Effect of Overexpression of the HAC1 Genes from Pichia pastoris and Saccharomyces cerevisiae on the Heterologous Phytase and Xylanase Production by P. pastoris

2019 ◽  
Vol 35 (6) ◽  
pp. 57-66
Author(s):  
L.N. Borshchevskaya ◽  
A.N. Kalinina ◽  
S.P. Sineoky ◽  
M.D. Kashirskaya
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pichia Pastoris ◽  
Xylanase Activity ◽  
Ministry Of Education ◽  
Transcription Activator ◽  
Xylanase Production ◽  
Codon Composition ◽  
The Russian Federation ◽  
Encoding Genes

Effect of overexpression of the HAC1 genes from Pichia pastoris and Saccharomyces cerevisiae on the production of heterologous enzymes, Escherichia coli phytase and Paenibacillus brasilensis xylanase, in P. pastoris cells has been studied. Codon composition of the phytase and xylanase encoding genes was optimized, and the genes were expressed in P. pastoris under the control of AOX1 promoter. The obtained multi-copy strains produced in vitro 927 U/mL phytase and 1,401 U/ml xylanase activity. Overexpression of the HAC1 gene from P. pastoris was shown to increase the phytase and xylanase production by 46% and 41%, respectively. Overexpression of HAC1 from S. cerevisiae increased the phytase production by 28% and xylanase by 20%. Data obtained could be helpful in the construction of industrial enzyme-producing strains based on P. pastoris. phytase, xylanase, Нас1р transcription activator, UPR, Pichia pastoris, Saccharomyces cerevisiae The work was carried out using Multipurpose Scientific Installation All-Russian Collection of Industrial Microorganisms National Bioresource Center, NRC «Kurchatov Institute» -GosNIIgenetika. The authors are grateful to A.V. Nikulin (Sintol LLC, Russia) for the assistance in the real-time PCR experiments. The work was financially supported by the Ministry of Education and Science of the Russian Federation (Unique Identifiers of the Projects are RFMEFI57917X0145 and RFMEFI60717X0180).

Increase in the Production of Endo-1,4-β-Xylanase from Paenibacillus brasilensis in Pichia pastoris

2019 ◽  
Vol 35 (6) ◽  
pp. 30-38 ◽  
Author(s):  
L.N. Borshchevskaya ◽  
T.L. Gordeeva ◽  
S.P. Sineoky
Keyword(s):  
Pichia Pastoris ◽  
Target Gene ◽  
Heterologous Protein ◽  
Ministry Of Education ◽  
Kurchatov Institute ◽  
Xylanase Production ◽  
Resource Center ◽  
Recombination System ◽  
Codon Composition ◽  
Pichia Pastoris Yeast

A Pichia pastoris yeast strain producing endo-l,4-β-xylanase from Paenibacillus brasilensis with an activity of 54,400 U/mL after 140 h of fermentation in a laboratory fermenter has been obtained. A number of approaches were used to increase the level of the xylanase production in this strain: optimization of the target gene codon composition, multiple integration of the expression cassette into the recipient strain chromosome using the Cre-lox recombination system, and also improving the heterologous protein folding via the overexpression of the HAC1i gene from Pichia pastoris. xylanase, xylan, Cre-lox system, HAC1p transcriptional activator, multicopy strain, Paenibacillus brasilensis, Pichia pastoris The work was performed with the financial support of the Ministry of Education and Science of Russia (Unique Project Identifier RFMEFI60717X0180) using the Multipurpose Scientific Installation of «All-Russian Collection of Industrial Microorganisms» National Bio-Resource Center, NRC «Kurchatov Institute» - GosNIIgenetika.

