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yeast glycosylation

Sha Xu, Ge-Yuan Zhang, Huijie Zhang, Toshihiko Kitajima, Hideki Nakanishi, Xiao-Dong Gao
BACKGROUND: To humanize yeast N-glycosylation pathways, genes involved in yeast specific hyper-mannosylation must be disrupted followed by the introduction of genes catalyzing the synthesis, transport, and addition of human sugars. However, deletion of these genes, for instance, OCH1, which initiates hyper-mannosylation, could cause severe defects in cell growth, morphogenesis and response to environmental challenges. RESULTS: In this study, overexpression of RHO1, which encodes the Rho1p small GTPase, is confirmed to partially recover the growth defect of Saccharomyces cerevisiae Δalg3Δoch1 double mutant strain...
October 21, 2016: Microbial Cell Factories
Lasse van Wijlick, René Geissen, Jessica S Hilbig, Quentin Lagadec, Pilar D Cantero, Eugen Pfeifer, Mateusz Juchimiuk, Sven Kluge, Stephan Wickert, Paula Alepuz, Joachim F Ernst
In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation...
October 2016: PLoS Genetics
Amalia E Yanni, Nikoleta S Stamataki, Panagiotis Konstantopoulos, Maria Stoupaki, Athanasios Abeliatis, Irene Nikolakea, Despoina Perrea, Vaios T Karathanos, Nikolaos Tentolouris
PURPOSE: Chromium (Cr)-enriched yeast supplementation to whole wheat bread (WWCrB) has been shown to ameliorate postprandial glycemic response in healthy subjects. The present study investigates the long-term benefit of WWCrB consumption for patients with type-2 diabetes mellitus (T2DM). METHODS: Thirty patients with T2DM were randomly assigned to a group receiving WWCrB or the plain whole wheat bread (WWB) group. Plasma glucose, insulin, glycosylated hemoglobin (HbA1c) and insulin resistance were determined, and oral glucose tolerance test (OGTT) was performed at the beginning and the end of the dietary intervention, which lasted for 12 weeks...
October 12, 2016: European Journal of Nutrition
Zhangjiang He, Linli Luo, Nemat O Keyhani, Xiaodong Yu, Shenghua Ying, Yongjun Zhang
Protein O-mannosyltransferases (Pmts) belong to a highly conserved protein family responsible for the initiation of O-glycosylation of many proteins. Pmts contain one dolichyl-phosphate-mannose-protein mannosyltransferases (PMT) domain and three MIR motifs (mannosyltransferase, inositol triphosphate, and ryanodine receptor) that are essential for activity in yeast. We report that in the insect fungal pathogen, Beauveria bassiana, deletion of the C-terminal Pmt1 MIR-containing region (Pmt1∆ (311-902)) does not alter O-mannosyltransferase activity, but does increase total cell wall protein O-mannosylation levels and results in phenotypic changes in fungal development and cell wall stability...
October 8, 2016: Applied Microbiology and Biotechnology
Manuel Nieto-Domínguez, Alicia Prieto, Beatriz Fernández de Toro, Francisco Javier Cañada, Jorge Barriuso, Zach Armstrong, Stephen G Withers, Laura I de Eugenio, María Jesús Martínez
BACKGROUND: Glycosides are compounds displaying crucial biological roles and plenty of applications. Traditionally, these molecules have been chemically obtained, but its efficient production is limited by the lack of regio- and stereo-selectivity of the chemical synthesis. As an interesting alternative, glycosidases are able to catalyze the formation of glycosides in a process considered green and highly selective. In this study, we report the expression and characterization of a fungal β-xylosidase in Pichia pastoris...
October 4, 2016: Microbial Cell Factories
Sheng-Tao Li, Ning Wang, Sha Xu, Jian Yin, Hideki Nakanishi, Neta Dean, Xiao-Dong Gao
BACKGROUND: Asparagine (N)-linked glycosylation begins with a stepwise synthesis of the dolichol-linked oligosaccharide (DLO) precursor, Glc3Man9GlcNAc2-PP-Dol, which is catalyzed by a series of endoplasmic reticulum membrane-associated glycosyltransferases. Yeast ALG1 (asparagine-linked glycosylation 1) encodes a β-1, 4 mannosyltransferase that adds the first mannose onto GlcNAc2-PP-Dol to produce a core trisaccharide Man1GlcNAc2-PP-Dol. ALG1 is essential for yeast viability, and in humans mutations in the ALG1 cause congenital disorders of glycosylation known as ALG1-CDG...
