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https://www.readbyqxmd.com/read/28222737/dynamic-genome-scale-metabolic-modeling-of-the-yeast-pichia-pastoris
#1
Francisco Saitua, Paulina Torres, José Ricardo Pérez-Correa, Eduardo Agosin
BACKGROUND: Pichia pastoris shows physiological advantages in producing recombinant proteins, compared to other commonly used cell factories. This yeast is mostly grown in dynamic cultivation systems, where the cell's environment is continuously changing and many variables influence process productivity. In this context, a model capable of explaining and predicting cell behavior for the rational design of bioprocesses is highly desirable. Currently, there are five genome-scale metabolic reconstructions of P...
February 21, 2017: BMC Systems Biology
https://www.readbyqxmd.com/read/28218973/expression-of-rabies-virus-glycoprotein-in-the-methylotrophic-yeast-pichia-pastoris
#2
Safa Ben Azoun, Meriem Ben Zakour, Soufien Sghaier, Héla Kallel
Rabies is a fatal disease that can be prevented by vaccination. Different approaches were investigated to develop novel human rabies vaccines with improved features compared to the current available vaccines, among them is the use of heterologous gene expression technology. Here, we describe the expression of the surface rabies virus glycoprotein (RABV-G), which is the major antigen responsible for the induction of protective immunity, in Pichia pastoris. Six transformants were selected according to their gene copy number as determined by real time qPCR...
January 2017: Biotechnology and Applied Biochemistry
https://www.readbyqxmd.com/read/28209146/heterologous-expression-of-aspergillus-aculeatus-endo-polygalacturonase-in-pichia-pastoris-by-high-cell-density-fermentation-and-its-application-in-textile-scouring
#3
Dede Abdulrachman, Paweena Thongkred, Kanokarn Kocharin, Monthon Nakpathom, Buppha Somboon, Nootsara Narumol, Verawat Champreda, Lily Eurwilaichitr, Antonius Suwanto, Thidarat Nimchua, Duriya Chantasingh
BACKGROUND: Removal of non-cellulosic impurities from cotton fabric, known as scouring, by conventional alkaline treatment causes environmental problems and reduces physical strength of fabrics. In this study, an endo-polygalacturonase (EndoPG) from Aspergillus aculeatus produced in Pichia pastoris was evaluated for its efficiency as a bioscouring agent while most current bioscouring process has been performed using crude pectinase preparation. RESULTS: The recombinant EndoPG exhibited a specific activity of 1892...
February 16, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/28201611/secretion-of-functional-formate-dehydrogenase-in-pichia-pastoris
#4
Michelle Takacs, Olga V Makhlynets, Patricia L Tolbert, Ivan V Korendovych
No abstract text is available yet for this article.
February 15, 2017: Protein Engineering, Design & Selection: PEDS
https://www.readbyqxmd.com/read/28195395/heterologous-expression-of-helicoverpa-armigera-cytochrome-p450-cyp6b7-in-pichia-pastoris-and-interactions-of-cyp6b7-with-insecticides
#5
Chunqing Zhao, Genmiao Song, Hongxia Duan, Tao Tang, Chen Wang, Lihong Qiu
BACKGROUND: Previous studies indicated that constitutive overexpression of cytochrome P450 CYP6B7 was involved in fenvalerate resistance in Helicoverpa armigera. In this study, the CYP6B7 gene from H. armigera (namely HaCYP6B7), was heterologously expressed in Pichia pastoris GS115. A vector pPICZA-HaCYP6B7 was constructed and transformed into P. pastoris GS115, the transformant of pPICZA-HaCYP6B7-GS115 was then cultured and induced by 1% (v/v) methanol and the heterologous expression of HaCYP6B7 protein in P...
