scholarly journals Improved Production ofAspergillus usamiiendo-β-1,4-Xylanase inPichia pastorisvia Combined Strategies

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Jianrong Wang ◽  
Yangyuan Li ◽  
Danni Liu
Keyword(s):  
Pichia Pastoris ◽  
Cell Viability ◽  
Recombinant Proteins ◽  
Recombinant Strain ◽  
Xylanase Activity ◽  
Vitreoscilla Hemoglobin ◽  
Wild Type ◽  
Methanol Induction ◽  
Aspergillus Usamii ◽  
Induction Temperature

A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase fromAspergillus usamii(A. usamii) inPichia pastoris(P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene fromA. usamiiwas optimized forP. pastorisand expressed inP. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression ofxynB-opt, theVitreoscilla hemoglobin(VHb) gene was transformed to the recombinant strain containingxynB-opt. The results showed that recombinant strain harboring thexynB-optandVHb(named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHbin 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHbwas optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHbreached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins inP. pastoris.

scholarly journals The mechanisms of fructose 1,6 bisphosphatase degradation in methylotrophic yeasts Pichia pastoris

2018 ◽  
Vol 22 ◽  
pp. 235-239
Author(s):  
O. V. Dmytruk ◽  
N. V. Bulbotka ◽  
A. A. Sibirny
Keyword(s):  
Pichia Pastoris ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Specific Activity ◽  
Wild Type ◽  
Methylotrophic Yeasts ◽  
Methanol Induction ◽  
Fructose 1,6 Bisphosphatase ◽  
In The Wild

Aim. The study of the mechanisms of fructose-1,6-bisphosphatase degradation in methylotrophic yeasts Pichia pastoris. Methods. Methods of determination the specific activity of fructose-1,6-bisphosphatase in the wild type and mutant strains of methylotrophic yeast P. pastoris after shifting cells from the medium with methanol into the medium with glucose were used. The study of fructose-1,6-bisphosphatase protein degradetion was performed by Western blot analysis. Results. The changes of the specific activity of fructose-1,6-bisphosphatase in the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of P. pastoris in short-term and long-term induction with methanol, and with or without the addition of the MG132 (proteasome degradation inhibitor) was investigated. Degradation of fructose‑1,6‑bisphosphatase by the Western blot analysis in GS200, SMD1163 and Δgss1 strains was studied. Conclusions. It was shown that the duration of cell incubation on methanol has no particular effect on the inactivation of the enzyme. The effect of the proteasome inhibitor MG132 was insignificant. Catabolic inactivation of cytosolic and peroxisomal enzymes is damaged in the Δgss1 mutant as glucose signaling is impaired. Fructose-1,6-bisphosphatase degrades by a vacuolar pathway, regardless of the duration of methanol induction, which correlates with the activity data of this enzyme. Keywords: fructose-1,6-bisphosphatase, yeasts, Pichia pastoris, methanol, autophagy.

scholarly journals Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris

2004 ◽  
Vol 70 (6) ◽  
pp. 3370-3376 ◽  
Author(s):  
Jung Hee Woo ◽  
Yuan Yi Liu ◽  
Scott Stavrou ◽  
David M. Neville
Keyword(s):  
T Cell ◽  
Cell Growth ◽  
Pichia Pastoris ◽  
Yeast Extract ◽  
Consumption Rate ◽  
Expression System ◽  
Tolerance Induction ◽  
Methanol Induction ◽  
Cell Expression ◽  
Induction Temperature

ABSTRACT The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G4S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut+ ) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15�C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris.

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

Methanol Induction Optimization for Recombinant Human Consensus Interferon Mutant Production in Pichia pastoris

2010 ◽  
Vol 150 ◽  
pp. 540-540
Author(s):  
Dan Wu ◽  
Ju Chu ◽  
Yu-You Hao ◽  
Yong-Hong Wang ◽  
Ying-Ping Zhuang ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methanol Induction ◽  
Consensus Interferon

scholarly journals Synergistic Effect of Different Plant Cell Wall–Degrading Enzymes Is Important for Virulence of Fusarium graminearum

2017 ◽  
Vol 30 (11) ◽  
pp. 886-895 ◽  
Author(s):  
Maria Chiara Paccanaro ◽  
Luca Sella ◽  
Carla Castiglioni ◽  
Francesca Giacomello ◽  
Ana Lilia Martínez-Rocha ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fusarium Graminearum ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Xylanase Activity ◽  
Cellulase Activity ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Xylanase Production ◽  
Significant Difference

Endo-polygalacturonases (PGs) and xylanases have been shown to play an important role during pathogenesis of some fungal pathogens of dicot plants, while their role in monocot pathogens is less defined. Pg1 and xyr1 genes of the wheat pathogen Fusarium graminearum encode the main PG and the major regulator of xylanase production, respectively. Single- and double-disrupted mutants for these genes were obtained to assess their contribution to fungal infection. Compared with wild-type strain, the ∆pg mutant showed a nearly abolished PG activity, slight reduced virulence on soybean seedlings, but no significant difference in disease symptoms on wheat spikes; the ∆xyr mutant was strongly reduced in xylanase activity and moderately reduced in cellulase activity but was as virulent as wild type on both soybean and wheat plants. Consequently, the ΔpgΔxyr double mutant was impaired in xylanase, PG, and cellulase activities but, differently from single mutants, was significantly reduced in virulence on both plants. These findings demonstrate that the concurrent presence of PG, xylanase, and cellulase activities is necessary for full virulence. The observation that the uronides released from wheat cell wall after a F. graminearum PG treatment were largely increased by the fungal xylanases suggests that these enzymes act synergistically in deconstructing the plant cell wall.

Cloning and Expression of a Novel Xylanase Gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris

2012 ◽  
Vol 167 (8) ◽  
pp. 2198-2211 ◽  
Author(s):  
Huimin Zhang ◽  
Minchen Wu ◽  
Jianfang Li ◽  
Shujuan Gao ◽  
Yanjun Yang
Keyword(s):  
Pichia Pastoris ◽  
Cloning And Expression ◽  
Xylanase Gene ◽  
Aspergillus Usamii

scholarly journals In Vivo Functional Assay of a Recombinant Aquaporin in Pichia pastoris

2006 ◽  
Vol 72 (2) ◽  
pp. 1507-1514 ◽  
Author(s):  
Mark J. Daniels ◽  
Malcolm R. Wood ◽  
Mark Yeager
Keyword(s):  
Pichia Pastoris ◽  
Linear Relationship ◽  
Osmotic Shock ◽  
Water Channel ◽  
Yeast Cells ◽  
Mercury Chloride ◽  
Functional Assay ◽  
Channel Activity ◽  
Wild Type

ABSTRACT The water channel protein PvTIP3;1 (α-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.

Sign in / Sign up

Export Citation Format

Share Document