September 23, 2016: Biochimica et Biophysica Acta
Osamu Yamada, Masayuki Machida, Akira Hosoyama, Masatoshi Goto, Toru Takahashi, Taiki Futagami, Youhei Yamagata, Michio Takeuchi, Tetsuo Kobayashi, Hideaki Koike, Keietsu Abe, Kiyoshi Asai, Masanori Arita, Nobuyuki Fujita, Kazuro Fukuda, Ken-Ichi Higa, Hiroshi Horikawa, Takeaki Ishikawa, Koji Jinno, Yumiko Kato, Kohtaro Kirimura, Osamu Mizutani, Kaoru Nakasone, Motoaki Sano, Yohei Shiraishi, Masatoshi Tsukahara, Katsuya Gomi
Awamori is a traditional distilled beverage made from steamed Thai-Indica rice in Okinawa, Japan. For brewing the liquor, two microbes, local kuro (black) koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae are involved. In contrast, that yeasts are used for ethanol fermentation throughout the world, a characteristic of Japanese fermentation industries is the use of Aspergillus molds as a source of enzymes for the maceration and saccharification of raw materials. Here we report the draft genome of a kuro (black) koji mold, A...
September 20, 2016: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes
Jihye Kim, Heajin Park, Byung Tae Park, Hye Seong Hwang, Jae Il Kim, Dae Kyong Kim, Ha Hyung Kim
Recombinant human GM-CSF (rhGM-CSF) from yeast has been clinically applied to immunosuppressed patients. The production of suspension-cultured rice-cell-derived rhGM-CSF (rrhGM-CSF), which has a longer blood clearance time and the same bioactivity as yeast-derived rhGM-CSF, and the analysis of its N-glycans have been reported recently. However, there are no previous reports of the O-glycosylation of rhGM-CSF from plant cells, and so this study investigated O-glycans, O-glycosylation sites, and their structural role in rrhGM-CSF...
October 14, 2016: Biochemical and Biophysical Research Communications
Nutan Mhetras, Susan Liddell, Digambar Gokhale
This paper reports on the production of β-xylosidase from an unexplored yeast, Pseudozyma hubeinsis. The expression of this enzyme could be induced by beech wood xylan when the yeast was grown at 27 °C. The enzyme was purified to homogeneity as a glycoprotein with 23 % glycosylation. The purification protocol involved ammonium sulphate precipitation, QAE-Sephadex A50 ion exchange chromatography and sephacryl-200 column chromatography which resulted in 8.3-fold purification with 53.12 % final recovery. The purified enzyme showed prominent single band on SDS-PAGE...
December 2016: AMB Express
Mengying Li, Xinyu Liu, Zhixi Liu, Yi Sun, Muxing Liu, Xiaoli Wang, Haifeng Zhang, Xiaobo Zheng, Zhengguang Zhang
N-linked glycosylation is a way of glycosylation for newly synthesized protein, which plays a key role in the maturation and transport of proteins. Glycoside hydrolases (GHs) are essential in this process, and are involved in processing of N-linked glycoproteins or degradation of carbohydrate structures. Here, we identified and characterized MoGls2 in Magnaporthe oryzae, which is a yeast glucosidase II homolog Gls2 and is required for trimming the final glucose in N-linked glycans and normal cell wall synthesis...
2016: PloS One
Michael W Traxlmayr, Jonathan D Kiefer, Raja R Srinivas, Elisabeth Lobner, Alison W Tisdale, Naveen K Mehta, Nicole J Yang, Bruce Tidor, K Dane Wittrup
The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold due to its small size (7 kDa), high thermal stability (Tm of 98degC) and absence of cysteines and glycosylation sites. However, as a DNA-binding protein Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to non-specific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability...
August 30, 2016: Journal of Biological Chemistry
Min Shu, Wei Shen, Shihui Yang, Xiaojuan Wang, Fei Wang, Yaping Wang, Lixin Ma
A novel serine protease from Trichoderma koningii (SPTK) was synthesized and expressed in Pichia pastoris. The recombinant SPTK was completely inhibited by phenyl methyl sulfonyl fluoride (PMSF), suggesting that SPTK belonged to the subgroup of serine proteases. The optimum pH and temperature for the recombinant SPTK reaction were 6.0 and 55°C, respectively. SPTK performed a tolerance to most organic solvents and metal ions, and the addition of Triton X-100 exhibited an activation of SPTK up to 243% of its initial activity but SDS strongly inhibited...
October 2016: Enzyme and Microbial Technology
Juan C Aon, Jianxin Sun, Julie M Leighton, Edward R Appelbaum
BACKGROUND: In this study we examine the integrity of the cell wall during scale up of a yeast fermentation process from laboratory scale (10 L) to industrial scale (10,000 L). In a previous study we observed a clear difference in the volume fraction occupied by yeast cells as revealed by wet cell weight (WCW) measurements between these scales. That study also included metabolite analysis which suggested hypoxia during scale up. Here we hypothesize that hypoxia weakens the yeast cell wall during the scale up, leading to changes in cell permeability, and/or cell mechanical resistance, which in turn may lead to the observed difference in WCW...
