Comparative Characteristics of Phytases from Citrobacter freundii and Yersinia intermedia Expressed in Ogataea polymorpha and Pichia pastoris Methylotrophic Yeasts

2019 ◽  
Vol 35 (6) ◽  
pp. 51-56
Author(s):  
M.D. Kashirskaya ◽  
M.N. Lazareva ◽  
A.R. Lapteva ◽  
V.Yu. Dobrynin ◽  
T.L. Gordeeva ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Hansenula Polymorpha ◽  
Methylotrophic Yeast ◽  
Citrobacter Freundii ◽  
Unique Identifier ◽  
Recombinant Phytase ◽  
The Stability ◽  
First Time ◽  
Yersinia Intermedia ◽  
Ogataea Polymorpha

The genes for bacterial phytases from Citrobacter freundii and Yersinia intermedia were expressed for the first time in a thermotolarant yeast Ogataea polymorpha. A comparative analysis of the properties of recombinant phytases produced by Ogataea polymorpha and Pichia pastoris yeasts was carried out. It was shown that the stability, pH and temperature profiles of the enzyme activities are the same regardless of the host strain. It was proved that O. polymorpha yeast can be used to create producers of feed enzymes and to develop a technology for their cultivation at temperatures above 37 °C. The prospects of using the O. polymorpha yeast for these purposes were evaluated. Ogataea (Hansenula) polymorpha, Pichia pastoris, methylotrophic yeast, thermal tolerance, producer, recombinant phytase The work was financially supported by the Ministry of Science and Higher Education of RF (Project Unique Identifier RFMEFI57917X0145) using the Multipurpose Scientific Installation of All-Russian National Collection of Industrial Microorganisms National Bioresource Center, NRC «Kurchatov Institute»-GosNIIgenetika.

scholarly journals Improving the heterologous production of fungal peroxygenases through an episomal Pichia pastoris promoter and signal peptide shuffling system

2020 ◽  
Author(s):  
Pascal Püllmann ◽  
Martin J. Weissenborn
Keyword(s):  
Pichia Pastoris ◽  
High Throughput ◽  
Signal Peptide ◽  
Protein Production ◽  
Hansenula Polymorpha ◽  
Methylotrophic Yeast ◽  
Enzyme Engineering ◽  
Signal Peptides ◽  
Heterologous Production ◽  
Test Set

ABSTRACTFungal Peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and -signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. 11 endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various co-feeding, derepression and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.

Prospects for the Use of Methylotrophic Yeast in the Creation of Industrial Producers of Feed Enzymes

2019 ◽  
Vol 35 (6) ◽  
pp. 12-20
Author(s):  
S.P. Sineoky
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
Methylotrophic Yeast ◽  
Mycelial Fungi ◽  
Recombinant Enzymes ◽  
Unique Identifier ◽  
Genes Encoding ◽  
Feed Enzymes ◽  
The Creation ◽  
Yeast Genes

In recent years, mycelial fungi have faced competition from recombinant yeast producers in the production of feed enzymes. An intensive study on genetic diversity identified yeast genes encoding feed enzymes the specific activity of which is much higher than that in mycelial fungi. In addition, these genes were expressed in yeast much more efficiently than in mycelial fungi. The use of yeast recombinant producers allowed expanding the production of a line of industrial enzymes with a significant reduction in their cost. The advantages of yeast producers of recombinant enzymes include the possibility of obtain monoenzymes that are part of various enzyme complexes used for different purposes. Pichia pastoris methylotrophic yeast is the most attractive subject for the creation of recombinant protein-producing strains. feed enzymes, Pichia pastoris, phytase, xylanase, β-glucanase, AOX1 promoter The work was carried out using the equipment of the Multipurpose Scientific Installation of «All-Russian Collection of Industrial Microorganisms» National Bio-Resource Center, NRC «Kurchatov Institute» - GOSNIIGENETIKA The work was funded by the Ministry of Education and Science of Russia (Project Unique Identifier RFMEFI60717X0180).

scholarly journals Use of the Yeast Pichia pastoris as an Expression Host for Secretion of Enterocin L50, a Leaderless Two-Peptide (L50A and L50B) Bacteriocin from Enterococcus faecium L50

2010 ◽  
Vol 76 (10) ◽  
pp. 3314-3324 ◽  
Author(s):  
Antonio Basanta ◽  
Beatriz Gómez-Sala ◽  
Jorge Sánchez ◽  
Dzung B. Diep ◽  
Carmen Herranz ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Enterococcus Faecium ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Secretion Signal ◽  
Expression Host ◽  
Sec System ◽  
Alcohol Oxidase Promoter ◽  
The Individual ◽  
First Time

ABSTRACT In this work, we report the expression and secretion of the leaderless two-peptide (EntL50A and EntL50B) bacteriocin enterocin L50 from Enterococcus faecium L50 by the methylotrophic yeast Pichia pastoris X-33. The bacteriocin structural genes entL50A and entL50B were fused to the Saccharomyces cerevisiae gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s ) and cloned, separately and together (entL50AB), into the P. pastoris expression and secretion vector pPICZαA, which contains the methanol-inducible alcohol oxidase promoter (PAOX1) to express the fusion genes. After transfer into the yeast, the recombinant plasmids were integrated into the genome, resulting in three bacteriocinogenic yeast strains able to produce and secrete the individual bacteriocin peptides EntL50A and EntL50B separately and together. The secretion was efficiently directed by MFα1s through the Sec system, and the precursor peptides were found to be correctly processed to form mature and active bacteriocin peptides. The present work describes for the first time the heterologous expression and secretion of a two-peptide non-pediocin-like bacteriocin by a yeast.

