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Tsung-Lin Li, Ning-Shian Hsu, Yung-Lin Wang, Kuan-Hung Lin, Chi-Fon Chang, Syue-Yi Lyu, Li-Jen Hsu, Yu-Chen Liu, Chin-Yuan Chang, Chang-Jer Wu
Given energetic carbene and uneven electronegativity, thiazolium that mesomerizes to congeners other than carbene in the low effective dielectric binding site is theoretically plausible but never reported. Here we report 9 subatomic-resolution crystal structures of TKps from Pichia stipitis, where thiazolium displays an extraordinary ring-bending effect. These bent thiazolium congeners are correlated to non-Kekulé diradicals due to no gain/loss of electrons. In conjunction with biophysical/biochemical analyses, we conclude that the ring bending is a result of tautomerization of thiazolium with its non-Kekulé diradicals exclusively in the binding site of TKps...
December 15, 2017: Angewandte Chemie
Veeresh Juturu, Jin Chuan Wu
Pichia pastoris is a well-known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent-free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site-specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α-factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co-expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding...
December 13, 2017: Chembiochem: a European Journal of Chemical Biology
Hai Du, Zhewei Song, Yan Xu
This study aimed to identify specific microorganisms related to the formation of precursors of ethyl carbamate (EC) in the solid-state fermentation of a Chinese Moutai-flavor liquor. The EC content was significantly correlated with the urea content during the fermentation process (R2 = 0.772, P < 0.01). Differences in urea production and degradation were found between members of the microbiota in the fermentation (at both species and functional gene levels) by metatranscriptomic sequencing and culture-dependent analysis...
December 13, 2017: Journal of Agricultural and Food Chemistry
Jinjia Wang, Xiaolong Wang, Lei Shi, Yuanxing Zhang, Xiangshan Zhou, Menghao Cai
High oxygen consumption and heat release caused by methanol catabolism usually bring difficulties to industrial scale-up and cost for protein expression driven by methanol-induced AOX1 promoter in Pichia pastoris. Here, reduced methanol feeding levels were investigated for expression of insulin precursor in a trans-acting elements engineered P. pastoris strain MF1-IP. Insulin precursor expression level reached 6.69 g/(L supernatant) at the methanol feeding rate of 6.67 mL/(h·L broth), which was 59% higher than that in the wild-type strain WT-IP at the methanol feeding rate of 12 mL/(h·L broth)...
December 12, 2017: Journal of Industrial Microbiology & Biotechnology
Bing Liu, Åke Olson, Miao Wu, Anders Broberg, Mats Sandgren
Lytic polysaccharide monooxygenases (LPMO) are important redox enzymes produced by microorganisms for the degradation of recalcitrant natural polysaccharides. Heterobasidion irregulare is a white-rot phytopathogenic fungus that causes wood decay in conifers. The genome of this fungus encodes 10 putative Auxiliary Activity family 9 (AA9) LPMOs. We describe the first biochemical characterization of H. irregulare LPMOs through heterologous expression of two CBM-containing LPMOs from this fungus (HiLPMO9H, HiLPMO9I) in Pichia pastoris...
2017: PloS One
Jing Leng, Xuchuan Liu, Chunyong Zhang, Renjun Zhu, Huaming Mao
A total of 6,219 positive clones were obtained by constructing a BAC library of uncultured ruminal fungi of gayal, and two clones (xynF1 and eglF2) with lignocellulolytic enzyme activity were selected. The sequencing results showed that xynF1 and eglF2 had 903-bp, and 1,995-bp, open reading frames likely to encode β-xylanase (XynF1) and β-glucosidase (EglF2), respectively. The amino acid sequence of XynF1 had 99% coverage and 95% homology to the endo-β-1,4-xylanase encoded by the cellulase gene of Orpinomyces sp...
December 8, 2017: Journal of General and Applied Microbiology
Sergio Calderon-Salais, Prisiliana Velazquez-Bernardino, Victor E Balderas-Hernandez, Ana P Barba-de la Rosa, Antonio De Leon-Rodriguez
Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135-164aa) of human calreticulin (hCALR) and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a multi-copy strain was obtained using an in vitro multimerization approach...
