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.

scholarly journals Modified protocol for quick and large-scale transformation ofPichia pastoris

2018 ◽  
Author(s):  
Ravinder Kumar
Keyword(s):  
Pichia Pastoris ◽  
Cell Density ◽  
Large Scale ◽  
Methylotrophic Yeast ◽  
Protein S ◽  
Scale Transformation ◽  
Biological Research ◽  
Expression And Purification ◽  
Competent Cells ◽  
Resource Space

AbstractOver the last couple of decades, methylotrophic yeast,Pichia pastorisemerges as an important yeast species owing to its increasing application in industry and basic biological research. Transformation ofPichia pastoriscells for the introduction of the gene of interest is common practice for expression and purification of a heterologous protein(s). Presently available protocol ofPichia pastoristransformation involves preparation of competent cells and followed by their transformation. Preparation of competent cells requires growth of cells to certain cell density which requires lots of resource, space, time and efforts. This limits the number of transformations that can be performed by an individual at a time. In the present paper, I will describe a modification in the available protocol which makesP. pastoristransformation hassle free. The present procedure does not require growth of pre-culture or growth of cells to certain cell density rather cells are grown in a patch on YPD plate(s) and rest procedure is performed in small eppendrof tubes which allow a large number of transformations in quickest possible time with minimal resource and efforts. In the end, I also compare various protocols in tabular form which allows the user to choose best suitable procedure depending on the available resource, time, number of transformations, requirement, and efforts. The present modified protocol does not require big centrifuge and shaker which further makes this procedure more useful. I believe that present protocol of transformation with its many unique features will be really helpful to those working withP. pastoris.

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 Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

2015 ◽  
Vol 11 ◽  
pp. 1741-1748 ◽  
Author(s):  
Martina Geier ◽  
Christoph Brandner ◽  
Gernot A Strohmeier ◽  
Mélanie Hall ◽  
Franz S Hartner ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methanol Oxidation ◽  
Large Scale ◽  
Methylotrophic Yeast ◽  
Cofactor Regeneration ◽  
Regeneration System ◽  
Nadh Regeneration ◽  
Whole Cell ◽  
Knock Out ◽  
In The Wild

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.

scholarly journals Cloning and expression of human granulocyte-macrophage colony stimulating factor (hGM-CSF) in Pichia pastoris

2013 ◽  
Vol 16 (3) ◽  
pp. 22-29
Author(s):  
Lien Thi Kim Phan ◽  
Truong Tat Dang ◽  
Hieu Van Tran
Keyword(s):  
Pichia Pastoris ◽  
Shuttle Vector ◽  
Methylotrophic Yeast ◽  
Cloning And Expression ◽  
Colony Stimulating Factor ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Cloned Gene ◽  
Stimulating Factor

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein, belongs to the family of colony stimulating factors (CSFs) that regulate proliferation and differentiation of hematopoietic cells. Recombinant hGM-CSF (rhGM-CSF) is one of FDA-appoved, therapeutic recombinant proteins that have been successfully used to treat many diseases like neutropenia, leukemia, or in combination with chemical therapy, etc. In this study, we reported the production of rhGM-CSF in methylotrophic yeast Pichia pastoris through secretory expression using the inducible AOX1 promoter. The gene hgm-csf encoding for hGM-CSF comprising of 415 bps was isolated using PCR reaction with two primers hGM-F and hGM-R bearing XhoI and NotI restriction sites, respectively. After double digesting with these enzymes, the hgm-csf fragment was cloned into pPICZαA shuttle vector, between the XhoI and NotI sites. Recombinant vector containing the gene hgm-csf, termed pPICZαA/hGMCSF, under the control of promoter AOX has the ability to express hGM-CSF in P. pastoris. The accuracy of cloning strategy was confirmed by digestion with REs, PCR and sequencing. Sequencing alignment showed that the cloned gene completely homologous to those published on Genebank. SacI linearized pPICZαA/hGM-CSF was transformed into P. pastoris X33 strain to establish the recombinant P. pastoris X33::hgm-csf. In methanol-contained, inducing BMMY medium, the recombinant X33 cells expressed and secreted hGM-CSF into the supernatant. The secreted hGM-CSF migrated as a band at about 20 kDa, a diffuse band at the range of 29 to 35 kDa, indicating differentially glycosylated forms, and a band at 14,7kDa which is a nonglycosylated form on SDS-PAGE gel. This result was confirmed by Western blot with specific antibodies.

scholarly journals Design of Thermostable Beta-Propeller Phytases with Activity over a Broad Range of pHs and Their Overproduction by Pichia pastoris

2010 ◽  
Vol 76 (19) ◽  
pp. 6423-6430 ◽  
Author(s):  
José M. Viader-Salvadó ◽  
Juan A. Gallegos-López ◽  
J. Gerardo Carreón-Treviño ◽  
Miguel Castillo-Galván ◽  
Arturo Rojo-Domínguez ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Residual Activity ◽  
Amino Acid Sequences ◽  
Feed Additives ◽  
Total Charge ◽  
Terminal Sequence ◽  
Consensus Approach ◽  
Amino Terminal ◽  
Ph Range ◽  
Amino Terminal Sequence

ABSTRACT Thermostable phytases, which are active over broad pH ranges, may be useful as feed additives, since they can resist the temperatures used in the feed-pelleting process. We designed new beta-propeller phytases, using a structure-guided consensus approach, from a set of amino acid sequences from Bacillus phytases and engineered Pichia pastoris strains to overproduce the enzymes. The recombinant phytases were N-glycosylated, had the correct amino-terminal sequence, showed activity over a pH range of 2.5 to 9, showed a high residual activity after 10 min of heat treatment at 80°C and pH 5.5 or 7.5, and were more thermostable at pH 7.5 than a recombinant form of phytase C from Bacillus subtilis (GenBank accession no. AAC31775). A structural analysis suggested that the higher thermostability may be due to a larger number of hydrogen bonds and to the presence of P257 in a surface loop. In addition, D336 likely plays an important role in the thermostability of the phytases at pH 7.5. The recombinant phytases showed higher thermostability at pH 5.5 than at pH 7.5. This difference was likely due to a different protein total charge at pH 5.5 from that at pH 7.5. The recombinant beta-propeller phytases described here may have potential as feed additives and in the pretreatment of vegetable flours used as ingredients in animal diets.

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