New Recombinant Phytase from Kosakonia sacchari: characteristic and biotechnological potential

2019 ◽  
Vol 35 (4) ◽  
pp. 33-41
Author(s):  
L.N. Borshchevskaya ◽  
A.N. Kalinina ◽  
N.V. Bulushova ◽  
S.P. Syneoky ◽  
S.P. Voronin ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
Ministry Of Education ◽  
Maximum Reaction Rate ◽  
Biotechnological Potential ◽  
Resource Center ◽  
Codon Composition ◽  
Maximum Reaction ◽  
Wide Range ◽  
Recombinant Phytase

A DNA sequence from Kosakonia sacchari that according to automated computer analysis is believed to correspond to a gene for histidine acid phytase has been selected from the GenBank database. The sequence was optimized for codon composition, synthesized, cloned and expressed in Pichia pastoris. Main characteristics of the purified recombinant enzyme were determined. It was established that the values of pH=4.5 and temperature of 50 °C are optimal for the phytase functioning. The values of specific activity, Michaelis constant (Km) and maximum reaction rate (Vmax) with phytate as a substrate were 1470 U/mg, 193 uM and 2167 umol/(min ∙ mg), respectively. It was shown that the enzyme was characterized by a wide range of working pH. Therefore, the properties of a new recombinant phytase allow us to consider it as a high-potential enzyme for agrobiotechnology. histidine acid phosphatases, Kosakonia sacchari phytase, Pichia pastoris The work was financially supported by the Ministry of Education and Science of Russian Federation (Unique Project Identifier RFMEFI57917X0145) and was carried out using the Multipurpose Scientific Installation of National Bio-resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA.

Identification of Schizosaccharomyces pombe transcription factor PGA4, which binds cooperatively to Saccharomyces cerevisiae GAL4-binding sites

1990 ◽  
Vol 10 (4) ◽  
pp. 1432-1438
Author(s):  
D M Ruden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Schizosaccharomyces Pombe ◽  
Binding Sites ◽  
Transcription Activator ◽  
Integral Number ◽  
Dna Binding Site ◽  
Adh1 Gene

When the DNA-binding site for the Saccharomyces cerevisiae transcription activator GAL4 is placed upstream of the Schizosaccharomyces pombe ADH1 TATA box, transcription of the ADH1 gene is activated in S. pombe in vivo by an endogenous transcription factor. In vitro studies show that this S. pombe protein, PGA4, binds specifically to DNA containing a GAL4 site and that when two GAL4 sites are present, this protein binds cooperatively. Cooperating binding of PGA4 to DNA is favored if the GAL4 sites are separated by an integral number of turns of the DNA helix.

scholarly journals Statistical optimization of xylanase production, using different agricultural wastes by Aspergillus oryzae MN894021, as a biological control of faba bean root diseases

2020 ◽  
Vol 30 (1) ◽  
Author(s):  
Sherien M. M. Atalla ◽  
Nehad E. Ahmed ◽  
Hassan M. Awad ◽  
Nadia G. El Gamal ◽  
Aliaa R. El Shamy
Keyword(s):  
Biological Control ◽  
Antifungal Activity ◽  
Rice Straw ◽  
Sea Water ◽  
Xylanase Activity ◽  
Agricultural Wastes ◽  
Fungal Isolate ◽  
Main Body ◽  
Xylanase Production

Abstract Background Xylanase enzyme plays an important role in nature as being a part of protecting the environment from pollution. It has also various industrial applications. Main body of abstract Marine fungal isolate was recovered from red sea water at Sharm El-Sheikh province, Egypt, and tested for xylanase activity, using different agricultural wastes as a substrate. It was found that rice straw was the best substrate for xylanase production (0.37 U/ml). Thus, it was subjected for identification by 18S rDNA gene. The phylogenetic analysis results indicated that this fungal isolate belonging to Aspergillus species with a similarity of 99% and named as A. oryzae SS_RS-SH (MN894021). The regular two-level factorial design was used to optimize the important medium components, which significantly affected the xylanase production. The model in equation suggested optimal conditions of 2% of rice straw, 8 g/l of yeast extract, 4 g/l of (NH4)2SO4, 2 g/l K2HPO4, and 2.5 g/l MgSO4.7H2O for a maximum xylanase yield. The antifungal activity of crude xylanase on mycelial growth of some pathogenic fungi isolated from different hosts was investigated. The results showed that xylanase T1 had a potent antifungal activity than control. Greenhouse experiments indicated that all treatments with xylanase at different concentrations significantly decreased infection occurrence of beans, which have been effectively infected with root rot pathogens, compared to unprocessed control treatments. Short conclusion Xylanase yield increased 2.43-folds than initial screening. The xylanase had a potential antifungal activity both in vitro and under greenhouse conditions. The outcome of this study ensured that this fungal strain could be used as biological control for plant disease.

