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

Production of foreign proteins in the yeast Pichia pastoris

1995 ◽  
Vol 73 (S1) ◽  
pp. 891-897 ◽  
Author(s):  
James M. Cregg ◽  
David R. Higgins
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Culture Broth ◽  
Heterologous Gene ◽  
Foreign Gene ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

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 Integration Stability of sHBsAg-Multi Expression Cassettes in Pichia pastoris GS115 during Methanol Induction

2020 ◽  
Vol 27 (4) ◽  
pp. 283
Author(s):  
Patricia Gita Naully ◽  
Neni Nurainy ◽  
Elvi Restiawaty ◽  
Dessy Natalia ◽  
Debbie Soefie Retnoningrum ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Hepatitis B ◽  
Vaccine Development ◽  
Expression Cassette ◽  
Foreign Gene ◽  
Pastoris Gs115 ◽  
Methanol Induction ◽  
Small Surface ◽  
Major Health Problem ◽  
Replacement Method

Hepatitis B is the major health problem worldwide including in Indonesia. Vaccination is the best prevention strategy for the disease. For the purpose of vaccine development and to decrease drug import, production of Hepatitis B Virus (HBV) small surface antigen (sHBsAg) from Indonesian HBV subtype is needed. The recombinant protein production can be conducted by integrating multi expression cassettes of sHBsAg gene in Pichia pastoris chromosome using gene replacement method. Such integration method turns out to allow loss of foreign gene from chromosome by excisional recombination-mediated looping out. This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium. The methanol induction was conducted twice at 63-h and 75-h. Integration stability determination was conducted qualitatively using PCR and quantitatively using qPCR absolute quantification. A band of 208 bp with similar intensity was observed after amplification of genomic DNA. All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction.

scholarly journals Exploring differentially expressed key genes related to development of follicle by RNA-seq in Peking ducks (Anas Platyrhynchos)

2018 ◽  
Author(s):  
Jindong Ren ◽  
Changsen Sun ◽  
Li Chen ◽  
Jianhong Hu ◽  
Xuetao Huang ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Expression Patterns ◽  
Follicular Development ◽  
Egg Laying ◽  
Differentially Expressed ◽  
Transcriptional Analysis ◽  
Follicle Development ◽  
Hormone Synthesis ◽  
Key Genes ◽  
Reproductive Phases

AbstractDuck follicle enter different reproductive phases throughout life, and follicle gene expression patterns differ according to these phases. In particular, differentially expressed genes and related to development of follicle (mRNAs) play an important role to explore the key genes in this process; however, the expression profiles of these genes remain unclear. In this study, transcriptome sequencing was used to investigate the expression levels of duck ovarian genes, and comparative transcriptional analysis was carried out to identify differential genes, cluster them into groups and function identification. The results showed differential expression of 593 coding genes between young and laying ducks, and of 518 coding genes between laying and old ducks. In further GO analysis, 35 genes from the comparison bewtween old ducks and laying ducks have significant been changed involved in hormones related to follicle development. They include up-regulated genes StAR, CYP17, EPOX, 3β-HSD, CYP1B1 CYP19A1 and down-regulated genes SR-B1 in laying ducks hormone synthesis than old ducks. Among which EPOX is a key gene for time special highly expression during egg laying stage, and other key regulatory genes’ highly expression showed in young and laying stage and lower expression showing with follicular development stopping. Therefore, EPOX is key regulator for duck follicle development in laying period, when its expression level decrease 98% the follicular development will stopping in duck life cycle.

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.

scholarly journals Genome-scale transcriptional analysis reveals key genes associated with the development of type II diabetes in mice

2017 ◽  
Vol 13 (3) ◽  
pp. 1044-1150 ◽  
Author(s):  
Yuchi Zhang ◽  
Dongwei Han ◽  
Pengyang Yu ◽  
Qijing Huang ◽  
Pengling Ge
Keyword(s):  
Type Ii Diabetes ◽  
Transcriptional Analysis ◽  
Type Ii ◽  
Diabetes In Mice ◽  
Key Genes ◽  
Genome Scale
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