Enhancement of alkaline phytase production in Pichia pastoris: Influence of gene dosage, sequence optimization and expression temperature

2012 ◽  
Vol 84 (2) ◽  
pp. 247-254 ◽  
Author(s):  
Mimi Yang ◽  
Steven C. Johnson ◽  
Pushpalatha P.N. Murthy
Keyword(s):  
Pichia Pastoris ◽  
Gene Dosage ◽  
Phytase Production ◽  
Sequence Optimization ◽  
Alkaline Phytase

Medium Optimization for the Production of Phytase by Recombinant Pichia pastoris Grown on Glycerol

2011 ◽  
Vol 9 (1) ◽  
Author(s):  
Min Liu ◽  
Gabriel Potvin ◽  
Yiru Gan ◽  
Zhanbin Huang ◽  
Zisheng Zhang
Keyword(s):  
Pichia Pastoris ◽  
Phytase Production ◽  
Medium Components ◽  
Gap Promoter ◽  
Recombinant Pichia Pastoris ◽  
Burman Design ◽  
Statistical Designs ◽  
Plackett Burman Design ◽  
Central Composite ◽  
Three Components

Based on statistical designs, minimal salts medium, commonly used for yeast cultivation, was optimized to maximize GAP promoter-mediated phytase production by recombinant Pichia pastoris grown on glycerol. A Plackett-Burman design was followed to screen medium components to determine those that significantly affected phytase production. Of the 8 components studied, the concentrations of K2SO4, CaSO4•2H2O and MgSO4•7H2O were identified as having a significant effect. These three components were subsequently optimized by response surface methodology using a central composite design. The optimal concentrations of the three components, leading to a maximal extracellular phytase activity of 161.64 U/ml, were K2SO4 13.25g/l, CaSO4•2H2O 1.03g/l and MgSO4•7H2O 17.94g/l. The activity measured in cultures using optimized growth medium is significantly higher than the 73.31 U/ml measured in cultures using standard minimal salts media. The theoretical phytase yields predicted by the developed model were very close to experimentally obtained values.

Effect of cooperation of chaperones and gene dosage on the expression of porcine PGLYRP-1 in Pichia pastoris

2016 ◽  
Vol 100 (12) ◽  
pp. 5453-5465 ◽  
Author(s):  
Jun Yang ◽  
Zhipeng Lu ◽  
Jiawei Chen ◽  
Pinpin Chu ◽  
Qingmei Cheng ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Gene Dosage

scholarly journals High-Level Production of a Thermostable Mutant of Yarrowia lipolytica Lipase 2 in Pichia pastoris

2019 ◽  
Vol 21 (1) ◽  
pp. 279
Author(s):  
Qinghua Zhou ◽  
Zhixin Su ◽  
Liangcheng Jiao ◽  
Yao Wang ◽  
Kaixin Yang ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Yarrowia Lipolytica ◽  
Large Scale ◽  
Gene Dosage ◽  
Life Time ◽  
Industrial Applications ◽  
Culture Conditions ◽  
Scale Production ◽  
Large Scale Production ◽  
High Level

As a promising biocatalyst, Yarrowia lipolytica lipase 2 (YlLip2) is limited in its industrial applications due to its low thermostability. In this study, a thermostable YlLip2 mutant was overexpressed in Pichia pastoris and its half-life time was over 30 min at 80 °C. To obtain a higher protein secretion level, the gene dosage of the mutated lip2 gene was optimized and the lipase activity was improved by about 89%. Then, the YlLip2 activity of the obtained strain further increased from 482 to 1465 U/mL via optimizing the shaking flask culture conditions. Subsequently, Hac1p and Vitreoscilla hemoglobin (VHb) were coexpressed with the YlLip2 mutant to reduce the endoplasmic reticulum stress and enhance the oxygen uptake efficiency in the recombinant strains, respectively. Furthermore, high-density fermentations were performed in a 3 L bioreactor and the production of the YlLip2 mutant reached 9080 U/mL. The results demonstrated that the expression level of the thermostable YlLip2 mutant was predominantly enhanced via the combination of these strategies in P. pastoris, which forms a consolidated basis for its large-scale production and future industrial applications.

scholarly journals Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Javier Garrigós-Martínez ◽  
Miguel Angel Nieto-Taype ◽  
Arnau Gasset-Franch ◽  
José Luis Montesinos-Seguí ◽  
Xavier Garcia-Ortega ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Rate ◽  
Pichia Pastoris ◽  
Specific Growth Rate ◽  
Specific Growth ◽  
Gene Dosage ◽  
Fed Batch ◽  
Komagataella Phaffii ◽  
Production Kinetics ◽  
Kinetics Of

Abstract Background The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear. Results The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities. Conclusions PAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression—heterologous genes included—, thus directly influencing the production kinetics of recombinant protein.

Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes

2010 ◽  
Vol 89 (4) ◽  
pp. 1127-1135 ◽  
Author(s):  
Taicheng Zhu ◽  
Meijin Guo ◽  
Yingping Zhuang ◽  
Ju Chu ◽  
Siliang Zhang
Keyword(s):  
Pichia Pastoris ◽  
Gene Dosage ◽  
Transcriptional Analysis ◽  
Foreign Gene ◽  
Key Genes

scholarly journals Co-feeding strategy to enhance phytase production in Pichia pastoris

2014 ◽  
Vol 13 (21) ◽  
pp. 2181-2187 ◽  
Author(s):  
Jean Bernard Ndayambaje, ◽  
Meenakshisundaram S.
Keyword(s):  
Pichia Pastoris ◽  
Feeding Strategy ◽  
Phytase Production

scholarly journals Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system

2019 ◽  
Vol 19 (6) ◽  
Author(s):  
Shinobu Takagi ◽  
Noriko Tsutsumi ◽  
Yuji Terui ◽  
XiangYu Kong ◽  
Hiroya Yurimoto ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Expression System ◽  
Constitutive Expression ◽  
Free Expression ◽  
Aox1 Promoter ◽  
Phytase Production ◽  
Model Case ◽  
Komagataella Phaffii ◽  
Gap Promoter ◽  
Important Region

ABSTRACT The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1—a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

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