scholarly journals Identification of Schizosaccharomyces pombe transcription factor PGA4, which binds cooperatively to Saccharomyces cerevisiae GAL4-binding sites.

1990 ◽  
Vol 10 (4) ◽  
pp. 1432-1438 ◽  
Author(s):  
D M Ruden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Schizosaccharomyces Pombe ◽  
Binding Sites ◽  
Transcription Activator ◽  
Integral Number ◽  
Dna Binding Site ◽  
Adh1 Gene

When the DNA-binding site for the Saccharomyces cerevisiae transcription activator GAL4 is placed upstream of the Schizosaccharomyces pombe ADH1 TATA box, transcription of the ADH1 gene is activated in S. pombe in vivo by an endogenous transcription factor. In vitro studies show that this S. pombe protein, PGA4, binds specifically to DNA containing a GAL4 site and that when two GAL4 sites are present, this protein binds cooperatively. Cooperating binding of PGA4 to DNA is favored if the GAL4 sites are separated by an integral number of turns of the DNA helix.

An ancient oxidoreductase making differential use of its cofactors

2014 ◽  
Vol 395 (7-8) ◽  
pp. 855-869 ◽  
Author(s):  
Doreen Blüher ◽  
Annekathrin Reinhardt-Tews ◽  
Martin Hey ◽  
Hauke Lilie ◽  
Ralph Golbik ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kluyveromyces Lactis ◽  
Protein A ◽  
Negative Regulator ◽  
Transcription Activator ◽  
Metabolic State ◽  
Multiple Signals ◽  
Negative Effect

Abstract Many transcription factors contribute to cellular homeostasis by integrating multiple signals. Signaling via the yeast Gal80 protein, a negative regulator of the prototypic transcription activator Gal4, is primarily regulated by galactose. ScGal80 from Saccharomyces cerevisiae has been reported to bind NAD(P). Here, we show that the ability to bind these ligands is conserved in KlGal80, a Gal80 homolog from the distantly related yeast Kluyveromyces lactis. However, the homologs apparently have diverged with respect to response to the dinucleotide. Strikingly, ScGal80 binds NAD(P) and NAD(P)H with more than 50-fold higher affinity than KlGal80. In contrast to ScGal80, where NAD is neutral, NAD and NADP have a negative effect in KlGal80 on its interaction with a KlGal4-peptide in vitro. Swapping a loop in the NAD(P) binding Rossmann-fold of ScGal80 into KlGal80 increases the affinity for NAD(P) and has a significant impact on KlGal4 regulation in vivo. Apparently, dinucleotide binding allows coupling of the metabolic state of the cell to regulation of the GAL/LAC genes. The particular sequences involved in binding determine how exactly the metabolic state is sensed and integrated by Gal80 to regulate Gal4.