December 5, 2017: Protein Expression and Purification
Roland Prielhofer, Juan J Barrero, Stefanie Steuer, Thomas Gassler, Richard Zahrl, Kristin Baumann, Michael Sauer, Diethard Mattanovich, Brigitte Gasser, Hans Marx
BACKGROUND: State-of-the-art strain engineering techniques for the host Pichia pastoris (syn. Komagataella spp.) include overexpression of homologous and heterologous genes, and deletion of host genes. For metabolic and cell engineering purposes the simultaneous overexpression of more than one gene would often be required. Very recently, Golden Gate based libraries were adapted to optimize single expression cassettes for recombinant proteins in P. pastoris. However, an efficient toolbox allowing the overexpression of multiple genes at once was not available for P...
December 8, 2017: BMC Systems Biology
Jing Chen, Haibin Li, Tao Wang, Shuyang Sun, Jia Liu, Jianhua Chen
Thymosin alpha 1 (Tα1) is an important immunomodulating agent with various clinical applications. The natural form of Tα1 is Nα -acetylated, which was supposed to be related to in vivo stability of the hormone. In this study, fusion protein Tα1-HSA was constructed and expressed in Pichia pastoris. RimJ, a Nα -acetyltransferase from E.coli, was also overexpressed and purified to homogeneity. In vitro acetylation of Tα1-HSA in the presence of RimJ and acetyl coenzyme A resulted in Nα -acetyl Tα1-HSA. The Nα -acetylation was determined by LC-MS/MS...
November 10, 2017: Oncotarget
Raminta Venskutonytė, Karin Elbing, Karin Lindkvist-Petersson
Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein...
2018: Methods in Molecular Biology
L M Broissin-Vargas, R Snell-Castro, J J Godon, O González-Ríos, M L Suárez-Quiroz
AIMS: The purpose of this study was to evaluate the effect of warehouse storage conditions on the composition of the fungal community of green coffee beans (GCB) that were stored in jute sacks for one year. METHODS AND RESULTS: Molecular characterization of the fungal community composition and population dynamics obtained by Q-PCR, CE-SSCP (Simpson's diversity index D), and Illumina MiSeq sequencing indicated that Saccharomycetales dominated during the first 6 months of storage period with species as Meyerozyma guilliermondii and Pichia kluyveri...
December 7, 2017: Journal of Applied Microbiology
C Grangeteau, V David, H Alexandre, M Guilloux-Benatier, S Rousseaux
Although differences in yeast biodiversity have often been found between vineyards subjected to organic protection or conventional protection, little is known about the effect of copper and sulfur fungicides (the only fungicides allowed in organic farming) on yeast populations. The sensitivity to copper and sulfur of 158 yeast isolates of seven different species (Aureobasidium pullulans, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Metschnikowia sp., Pichia membranifaciens, Saccharomyces cerevisiae and Starmerella bacillaris) was evaluated...
November 29, 2017: FEMS Yeast Research
Hao Tan, Renyun Miao, Tianhai Liu, Lufang Yang, Yumin Yang, Chunxiu Chen, Jianrong Lei, Yuhui Li, Jiabei He, Qun Sun, Weihong Peng, Bingcheng Gan, Zhongqian Huang
A new cellulolytic strain of Chryseobacterium genus was screened from the dung of a cattle fed with cereal straw. A putative cellulase gene (cbGH5) belonging to glycoside hydrolase family 5 subfamily 46 (GH5_46) was identified and cloned by degenerate PCR plus genome walking. The CbGH5 protein was overexpressed in Pichia pastoris, purified and characterized. It is the first bifunctional cellulase-xylanase reported in GH5_46 as well as in Chryseobacterium genus. The enzyme showed an endoglucanase activity on carboxymethylcellulose of 3237 μmol min-1  mg-1 at pH 9, 90 °C and a xylanase activity on birchwood xylan of 1793 μmol min-1  mg-1 at pH 8, 90 °C...
December 4, 2017: Microbial Biotechnology
Thomas Kickenweiz, Anton Glieder, Jin Chuan Wu
Cellulose is a highly available and renewable carbon source in nature. However, it cannot be directly metabolized by most microbes including Komagataella phaffii (formerly Pichia pastoris), which is a frequently employed host for heterologous protein expression and production of high-value compounds. A K. phaffii strain was engineered that constitutively co-expresses an endoglucanase and a β-glucosidase both from Aspergillus niger and an exoglucanase from Trichoderma reesei under the control of bidirectional promoters...