scholarly journals Heterologous Expression of Histidine Acid Phytase From Pantoea sp. 3.5.1 in Methylotrophic Yeast Pichia Pastoris

2020 ◽  
Vol 14 (1) ◽  
pp. 179-189
Author(s):  
Aliya Suleimanova ◽  
Daria Bulmakova ◽  
Margarita Sharipova
Keyword(s):  
Pichia Pastoris ◽  
Large Scale ◽  
Residual Activity ◽  
Methylotrophic Yeast ◽  
Feed Additives ◽  
Cloning And Expression ◽  
Small Scale ◽  
Excellent Thermal Stability ◽  
Inulinase Gene ◽  
Recombinant Phytase

Background and Objective: The major storage form of phosphorus in plant-derived feed is presented by phytates and not digested by animals. Phytases are able to hydrolyze phytates and successfully used as feed additives. Nevertheless, nowadays, there is a constant search of new phytases and expression systems for better production of these enzymes. In this study, we describe cloning and expression of gene encoding histidine acid phytase from Pantoea sp. 3.5.1 using methylotrophic yeast Pichia pastoris as the host. Methods: The phytase gene was placed under the control of the methanol-inducible AOX1 promoter and expressed in P. pastoris. Experiments of small-scale phytase expression and activity assays were used to test recombinant colonies. Four different signal peptides were screened for better secretion of phytase by P. pastoris. After 36 h of methanol induction in shake flasks, the maximum extracellular phytase activity (3.2 U/ml) was observed in P. pastoris strain with integrated construct based on pPINK-HC vector and Kluyveromyces maxianus inulinase gene signal sequence. This phytase was isolated and purified using affinity chromatography. Results: Recombinant phytase was a glycosylated protein, had a molecular weight of around 90 kDa and showed maximum activity at pH 4.0 and at 50°C. Recombinant phytase had excellent thermal stability – it retained high residual activity (100% ± 2%) after 1 hour of heat treatment at 70°C. Conclusion: The enhanced thermostability of the recombinant phytase, its expression provided by strong inducible promotor and the effectively designed expression cassette, the simple purification procedure of the secreted enzyme, and the possibility of large-scale expression make the foundation for further production of this bacterial phytase in P. pastoris at an industrial scale.

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

2014 ◽  
Vol 1841 (2) ◽  
pp. 215-226 ◽  
Author(s):  
Lisa Klug ◽  
Pablo Tarazona ◽  
Clemens Gruber ◽  
Karlheinz Grillitsch ◽  
Brigitte Gasser ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

scholarly journals Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Allyl Alcohol ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Direct Selection ◽  
Toxic Exposure ◽  
Peroxisome Biogenesis ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

scholarly journals Optimisation of Recombinant Myrosinase Production in Pichia pastoris

2021 ◽  
Vol 22 (7) ◽  
pp. 3677
Author(s):  
Zuzana Rosenbergová ◽  
Kristína Kántorová ◽  
Martin Šimkovič ◽  
Albert Breier ◽  
Martin Rebroš
Keyword(s):  
Pichia Pastoris ◽  
Plant Defence ◽  
Plant Secondary Metabolites ◽  
Specific Productivity ◽  
Fermentation Conditions ◽  
Threefold Increase ◽  
Dry Cell Weight ◽  
First Time ◽  
Hydrolysis Of ◽  
Dry Cell

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (MutS) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/Lmedium/h was achieved, which was 102.5-fold higher compared to flask cultivations.

scholarly journals A Novel Approach to Fabricate Foam Ceramics from Steel Slag

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Yu Zheng ◽  
Xudong Luo ◽  
Jinlong Yang ◽  
Wenlong Huo ◽  
Chi Kang
Keyword(s):  
Compressive Strength ◽  
Ph Value ◽  
Steel Slag ◽  
Raw Material ◽  
Solid Loading ◽  
Insulation Material ◽  
Ceramic Foams ◽  
Novel Approach ◽  
The Stability ◽  
First Time

A novel approach is used for fabricating steel slag foam ceramics based on the particle-stabilized foaming method. In this work, steel slag was used as the raw material and propyl gallate (PG) was used as the surface modifier. For the first time, steel slag ceramic foams were successfully fabricated based on particle-stabilized foams. The results show that the stability of the ceramic foams was closely related to the pH value and PG concentration. The porosity and compressive strength could be controlled by changing the solid loading of steel slag and sintering temperature. The porosity of steel slag foam ceramics ranged from 85.6% to 62.53%, and the compressive strength was from 1.74 MPa to 10.42 MPa. The thermal conductivity of steel slag foam ceramics was only 0.067 W (m·K)−1, which shows that it could be used as a thermal insulation material.

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