February 13, 2017: Pest Management Science
https://www.readbyqxmd.com/read/28193329/characterization-and-crystal-structure-of-a-thermostable-glycoside-hydrolase-family-45-1-4-%C3%AE-endoglucanase-from-thielavia-terrestris
#6
Jian Gao, Jian-Wen Huang, Qian Li, Weidong Liu, Tzu-Ping Ko, Yingying Zheng, Xiansha Xiao, Chih-Jung Kuo, Chun-Chi Chen, Rey-Ting Guo
1,4-β-Endoglucanase is one of the most important biocatalysts in modern industries. Here, a glycoside hydrolase (GH) family 45 endoglucanase from thermophilic fungus Theilavia terrestris (TtCel45A) was expressed in Pichia pastoris. The recombinant protein shows optimal activity at 60°C, pH 4-5. The enzyme exhibits extraordinary thermostability that more than 80% activity was detected after heating at 80°C for 2.5h. The high resolution crystal structures of apo-form enzyme and that in complex with cellobiose and cellotetraose were solved to 1...
April 2017: Enzyme and Microbial Technology
https://www.readbyqxmd.com/read/28193216/a-high-throughput-expression-screening-platform-to-optimize-the-production-of-antimicrobial-peptides
#7
Christine Schreiber, Hagen Müller, Oliver Birrenbach, Moritz Klein, Doreen Heerd, Tobias Weidner, Denise Salzig, Peter Czermak
BACKGROUND: Antimicrobial peptides (AMPs) are promising candidates for the development of novel antibiotics, but it is difficult to produce sufficient quantities for preclinical and clinical studies due to their toxicity towards microbial expression hosts. To avoid laborious trial-and-error testing for the identification of suitable expression constructs, we have developed a small-scale expression screening platform based on a combinatorial plasmid library. RESULTS: The combinatorial library is based on the Golden Gate cloning system...
February 13, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28187141/role-of-n-linked-glycosylation-in-the-enzymatic-properties-of-a-thermophilic-gh-10-xylanase-from-aspergillus-fumigatus-expressed-in-pichia-pastoris
#8
Xiaoyu Chang, Bo Xu, Yingguo Bai, Huiying Luo, Rui Ma, Pengjun Shi, Bin Yao
N-Glycosylation is a posttranslational modification commonly occurred in fungi and plays roles in a variety of enzyme functions. In this study, a xylanase (Af-XYNA) of glycoside hydrolase (GH) family 10 from Aspergillus fumigatus harboring three potential N-glycosylation sites (N87, N124 and N335) was heterologously produced in Pichia pastoris. The N-glycosylated Af-XYNA (WT) exhibited favorable temperature and pH optima (75°C and pH 5.0) and good thermostability (maintaining stable at 60°C). To reveal the role of N-glycosylation on Af-XYNA, the enzyme was deglycosylated by endo-β-N-acetylglucosaminidase H (DE) or modified by site-directed mutagenesis at N124 (N124T)...
2017: PloS One
https://www.readbyqxmd.com/read/28177316/crystallization-of-a-fungal-lytic-polysaccharide-monooxygenase-expressed-from-glycoengineered-pichia-pastoris-for-x-ray-and-neutron-diffraction
#9
William B O'Dell, Paul D Swartz, Kevin L Weiss, Flora Meilleur
Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bonds via an oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 from Neurospora crassa (NcPMO-2) was heterologously expressed in Pichia pastoris to facilitate crystallographic studies of the fungal LPMO mechanism...
February 1, 2017: Acta Crystallographica. Section F, Structural Biology Communications
https://www.readbyqxmd.com/read/28173691/characterization-of-the-wild-type-and-truncated-form-of-a-neutral-gh10-xylanase-from-coprinus-cinereus-roles-of-c-terminal-basic-amino-acid-rich-extension-in-its-sds-resistance-thermostability-and-activity
#10
Hang Hu, Kaixiang Chen, Lulu Li, Liangkun Long, Shaojun Ding
A neutral xylanase (Ccxyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at C-terminus. In this study, the wild-type (Ccxyn) and C-terminus truncated xylanase (CcXynΔ5C) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism (CD) analysis indicated that both enzymes in general had a similar structure, but CcXynΔ5C contained less α-helices (42...