December 4, 2017: Applied Microbiology and Biotechnology
Deepti Yadav, Bibhuti Ranjan, Nokuthula Mchunu, Marilize Le Roes-Hill, Tukayi Kudanga
This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene (rSLAC) produced high titres of extracellular laccase (500±10U/l), which were further increased seven fold by pre-incubation at 80°C for 30min. The enzyme (∼38kDa) had an optimum activity at 80°C, but optimum pH varied with substrate used. Km values for ABTS, SGZ and 2,6-DMP were 142...
December 1, 2017: International Journal of Biological Macromolecules
An Bracke, David Hoogewijs, Sylvia Dewilde
Globins are among the best investigated proteins in biological and medical sciences and represent a prime tool for the study of the evolution of genes and the structure-function relationship of proteins. Here, we explore the recombinant expression of globins in three different expression systems: Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda. We expressed two different human globin types in these three expression systems: I) the well-characterized neuroglobin and II) the uncharacterized, circular permutated globin domain of the large chimeric globin androglobin...
December 1, 2017: Analytical Biochemistry
Yilei Wei, Yu Zhan, Pengfei Chen, Zhi Liu, Haohao Zhang, Dandan Liu, Jie Zhang, Min Yu, Wei Mo, Jun Zhang, Xiaoren Zhang
BACKGROUND: Receptor activator of NF-κB ligand (RANKL)/RANK signaling essentially functions within the skeletal system, particularly participating in osteoclastogenesis and bone resorption. In addition, this signaling pathway has also been shown to influence tumor progression as well as the development and function of the immune system. Therefore, blocking the interaction between RANKL and RANK is a new therapeutic approach to prevent bone-related diseases and cancer. RESULTS: The coding sequence encoding the extracellular domain of human RANK (RANK-N) was codon optimized for Pichia pastoris and cloned into the pPIC9K vector, and the recombinant plasmid was then transformed into P...
December 4, 2017: BMC Biotechnology
Wei Shu, Chao Li, Xiaoyun Liu, Jianye Xia, Yingping Zhuang
Isotope Dilution Mass Spectrometry (IDMS) is the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key is getting the corresponding fully uniformly(U) ¹³C-labeled metabolites to be measured. The conventional procedure for getting fully U ¹³C-labeled metabolites is through batch cultivation, but intracellular metabolites concentrations by this method are generally low. By applying U ¹³C-labeled glucose pulse, combined with fast sampling and quenching, mixture of fully U ¹³C-labeled intracellular metabolites was successfully extracted with higher concentration from Pichia pastoris G/DSEL fed with fully U ¹³C-labeled glucose as only carbon source...
November 25, 2017: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Raquel Quintilla, Anna Kolecka, Serge Casaregola, Heide M Daniel, Jos Houbraken, Markus Kostrzewa, Teun Boekhout, Marizeth Groenewald
Since food spoilage by yeasts causes high economic losses, fast and accurate identifications of yeasts associated with food and food-related products are important for the food industry. In this study the efficiency of the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify food related yeasts was evaluated. A CBS in-house MALDI-TOF MS database was created and later challenged with a blinded test set of 146 yeast strains obtained from food and food related products...
November 24, 2017: International Journal of Food Microbiology
Felipe Vilches, María Cristina Ravanal, Felipe Bravo-Moraga, Danilo Gonzalez-Nilo, Jaime Eyzaguirre
Penicillium purpurogenum secretes numerous lignocellulose-degrading enzymes, including four arabinofuranosidases and an exo-arabinanase. In this work, the biochemical properties of an endo-arabinanase (ABN1) are presented. A gene, coding for a potential ABN was mined from the genome. It includes three introns. The cDNA is 975 bp long and codes for a mature protein of 324 residues. The cDNA was expressed in Pichia pastoris. The enzyme is active on debranched arabinan and arabinooligosaccharides. In contrast to other characterized ABNs, inactive on p-nitrophenyl-α-L-arabinofuranoside (pNPAra), ABN1 is active on this substrate...
November 26, 2017: Carbohydrate Research
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