Bioethanol production from alkaline hydrogen peroxide pretreated Populus deltoides wood using hydrolytic enzymes of Bacillus stratosphericus N12(M) and Bacillus altitudinis Kd1(M) under different modes of separate hydrolysis and fermentation by monoculture and co-culture combinations of ethanologens

2016 ◽  
Vol 5 (02) ◽  
pp. 4810
Author(s):  
Nisha Sharma* ◽  
Nivedita Sharma
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Populus Deltoides ◽  
Ethanol Yield ◽  
Hydrolytic Enzyme ◽  
Xylanase Activity ◽  
Hydrolytic Enzymes ◽  
Cellulase Activity ◽  
Integrated Approach ◽  
Xylanase Production ◽  
Bacillus Altitudinis

An integrated approach was studied for in-house cellulase and xylanase production, from novel hyper hydrolytic enzyme producers and enzymatic hydrolysis of pretreated Populus deltoides wood into bioethanol. A xylanase producer Bacillus altitudinis Kd1 (M) and cellulase producerBacillus stratosphericus N12 (M) was isolated from soil. Optimization of process parameters led to an optimal xylanase activity of 96.25 IU at 300C and pH 5.5 and cellulase activity of 5.98 IU at 300C and pH 8.0. The NaOH+H2O2 pretreated biomass was hydrolysed using cellulase and xylanase producing 12.45 mg/g of reducing sugars. Further fermentation of lignocellulosic hydrolysate was performed using different yeasts viz. Saccharomyces cerevisiae I, Saccharomyces cerevisiae II, Pichia stipitis, Candida shehatae and Zymomonas mobilis and maximum 11.10 g/l ethanol yield achieved with co-culture of S. cerevisiae II + P. stipitis with fermentation efficiency of 43.52% under method IV of SHF. The results have significant implications and further applications regarding production of fuel ethanol from agricultural lignocellulosic waste.

Ca-, Mg- és K-sók hatása egyes nehézfémek Saccharomyces cerevisiae törzsekre gyakorolt toxicitására

2008 ◽  
Vol 57 (1) ◽  
pp. 161-175
Author(s):  
Nikoletta Tóth ◽  
Hamuda Hosam E. A. F. Bayoumi ◽  
Attila Palágyi ◽  
Mihály Kecskés
Keyword(s):  
Saccharomyces Cerevisiae

Az utóbbi években egyre több tanulmány született a mikroorganizmusok nehézfém akkumulációjáról. A mikroszervezetek nehézfémekkel szembeni tűrőképességére és nehézfém felvételére a bioremediációs hasznosíthatóságuk miatt egyre nagyobb figyelmet fordítanak. A mikroorganizmusok tulajdonságai nagyon jól hasznosíthatóak a talajszennyezés monitorozásánál. A toxikus nehézfémek komoly ökológiai problémát jelentenek környezetünkben, ezért kiemelkedő fontosságú a nehézfémekkel szennyezett talajok tisztítása. In vitro , két S. cerevisiae törzs (NSS5099 és NSS7002) nehézfémekkel szembeni toleranciáját vizsgáltuk. A két törzs szaporodási kinetikáját olyan táptalajon tanulmányoztuk, amelyhez 50 µM koncentrációban adtunk Cu 2+ -, Pb 2+ -, Cd 2+ - vagy Ni 2+ -ionokat. A vizsgált nehézfémek élesztőtörzsekre gyakorolt toxicitása csökkenő sorrendben: Cu 2+ > Pb 2+ > Cd 2+ > Ni 2+ . A 350 µM koncentrációjú Cu 2+ , Pb 2+ vagy Cd 2+ és 450 µM koncentrációjú Ni 2+ 48 órás inkubációt követően 50%-kal csökkentette az élősejtek számát. Amikor a nehézfémek táptalajba történő adagolása előtt 50 mM Ca(HCO 3 ) 2 , 75 mM MgSO 4 , vagy 150 mM K 2 SO 4 -ot adtunk a közeghez csökkent a nehézfémek sejtekre gyakorolt toxicitása, és több sejt maradt életben. A 350 és 450 µM koncentrációban lévő nehézfémek toxicitását a fémsók 40%-kal csökkentették. A kapott eredmények alapján az NSS7002 törzs sokkal alkalmasabbnak bizonyult a nehézfémekkel szennyezett talajok tisztítására, mint az NSS5099._

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