Koshmeyer-Peppas release model, the apparent diffusion coefficient, Dapp, and diffusional exponents, n, were Dapp (pre-K+) = 2.03 × 10−5, n = 0.4 and Dapp (post-K+) = 1.86 × 10−5, n = 0.33 respectively, indicative of Fickian transport. The Dapp (pre-Ca2+) = 3.90 × 10−6, n = 0.60 and Dapp (post-Ca2+) = 2.85 × 10−6, n = 0.85, respectively, indicative of case II and anomalous transport. Results indicate that divalent cations form cation-polyelectrolyte anion polymer complexes while monovalent ions do not. Temperature dependence of potassium ion release was shown to follow an Arrhenius-type relation with negative apparent activation energy of −19 ± 15 while calcium ion release was temperature independent over the physiologically relevant range (25–45 °C) studied. The negative apparent activation energy may be due to temperature dependent polymer swelling. No effect of polymer swelling on the diffusional exponent or rate constant was found suggesting polymer relaxation occurs independent of polymer swelling

4C29 : Crystal Structure of High-Affinity von Willebrand Factor A1 domain with Disulfide Mutation

Experimental Technique/Method:X-RAY DIFFRACTION Resolution:2.2 Classification:BLOOD CLOTTING Release Date:2014-01-08 Deposition Date:2013-08-16 Revision Date:2014-01-15#2014-01-29#2014-03-12 Molecular Weight:50017.11 Macromolecule Type:Protein Residue Count:430 Atom Site Count:3269 DOI:10.2210/pdb4c29/pdb Abstract: Activation by elongational flow of von Willebrand factor (VWF) is critical for primary hemostasis. Mutations causing type 2B von Willebrand disease (VWD), platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and platelet glycoprotein Ibα (GPIbα) for one another; however, the structural basis for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that structural changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα β-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state

4C2A : Crystal Structure of High-Affinity von Willebrand Factor A1 domain with R1306Q and I1309V Mutations in Complex with High Affinity GPIb alpha

Experimental Technique/Method:X-RAY DIFFRACTION Resolution:2.08 Classification:BLOOD CLOTTING Release Date:2014-01-08 Deposition Date:2013-08-16 Revision Date:2014-01-15#2014-03-12#2015-09-30 Molecular Weight:57503.9 Macromolecule Type:Protein Residue Count:506 Atom Site Count:3738 DOI:10.2210/pdb4c2a/pdb Abstract: Activation by elongational flow of von Willebrand factor (VWF) is critical for primary hemostasis. Mutations causing type 2B von Willebrand disease (VWD), platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and platelet glycoprotein Ibα (GPIbα) for one another; however, the structural basis for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that structural changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα β-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state

4C2B : Crystal Structure of High-Affinity von Willebrand Factor A1 domain with Disulfide Mutation in Complex with High Affinity GPIb alpha

Experimental Technique/Method:X-RAY DIFFRACTION Resolution:2.8 Classification:BLOOD CLOTTING Release Date:2014-01-08 Deposition Date:2013-08-16 Revision Date:2014-01-15#2014-01-29#2014-03-12 Molecular Weight:230057.11 Macromolecule Type:Protein Residue Count:2024 Atom Site Count:14703 DOI:10.2210/pdb4c2b/pdb Abstract: Activation by elongational flow of von Willebrand factor (VWF) is critical for primary hemostasis. Mutations causing type 2B von Willebrand disease (VWD), platelet-type VWD (PT-VWD), and tensile force each increase affinity of the VWF A1 domain and platelet glycoprotein Ibα (GPIbα) for one another; however, the structural basis for these observations remains elusive. Directed evolution was used to discover a further gain-of-function mutation in A1 that shifts the long range disulfide bond by one residue. We solved multiple crystal structures of this mutant A1 and A1 containing two VWD mutations complexed with GPIbα containing two PT-VWD mutations. We observed a gained interaction between A1 and the central leucine-rich repeats (LRRs) of GPIbα, previously shown to be important at high shear stress, and verified its importance mutationally. These findings suggest that structural changes, including central GPIbα LRR-A1 contact, contribute to VWF affinity regulation. Among the mutant complexes, variation in contacts and poor complementarity between the GPIbα β-finger and the region of A1 harboring VWD mutations lead us to hypothesize that the structures are on a pathway to, but have not yet reached, a force-induced super high affinity state

Additional file 1 of Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Additional file 1: Table S2. Yeast plasmids, strains, and primer sequences used to clone and validate the eccDNA ARS sequence

Additional file 5 of Autonomous replication sequences from the Amaranthus palmeri eccDNA replicon enable replication in yeast

Additional file 5: Figure S3. Cloning of the eccDNA autonomous replication sequence in a yeast system. A. Water control (no colonies) B. pRS305 non-replicating plasmid (LEU marker replication − no colonies). C. pRS315 replicating plasmid (LEU marker, CEN6/ARS -lawn of colonies). D. pRS305 + CS-ARS1 + CEN6 (pRS305 + CEN6 + ARS1 − few colonies). E. pRS315 + CS-ARS1 + CEN6 (pRS315ΔARS + ARS1 − few colonies)