2016: Microbial Cell Factories
Kerry R Love, Kartik A Shah, Charles A Whittaker, Jie Wu, M Catherine Bartlett, Duanduan Ma, Rachel L Leeson, Margaret Priest, Jonathan Borowsky, Sarah K Young, J Christopher Love
BACKGROUND: Pichia pastoris has emerged as an important alternative host for producing recombinant biopharmaceuticals, owing to its high cultivation density, low host cell protein burden, and the development of strains with humanized glycosylation. Despite its demonstrated utility, relatively little strain engineering has been performed to improve Pichia, due in part to the limited number and inconsistent frameworks of reported genomes and transcriptomes. Furthermore, the co-mingling of genomic, transcriptomic and fermentation data collected about Komagataella pastoris and Komagataella phaffii, the two strains co-branded as Pichia, has generated confusion about host performance for these genetically distinct species...
2016: BMC Genomics
Huimin Wang, Yan Yang, Lin Lin, Wenlong Zhou, Minzhi Liu, Kedi Cheng, Wei Wang
BACKGROUND: Glycosylation of flavonoids is a promising approach to improve the pharmacokinetic properties and biological activities of flavonoids. Recently, many efforts such as enzymatic biocatalysis and the engineered Escherichia coli biotransformation have increased the production of flavonoid glucosides. However, the low yield of flavonoid glucosides can not meet the increasing demand for human medical and dietary needs. Saccharomyces cerevisiae is a generally regarded as safe (GRAS) organism that has several attractive characteristics as a metabolic engineering platform for the production of flavonoid glucosides...
2016: Microbial Cell Factories
D Marquard, A Enders, G Roth, U Rinas, T Scheper, P Lindner
In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Pichia pastoris is one of the most promising protein expression systems. This yeast combines fast growth on simple media and important eukaryotic features such as glycosylation. In this work, the ISM technology was applied to Pichia pastoris cultivations for online monitoring of the cell concentration during cultivation. Different ISM settings were tested. The acquired images were analyzed with two image processing algorithms...
September 20, 2016: Journal of Biotechnology
Wei Xia, Xinxin Xu, Lichun Qian, Pengjun Shi, Yingguo Bai, Huiying Luo, Rui Ma, Bin Yao
BACKGROUND: β-Glucosidase is an important member of the biomass-degrading enzyme system, and plays vital roles in enzymatic saccharification for biofuels production. Candidates with high activity and great stability over high temperature and varied pHs are always preferred in industrial practice. To achieve cost-effective biomass conversion, exploring natural enzymes, developing high level expression systems and engineering superior mutants are effective approaches commonly used. RESULTS: A newly identified β-glucosidase of GH3, Bgl3A, from Talaromyces leycettanus JCM12802, was overexpressed in yeast strain Pichia pastoris GS115, yielding a crude enzyme activity of 6000 U/ml in a 3 L fermentation tank...
2016: Biotechnology for Biofuels
Genny Degani, Enrico Ragni, Pedro Botias, Davide Ravasio, Julia Calderon, Elena Pianezzola, Jose Manuel Rodriguez-Peña, Maria Antonietta Vanoni, Javier Arroyo, William A Fonzi, Laura Popolo
BACKGROUND: The cell wall is essential for the yeast to hypha (Y-H) transition that enables Candida albicans to invade human tissues and evade the immune system. The main constituent, β(1,3)-glucan, is remodeled by glucanosyltransferases of the GH72 family. Phr1p is responsible of glucan remodeling at neutral-alkaline pH and is essential for morphogenesis and virulence. Due to the pH-regulated expression of PHR1, the phr1Δ phenotype is manifested at pH > 6 and its severity increases with the rise in pH...
July 2, 2016: BMC Genomics
Eudoxie Dulary, Sven Potelle, Dominique Legrand, François Foulquier
Congenital Disorders of Glycosylation (CDG) are rare inherited diseases causing glycosylation defects responsible for severe growth and psychomotor retardations in patients. Whereas most genetic defects affect enzymes directly involved in the glycosylation process, like glycosyltransferases or sugar transporters, recent findings revealed the impact of gene mutations on proteins implicated in both Golgi vesicular trafficking and ion homeostasis. TMEM165 is one of these deficient Golgi proteins found in CDG patients whose function in the secretory pathway has been deduced from several recent studies using TMEM165 deficient mammalian cells or yeast cells deficient in Gtd1p, the yeast TMEM165 ortholog...
June 16, 2016: Tissue & Cell
Ting Luo, Thomas Krüger, Uwe Knüpfer, Lydia Kasper, Natalie Wielsch, Bernhard Hube, Andreas Kortgen, Michael Bauer, Evangelos J Giamarellos-Bourboulis, George Dimopoulos, Axel A Brakhage, Olaf Kniemeyer
During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map...
August 5, 2016: Journal of Proteome Research
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