February 7, 2017: Journal of Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28168874/deactivating-influence-of-3-o-glycosyl-substituent-on-anomeric-reactivity-of-thiomannoside-observed-in-oligomannoside-synthesis
#11
Jun Zhou, Siying Lv, Dan Zhang, Fei Xia, Wenhao Hu
It has been long recognized that, in chemical glycosylation, anomeric reactivity of glycosyl donor can be influenced greatly by protecting groups. As opposed to the effects of protecting groups, we report herein a finding on how O-glycosyl substituent can affect the reactivity of oligosaccharyl donor, which in turn should have impact on convergent assembly of oligosaccharide. During our synthetic efforts towards Pichia holstii oligomannoside, a type of α-1,3-linked dimannosyl thioglycosides displayed unexpected low reactivity towards the activation of NIS/TMSOTf, and this prompted us to perform a series of comparative reactivity studies, which attributed the donor deactivation to the presence of 3-O-glycosyl substituent, by comparison with O-acetyl group and O-glycosidic linkages at C-4/C-6 positions...
February 7, 2017: Journal of Organic Chemistry
https://www.readbyqxmd.com/read/28161122/acute-and-repeated-dose-28-days-toxicity-studies-in-rats-and-dogs-of-recombinant-batroxobin-a-snake-venom-thrombin-like-enzyme-expressed-from-pichia-pastoris
#12
Ok Hwan Kim, Kil-Sang Cho, Young Seomun, Jong-Tak Kim, Kwang-Hoe Chung
Recombinant batroxobin is a thrombin-like enzyme of Bothrops atrox moojeni venom. To evaluate its toxicological effect, it was highly expressed in Pichia pastorisand successfully purified to homogeneity from culture broth supernatant following Good Manufacturing Practice (GMP). The maximum tolerated dose of the recombinant batroxobin was examined in Sprague-Dawley (SD) rat and Beagle dogs following Good Laboratory Practice (GLP) regulations. The approximate lethal dose of recombinant batroxobin was 10 National Institute of Health (NIH) u/kg in male and female rats...
February 1, 2017: Toxicon: Official Journal of the International Society on Toxinology
https://www.readbyqxmd.com/read/28160205/rapid-screening-of-cellular-stress-responses-in-recombinant-pichia-pastoris-strains-using-metabolite-profiling
#13
Gregory D Tredwell, Rochelle Aw, Bryn Edwards-Jones, David J Leak, Jacob G Bundy
Heterologous protein production in the yeast Pichia pastoris can be limited by biological responses to high expression levels; the unfolded protein response (UPR) is a key determinant of the success of protein production in this organism. Here, we used untargeted NMR metabolic profiling (metabolomics) of a number of different recombinant strains, carried out in a miniaturized format suitable for screening-level experiments. We identified a number of metabolites (from both cell extracts and supernatants) which correlated well with UPR-relevant gene transcripts, and so could be potential biomarkers for future high-throughput screening of large numbers of P...
February 3, 2017: Journal of Industrial Microbiology & Biotechnology
https://www.readbyqxmd.com/read/28159727/a-p-coumaroyl-esterase-from-rhizoctonia-solani-with-a-pronounced-chlorogenic-acid-esterase-activity
#14
Annabel Nieter, Sebastian Kelle, Diana Linke, Ralf G Berger
Extracellular esterase activity was detected in submerged cultures of Rhizoctonia solani grown in the presence of sugar beet pectin or Tween 80. Putative type B feruloyl esterase (FAE) coding sequences found in the genome data of the basidiomycete were heterologously expressed in Pichia pastoris. Recombinant enzyme production on the 5-L bioreactor scale (RspCAE: 3245UL(-1)) exceeded the productivity of the wild type strain by a factor of 800. Based on substrate specificity profiling, the purified recombinant RspCAE was classified as a p-coumaroyl esterase (pCAE) with a pronounced chlorogenic acid esterase side activity...
January 31, 2017: New Biotechnology
https://www.readbyqxmd.com/read/28159612/a-prime-boost-vaccination-with-ha-dna-and-pichia-produced-ha-protein-elicits-a-strong-humoral-response-in-chickens-against-h5n1
#15
Anna Stachyra, Maria Pietrzak, Agnieszka Macioła, Anna Protasiuk, Monika Olszewska, Krzysztof Śmietanka, Zenon Minta, Anna Góra-Sochacka, Edyta Kopera, Agnieszka Sirko
Highly pathogenic avian influenza viruses cause severe disease and huge economic losses in domestic poultry and might pose a serious threat to people because of the high mortality rates in case of an accidental transmission to humans. The main goal of this work was to evaluate the immune responses and hemagglutination inhibition potential elicited by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens in chickens. A plasmid encoding hemagglutinin (HA) from the A/swan/Poland/305-135V08/2006 (H5N1) virus, or the recombinant HA protein produced in Pichia pastoris system, both induced H5 HA-specific humoral immune responses in chickens...
January 31, 2017: Virus Research
https://www.readbyqxmd.com/read/28153694/production-in-pichia-pastoris-antifungal-activity-and-crystal-structure-of-a-class-i-chitinase-from-cowpea-vigna-unguiculata-insights-into-sugar-binding-mode-and-hydrolytic-action
#16
Patrícia G Castro Landim, Tuana O Correia, Fredy D A Silva, Denise R Nepomuceno, Helen P S Costa, Humberto M Pereira, Marina D P Lobo, Frederico B M B Moreno, José Brandão-Neto, Suelen C Medeiros, Ilka M Vasconcelos, José T A Oliveira, Bruno L Sousa, Ito L Barroso-Neto, Valder N Freire, Cristina P S Carvalho, Ana C O Monteiro-Moreira, Thalles B Grangeiro
A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates...
January 30, 2017: Biochimie
https://www.readbyqxmd.com/read/28150747/methanol-independent-protein-expression-by-aox1-promoter-with-trans-acting-elements-engineering-and-glucose-glycerol-shift-induction-in-pichia-pastoris
#17
Jinjia Wang, Xiaolong Wang, Lei Shi, Fei Qi, Ping Zhang, Yuanxing Zhang, Xiangshan Zhou, Zhiwei Song, Menghao Cai
The alcohol oxidase 1 promoter (PAOX1) of Pichia pastoris is commonly used for high level expression of recombinant proteins. While the safety risk of methanol and tough process control for methanol induction usually cause problems especially in large-scale fermentation. By testing the functions of trans-acting elements of PAOX1 and combinatorially engineering of them, we successfully constructed a methanol-free PAOX1 start-up strain, in which, three transcription repressors were identified and deleted and, one transcription activator were overexpressed...
February 2, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28130769/secretory-expression-and-characterization-of-chinese-narcissus-gna-like-lectin-in-pichia-pastoris
#18
Xinyu Li, Pengpeng Zhang, Ning Zhang, Xiao Liang, Wenya Wang, Qipeng Yuan, Qiang Li
Narcissus tazetta lectin (NTL) is a GNA-like lectin, which has a wide potential application in medicine, agriculture, and glycobiology. In the present paper, the codon-optimized ntl gene was transformed into the yeast Pichia pastoris; SDS-PAGE gel and western blotting analysis revealed that the recombinant lectin was expressed successfully in Pichia yeast. The similarity between the recombinant NTL and the native NTL was confirmed by circular dichroism (CD) and hemagglutination assay further. In the 5-L scale fermentator, the protein yield was as high as 1...
January 27, 2017: Applied Biochemistry and Biotechnology
https://www.readbyqxmd.com/read/28130631/effects-of-glycerol-supply-and-specific-growth-rate-on-methanol-free-production-of-calb-by-p-pastoris-functional-characterisation-of-a-novel-promoter
#19
Verena Looser, Dominik Lüthy, Marcel Straumann, Katrin Hecht, Karel Melzoch, Karin Kovar
As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (q p) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source...
January 27, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28127815/novel-function-of-wsc-proteins-as-a-methanol-sensing-machinery-in-the-yeast-pichia-pastoris
#20
Shin Ohsawa, Hiroya Yurimoto, Yasuyoshi Sakai
Wsc family proteins are plasma membrane spanning sensor proteins conserved from yeasts to mammalian cells. We studied the functional roles of Wsc family proteins in the methylotrophic yeast Pichia pastoris, and found that PpWsc1 and PpWsc3 function as methanol-sensors during growth on methanol. PpWsc1 responds to a lower range of methanol concentrations than PpWsc3. PpWsc1, but not PpWsc3, also functions during high temperature stress, but PpWsc1 senses methanol as a signal that is distinct from high-temperature stress...
January 27, 2017: Molecular Microbiology
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